CN115381949A - Application of targeted inhibition of pigment epithelium-derived factor in promoting liver regeneration and improving liver injury - Google Patents

Abstract

The invention provides an application of targeted inhibition of Pigment Epithelium Derived Factor (PEDF) in promoting liver regeneration and improving liver injury. The invention discloses that PEDF plays a negative regulation role in the liver regeneration process for the first time, and the PEDF can be used as a novel target spot for diagnosis or treatment. The compound can be used as a target to develop a medicament for promoting liver regeneration, preventing, relieving and/or treating liver injury; the molecular marker can be used for diagnosing and prognostically evaluating the liver regeneration capability or the liver injury condition. Meanwhile, the inventor also screens and optimizes a targeted drug which can be targeted to PEDF and has particularly excellent in-vivo and in-vitro action effects.

Description

Targeted inhibition of pigment epithelium-derived factor in promoting liver regeneration and improving liver injury Applications

technical field

The invention belongs to the field of application of biotechnology drugs; more specifically, the invention relates to the application of targeted inhibition of pigment epithelium-derived factor (Pigment epithelium-derived factor, PEDF, also known as Serpinf1) in promoting liver regeneration and improving liver damage.

Background technique

The liver is one of the most functional and complex parenchymal organs in the body. Its main functions include regulating the balance of carbohydrates and lipids, participating in the metabolism and biotransformation of exogenous substances, the generation and excretion of bile, the storage of vitamins, Synthesis of secretory proteins, generation and elimination of coagulation substances, and plays an important role in specific and nonspecific immunity. As the center of human substance metabolism, the liver plays an important role in various physiological and pathological processes, and many diseases can also affect the liver.

Partial liver resection is currently the most common and effective method for treating liver tumors, and the strong regeneration capacity of normal liver tissue is the basis for effective liver resection. However, limited by the bottleneck of early diagnosis technology for liver tumors, most liver tumors have developed to the middle and late stages when they are first diagnosed. Due to the large tumor volume and many liver segments involved, the volume of the residual liver after resection cannot meet the metabolic needs of the human body. It can lead to life-threatening complications such as liver failure. In order to solve the problem of insufficient residual liver volume, a variety of surgical methods to stimulate rapid regeneration of the residual liver have been developed clinically, such as portal vein ligation (Portal Vein Ligation, PVL)/portal vein embolization (Portal Vein Embolization, PVE), combined liver separation and portal vein Ligated two-step liver resection (Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy, ALPPS).

However, the effectiveness of such procedures as described above is still controversial, and the risk of postoperative liver failure due to insufficient residual liver volume remains high. It is of great significance for the surgical treatment of liver cancer to study the pathways related to liver regeneration, explore the targets for regulating liver regeneration and develop safe and effective drugs to accelerate the regeneration of the residual liver in the perioperative period and reduce the incidence of liver failure.

Liver regeneration is a complex process regulated by different types of cells. Hepatocytes, immune cells, cholangiocytes, stellate cells, and liver sinusoidal endothelial cells (LSEC) are all involved in the regulation of liver regeneration.

Therefore, further research and development of new technical solutions for promoting liver regeneration and improving liver injury are needed in this field, in order to provide a new therapeutic approach for the clinic.

Contents of the invention

The purpose of the present invention is to provide the application of targeted inhibition of pigment epithelium-derived factor in promoting liver regeneration and improving liver damage.

In the first aspect of the present invention, an application of an inhibitor of pigment epithelium-derived factor (PEDF) is provided for: preparing a composition for promoting liver regeneration; or preparing a composition for preventing, alleviating and/or treating liver damage.

In a preferred example, the composition is also used to promote the proliferation of endothelial cells.

In a preferred example, the composition is also used to promote the ring-forming ability of endothelial cells;

In a preferred example, the composition is also used to stimulate the generation of new blood vessels in the residual liver or damaged liver; and/or

In a preferred example, the composition is also used to accelerate the recovery of liver-to-volume ratio and liver function after liver resection.

In another preferred example, the endothelial cells include (but not limited to): liver sinusoidal endothelial cells (LSEC), vascular endothelial cells (such as vein endothelial cells), and lymphatic endothelial cells.

In another preferred example, the inhibitor of pigment epithelium-derived factor includes (but not limited to): reagents for knocking out or silencing pigment epithelium-derived factor; binding molecules such as antibodies that specifically bind to pigment epithelium-derived factor; Chemical small molecule antagonists or inhibitors to pigment epithelium-derived factor; or agents that interfere with the interaction of pigment epithelium-derived factor with effector molecules or their receptors.

In another preferred example, the reagents for knocking out or silencing pigment epithelium-derived factors include (but are not limited to): interfering molecules that specifically interfere with the expression of genes encoding pigment epithelium-derived factors, CRISPR gene editing for pigment epithelium-derived factors Reagents, homologous recombination reagents or site-directed mutagenesis reagents for pigment epithelium-derived factors, the homologous recombination reagents or site-directed mutagenesis reagents perform loss-of-function mutations on pigment epithelium-derived factors.

In another preferred example, the interference molecule includes (but not limited to) shRNA, siRNA, miRNA, antisense nucleic acid, or a construct capable of forming the shRNA, siRNA, miRNA, antisense nucleic acid;

In another preferred example, the reagent for knocking out or silencing pigment epithelium-derived factors is an interfering molecule, targeting at positions 382-402, 323-343, 323-343, Position 246-266, position 447-467 or a combination thereof; preferably targeting position 382-402, position 323-343 or a combination thereof in the nucleotide sequence shown in SEQ ID NO:1.

In another preferred example, the inhibitor (such as interference molecule or sgRNA, etc.) is introduced into the target site (focus, such as postoperative liver) through an expression construct (expression vector); the expression construct includes: a viral vector , non-viral vectors; preferably, the viral vectors include (but not limited to): adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, and retroviral vectors.

In another preferred example, the liver injury includes liver dysfunction after liver surgery, liver injury caused by hepatitis, liver fibrosis, liver cirrhosis, end-stage liver disease, liver cancer, alcoholic liver disease, liver metabolic disease or liver failure;

In another preferred example, the liver surgery includes a treatment method based on the normal liver regeneration ability, by destroying the liver tissue at the lesion site, retaining the normal liver tissue and making it compensate for hyperplasia to exert normal liver function; more preferably Including but not limited to: traditional subtotal hepatectomy, secondary liver resection with portal vein ligation (Portal Vein Ligation, PVL), secondary liver resection combined with liver segmentation and portal vein ligation (Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy) , ALPPS), radiofrequency ablation, microwave ablation, cryoablation, hepatic arterial chemoembolization, hepatic arterial embolization radiotherapy, stereotactic radiotherapy.

In another aspect of the present invention, the use of reagents for specifically recognizing or amplifying pigment epithelium-derived factors is provided for the preparation of diagnostic reagents or kits for diagnosing or evaluating liver regeneration ability or liver injury; preferably, The reagents include (but are not limited to): binding molecules (such as antibodies or ligands) that specifically bind to the pigment epithelium-derived factor protein; primers that specifically amplify the pigment epithelium-derived factor gene; specifically recognize the pigment epithelium-derived factor gene or, a chip that specifically recognizes the gene of pigment epithelium-derived factor.

In another aspect of the present invention, a pharmaceutical composition or kit for promoting liver regeneration or preventing, alleviating and/or treating liver damage is provided, which includes an inhibitor of pigment epithelium-derived factor, and the inhibitor includes selected From: interfering molecules that specifically interfere with the expression of genes encoding pigment epithelium-derived factors, CRISPR gene editing reagents for pigment epithelium-derived factors, homologous recombination reagents or site-directed mutagenesis reagents for pigment epithelium-derived factors, said homologous recombination reagents or The site-directed mutagenesis reagent performs a loss-of-function mutation on the pigment epithelium-derived factor; preferably, the interfering molecule includes (but not limited to) shRNA, siRNA, miRNA, antisense nucleic acid, or can form the shRNA, siRNA, miRNA, antisense The construct of the sense nucleic acid; more preferably, the interfering molecule is targeted at the 382-402, the 323-343, the 246-266, and the 447-467 in the nucleotide sequence shown in SEQ ID NO:1 Or a combination thereof; preferably targeting at positions 382-402, 323-343 or a combination thereof in the nucleotide sequence shown in SEQ ID NO:1.

In another preferred example, the composition is a pharmaceutical composition, a bioactive preparation composition, a health product composition or a food composition.

In another preferred embodiment, the composition is a combination of one or more of inhibitors targeting PEDF gene (including biologically active agents, PEDF-related inhibitors and PEDF neutralizing antibodies) with pharmaceutically acceptable Mixed with the carrier of the present invention, a composition for stimulating liver regeneration after liver surgery and preventing, relieving and/or treating liver dysfunction after hepatectomy is obtained.

In another aspect of the present invention, a method for screening potential substances for promoting liver regeneration or preventing, alleviating and/or treating liver damage is provided, the method comprising: (1) treating an expression system with a candidate substance, which expresses Pigment epithelium-derived factor; and, (2) detecting the expression or activity of pigment epithelium-derived factor in the system; if the candidate substance is statistically down-regulated (significantly down-regulated, such as down-regulated by more than 10%, more than 20%, and 50%) above, above 80%, etc., or make it non-expressed or inactive) expression or activity of pigment epithelium-derived factor, then the candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating and/or treating liver damage.

In another preferred embodiment, the system in step (1) is an endothelial cell (culture) system; preferably, the endothelial cells include (but not limited to): liver sinusoidal endothelial cells (LSEC), vascular endothelial cells (such as vein endothelial cells), lymphatic endothelial cells; step (2) also includes: detecting the proliferation ability or ring-forming ability of endothelial cells in the system; if its proliferation ability or ring-forming ability is promoted (significantly promoted, such as If it is increased by more than 10%, more than 20%, more than 50%, more than 80% or higher), then the candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating and/or treating liver damage.

In another preferred example, step (1) includes: in the test group, adding candidate substances to the expression system; and/or, step (2) includes: detecting the expression of pigment epithelium-derived factor in the system or activity, or detect the proliferation ability or ring-forming ability of endothelial cells; and compare with the control group, wherein the control group is an expression system without adding the candidate substance; if the candidate substance statistically down-regulates the pigment epithelium If the expression or activity of derivative factors, or the proliferation ability or ring-forming ability of endothelial cells is statistically decreased, the candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating and/or treating liver damage.

In another preferred example, the candidate substances include (but are not limited to): regulatory molecules or their constructs designed for pigment epithelium-derived factors, their fragments or variants, their encoding genes or their upstream and downstream molecules or signaling pathways (such as shRNA, siRNA, gene editing practice, expression vector, recombinant viral or non-viral constructs, etc.), chemical small molecules (such as specific inhibitors or antagonists), interacting molecules, etc.

In another preferred embodiment, the system is selected from: cell system (such as cells or cell cultures expressing pigment epithelium-derived factors), subcellular (culture) system, solution system, tissue system, organ system or animal system .

In another preferred example, the method further includes: conducting further cell experiments and/or animal experiments on the obtained potential substances, so as to further select and determine the effects of promoting liver regeneration and preventing, alleviating and/or Substance useful in the treatment of liver damage.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Fig. 1A, Survival curves of littermate male C57 wild-type mice after ALPPS surgery given anti-PEDF antibody and placebo respectively.

Fig. 1B, the hepatic-to-body ratio recovery rate diagram of littermate male C57 wild-type mice after ALPPS operation after administration of anti-PEDF antibody and placebo respectively.

Fig. 2. Serum ALT and AST graphs of liver function test after anti-PEDF antibody and placebo were administered to male littermate C57 wild-type mice during the perioperative period of ALPPS.

Figure 3. Multiple immunofluorescent staining images of liver tissue in PEDF antibody group and placebo group on day 2 and day 6 after ALLPS I stage.

Figure 4. Western blot of LSEC-related markers LYVE and CD146 in liver tissue of PEDF antibody group and placebo group on day 2 and day 6 after ALLPS I stage.

Fig. 5. EDU proliferation test diagram of LSEC cultured in the medium supplemented with PEDF antibody and antibody dilution for 48 hours.

Fig. 6. Bright-field images of the ring-forming ability of LSEC cultured in the medium supplemented with PEDF antibody and antibody dilution for 6 hours.

Figure 7. Correlation between PEDF transcription level in liver tissue and postoperative ALT level in patients with hepatectomy.

Figure 8. The conditioned medium of hepatocytes with low PEDF transcription level promotes the ring-forming ability of HUVEC cells.

Figure 9. After adenovirus-mediated targeted interference with different sequences of PEDF in primary hepatocytes of patients undergoing hepatectomy, the CCK8 proliferation assay of HUVEC cells stimulated by the conditioned medium of the hepatocytes was detected.

FIG. 10 . After adenovirus-mediated targeted interference with different sequences of PEDF in primary hepatocytes of patients undergoing hepatectomy, the microscopic bright-field images of HUVEC cell ringing ability stimulated by the conditioned medium of the hepatocytes.

Detailed ways

Through in-depth research and analysis, the inventors revealed for the first time that pigment epithelium-derived factor (PEDF) plays a negative regulatory role in the liver regeneration process, and it can be used as a new target for diagnosis or treatment. Using it as a target, drugs for promoting liver regeneration, preventing, alleviating and/or treating liver damage can be developed; using it as a molecular marker can be used to diagnose and evaluate liver regeneration ability or liver damage. At the same time, the inventors also screened and optimized targeted drugs that can target PEDF and have particularly excellent in vivo and in vitro effects.

PEDF

PEDF, also known as Serpin F1, Gene ID: 5176, is a secreted glycoprotein and a member of the serine protease inhibitor superfamily. It is widely distributed in human tissues, especially abundantly expressed in liver and fat. Its main physiological functions include inhibiting angiogenesis, regulating lipid metabolism, anti-oxidation, anti-inflammation, anti-tumor, and nourishing nerves. Studies have found that PEDF can inhibit the growth of human liver cancer xenografts in nude mice by inhibiting tumor angiogenesis (Gao Yun et al.; pigment epithelium-derived factor inhibits the growth of human liver cancer xenografts in nude mice; National Artificial Liver Expert Forum; 2009). PEDF can also inhibit the invasion and metastasis of breast cancer cells by regulating epithelial-mesenchymal transition (Dan Zhou et al; Pigment epithelium-derived factor inhibits the invasion and metastasis of breast cancer cells by regulating epithelial-mesenchymal transition; Journal of Southern Medical University; 2018). In terms of regulating normal cell physiological functions, studies have shown that PEDF participates in the regulation of lipid metabolism in human HepG2 hepatocytes (Zhong Li et al.; Effect of pigment epithelium-derived factor on lipid metabolism in human HepG2 hepatocytes. Journal of Chongqing Medical University 2012), and can be passed Antioxidant effect alleviates oxidized low-density lipoprotein-induced human umbilical vein endothelial cell injury (Ma Shouyuan et al.; Pigment epithelium-derived factor alleviates oxidized low-density lipoprotein-induced human umbilical vein endothelial cell injury experiment; PLA Medical College Journal 2017; 38:155 -159). In addition, studies also believe that PEDF has functions in the nutrition and repair of nerves. It can be seen that the research on PEDF in this field has shown that it has multiple functions, but so far no researchers have associated it with liver regeneration and the promotion of recovery from liver damage. After in-depth research in vitro and in vivo, the inventors determined PEDF as the research target for liver regeneration.

The PEDF of the present invention may be naturally occurring, for example, it may be isolated or purified from mammals. In addition, the PEDF can also be artificially prepared, for example, the recombinant PEDF can be produced according to conventional genetic engineering recombination techniques, so as to be applied in experiments or clinics. When applicable, recombinant PEDF can be used. The PEDF includes full-length PEDF or its biologically active fragments. Preferably, the amino acid sequence of PEDF may be substantially the same as the sequence shown in SEQ ID NO:2; its nucleotide sequence may be substantially the same as the sequence shown in SEQ ID NO:1.

The amino acid sequence of PEDF formed by substitution, deletion or addition of one or more amino acid residues is also included in the present invention. PEDF or its biologically active fragments include a part of conservative amino acid replacement sequence, and the amino acid replacement sequence does not affect its activity or retains part of its activity. Appropriate substitution of amino acids is a well-known technique in the art, which can be readily performed and ensures that the biological activity of the resulting molecule is not altered. All biologically active fragments of PEDF that are expected to be available can be used in the present invention. Here, the biologically active fragment of PEDF refers to a polypeptide that can still maintain all or part of the functions of the full-length PEDF. Usually, the biologically active fragment retains at least 50% of the activity of the full-length PEDF. Under more preferred conditions, the active fragment can maintain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of the full-length PEDF.

Focusing on this PEDF target, the inventors conducted in-depth experimental demonstrations, including demonstrations at the cellular level and animal level, and determined the relationship between the PEDF gene and liver regeneration. According to the embodiments of the present invention, overexpression of the PEDF gene can inhibit liver regeneration after ALPPS and delay recovery of liver function after ALPPS. Targeted inhibition of PEDF expression, such as interfering with PEDF gene expression, using PEDF neutralizing antibodies and/or related preparations that inhibit its expression, can stimulate liver regeneration after ALPPS and accelerate the recovery of liver function after ALPPS.

In a more specific embodiment of the present invention, the inventors used primary cell proliferation detection in vitro, primary liver sinusoidal endothelial cell ring formation function detection in vitro, animal liver function detection in vivo, liver tissue immunofluorescence, liver tissue immunoblotting The role of PEDF gene in liver regeneration was determined by various experimental methods, as follows: 1. The use of PEDF neutralizing antibody can significantly improve the survival rate of animals after ALPPS; 2. Using PEDF neutralizing antibody can significantly increase the survival rate of animals after ALPPS. The liver-to-body ratio recovery rate of animals after ALPPS; 3. The use of PEDF neutralizing antibodies can significantly reduce the serum alanine aminotransferase (alanine aminotransferase, ALT) and aspartate aminotransferase (aspartateaminotransferase) of animals after ALPPS , AST) level; 4. The use of PEDF neutralizing antibody can significantly stimulate the proliferation of hepatic sinusoidal endothelial cells in the later stage of liver regeneration after ALPPS; The effect of promoting endothelial cell proliferation and ring formation can be significantly enhanced by PEDF neutralizing antibody; 6. Patients who underwent hepatectomy were divided into low, medium and high groups according to the PEDF transcription level in liver tissue, and the ALT level was detected after 3 days after operation. , the level of PEDF transcription is higher, and the ALT level is relatively higher at 3 days after operation; 7. After the primary hepatocytes were isolated from the liver tissues of the above patients and cultured in vitro, the conditioned medium of the hepatocytes was used to stimulate HUVEC cell. It was found that the hepatocyte conditioned medium with low PEDF transcription level can promote the ring-forming ability of HUVEC cells more than the hepatocyte conditioned medium with high PEDF transcription level; 8. Sorting human primary hepatocytes, and mediated target After interfering with PEDF, the conditioned medium of the hepatocytes can promote the proliferation and ring-forming ability of HUVEC cells.

From the above results, it can be seen that during ALPPS surgery, the use of PEDF neutralizing antibodies to inhibit PEDF can stimulate the proliferation of hepatic sinusoidal endothelial cells, accelerate the recovery rate of liver-to-body ratio, promote the recovery of liver function, and improve the survival rate of animals after ALPPS . Moreover, the level of PEDF transcription in liver tissue of patients undergoing hepatectomy is closely related to the recovery of postoperative liver function. Patients with low PEDF transcription level have relatively faster recovery of postoperative liver function, which may be related to the improvement of vascular endothelial cell function. After interfering with PEDF to suppress its expression in hepatocytes from patients undergoing hepatectomy, the conditioned medium of the hepatocytes can promote the proliferation and ring-forming ability of human umbilical cord endothelial cell line HUVEC cells (as a cell model of endothelial cells). Therefore, targeted inhibition of PEDF can promote liver regeneration and accelerate the recovery of liver function after hepatectomy, which provides a theoretical and clinical basis for the prevention, alleviation and/or treatment of liver dysfunction after hepatectomy.

Aiming at the above-mentioned functions of the PEDF gene, biologically active preparations targeted to interfere with the PEDF gene, PEDF-related inhibitors and neutralizing antibodies can be used as drugs to promote liver regeneration and prevent, relieve and/or treat liver function after liver surgery obstacle. PEDF can be used as a drug target for screening drugs that promote liver regeneration and prevent, relieve and/or treat liver dysfunction after liver surgery; PEDF can also be used as a target gene in gene therapy for the design and preparation of drugs that promote liver regeneration and Drugs and/or biological agents for the prevention, alleviation and/or treatment of liver dysfunction after liver surgery.

PEDF inhibitors and their applications

Based on the inventor's above-mentioned new discovery, the present invention provides a kind of purposes of the inhibitor of PEDF or its coded gene, be used for preparing and inhibiting and promoting liver regeneration and preventing, alleviating and/or treating liver damage (such as liver after hepatectomy) dysfunction) composition.

As used herein, the inhibitors of PEDF or its coding gene include down-regulators (such as expression down-regulators, activity down-regulators), antagonists, blockers, blockers, degradation agents, etc., and these terms are interchangeable use.

The inhibitor of PEDF or its coding gene refers to any inhibitor that can reduce the activity of PEDF, reduce the stability of PEDF or its coding gene, down-regulate the expression of PEDF, reduce the effective time of PEDF, or inhibit the transcription and translation of PEDF gene Substances, these substances can be used in the present invention as substances useful for down-regulating PEDF, so as to promote liver regeneration or prevent, alleviate or treat liver damage (such as liver dysfunction after hepatectomy). For example, the inhibitors include interfering RNA molecules or antisense nucleotides that specifically interfere with the expression of the PEDF gene; antibodies or ligands that specifically bind to the protein encoded by the PEDF gene; and so on.

As a particularly preferred mode of the present invention, the inhibitor is a PEDF-specific interfering RNA molecule (such as siRNA, shRNA, miRNA, etc.), and according to the sequence information of PEDF provided in the present invention, such an interfering RNA molecule can be prepared and obtained. RNA molecule. There is no particular limitation on the preparation method of the interfering RNA molecule, including but not limited to: chemical synthesis method, in vitro transcription method and so on. The interfering RNA can be delivered into cells by using appropriate transfection reagents, or can also be delivered into cells by various techniques known in the art. The inventor's research results have found that, for different segments of the PEDF gene, although molecules with certain interference capabilities can be obtained, the interference sequences targeting some specific segments have good specificity, and the targeting inhibition effect is especially good. excellent. Therefore, as the most preferred mode of the present invention, interfering molecules targeting positions 382-402 and 323-343 in the nucleotide sequence shown in SEQ ID NO: 1 are used as inhibitors. The preferred inhibitor properly down-regulates PEDF (without affecting other in vivo mechanisms of its production) without producing visible side effects, and effectively promotes liver regeneration.

As a particularly preferred mode of the present invention, the inhibitor is a neutralizing antibody specifically targeting PEDF (such as the polyclonal antibody in the embodiment of the present invention), which inhibits the function of PEDF at the protein level. The polyclonal antibody against PEDF can be prepared by conventional methods, for example, it can be obtained by introducing the PEDF protein into animals, for example, after mixing the PEDF protein with Freund's adjuvant in an appropriate ratio (such as 1:1) Immunize animals. The method of immunization can use subcutaneous injection of animals. The animal can be selected from rabbits, sheep, cattle and the like. For example, rabbits can be used to produce the polyclonal antibody: 2-3 months after the rabbit is immunized, the antiserum is harvested and purified from the rabbit's venous blood to obtain the specific polyclonal antibody.

According to the new discovery of the present invention, the hybridoma technology can also be used to prepare anti-PEDF monoclonal antibody. When the hybridoma is obtained, the hybridoma can be cultured and amplified in vitro according to a conventional animal cell culture method, so as to secrete anti-PEDF monoclonal antibody. As an embodiment, the anti-PEDF monoclonal antibody can be prepared by the following preparation methods: (1) provide mice pretreated with adjuvant; (2) inoculate the mouse intraperitoneally with the hybridoma cells and secrete the monoclonal antibody; (3) Extract ascites, separate and obtain the monoclonal antibody. Monoclonal antibodies isolated from ascitic fluid are further purified to obtain high-purity antibodies. The monoclonal antibody of the present invention can also be produced by recombinant methods or synthesized by a polypeptide synthesizer. Those skilled in the art understand that after obtaining the hybridoma cell line of the monoclonal antibody or knowing the monoclonal antibody through sequencing and other means, those skilled in the art can conveniently obtain the antibody.

As a preferred mode of the present invention, an anti-PEDF neutralizing antibody with excellent performance is provided. The antibody has high specificity for PEDF and does not bind to other proteins other than PEDF. Moreover, it can effectively block the effect of PEDF on inhibiting angiogenesis (without affecting other in vivo mechanisms of its production), and effectively promote liver regeneration.

As an optional mode of the present invention, the inhibitor may be a small molecule compound against PEDF. Those skilled in the art can use conventional screening methods in the art to screen such small molecule compounds. For example, in the embodiments of the present invention, combined with the regulation mechanism disclosed in the present invention, several optional screening methods are provided.

As a preferred mode of the present invention, the CRISPR/Cas (such as Cas9) system can be used for targeted gene editing, thereby knocking out the PEDF gene in the targeted disease region. A common method for knocking out the PEDF gene includes: co-transferring sgRNA or nucleic acid capable of forming the sgRNA, Cas9 mRNA or nucleic acid capable of forming the Cas9 mRNA into the targeted region or targeted cells. After the target site is determined, known methods can be used to introduce sgRNA and Cas9 into the cells. The nucleic acid that can form the sgRNA is a nucleic acid construct or an expression vector, or the nucleic acid that can form the Cas9mRNA is a nucleic acid construct or an expression vector, and these expression vectors are introduced into the cell, so that in the cell Form active sgRNA and Cas9 mRNA.

As an optional mode of the present invention, a method of homologous recombination can be used to specifically target PEDF to cause expression defect or lack of expression. Cre and loxp methods can also be used to selectively knock out relevant genes in the genome of cells, reduce their expression or inactivate them.

The above are some representative or preferred ways of down-regulating PEDF. After those skilled in the art understand the general scheme of the present invention, other methods known in the art can also be adopted to regulate PEDF, and these methods are also included in the present invention.

Applications related to diagnosis and prognosis evaluation

In the present invention, targets that play an important regulatory role in liver regeneration are revealed. Based on this new discovery of the inventors, PEDF can be used as a target for diagnosis or prognosis evaluation of liver regeneration ability or liver injury recovery: (i) type and differential diagnosis after liver injury; (ii) evaluate the relevant population ( Such as the population after liver surgery), drug efficacy, prognosis assessment, and selection of appropriate treatment methods. For example, people with abnormal gene expression of PEDF can be isolated, so that more targeted targeted therapy can be carried out.

By judging the expression or activity of PEDF in the sample to be evaluated, the prognosis of the subject providing the sample to be evaluated can be predicted, and an appropriate drug can be selected for treatment. Usually, a threshold value of PEDF expression can be specified, and when the expression of PEDF is higher than the specified threshold value, the regimen of inhibiting PEDF can be considered for treatment. The threshold is easy to determine for those skilled in the art, for example, after comparing the expression of PEDF in normal human cells or tissues with the expression of PEDF in test cells or tissues, the PEDF Threshold for expressing abnormalities. According to different measurement parameters, measurement instruments, etc., the specific value of the threshold may be different.

Various techniques known in the art can be used to detect the presence or absence and expression of the PEDF gene, and these techniques are included in the present invention. For example, existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc. can be used, and these methods can be used in combination.

The invention also provides reagents for detecting the presence or absence and expression of PEDF or its coding gene in the analyte. Preferably, when performing detection at the gene level, primers for specifically amplifying PEDF can be used; or probes for specifically recognizing PEDF can be used to determine the presence of the PEDF gene; when performing detection at the protein level, specific Antibodies or ligands that bind the protein encoded by PEDF were used to determine the expression of PEDF.

The method of detecting the expression of PEDF in an analyte by using an antibody specifically binding to PEDF is well known to those skilled in the art.

The design of specific probes for the PEDF gene is a technique well known to those skilled in the art, for example, preparing a probe that can specifically bind to a specific site on the PEDF gene, but not specific to other genes other than the PEDF gene Sexual binding, and the probe has a detectable signal.

The present invention also provides a kit for detecting the presence or absence and expression of the PEDF gene in an analyte, the kit comprising: a primer for specifically amplifying the PEDF gene; a probe for specifically recognizing the PEDF gene; or a specific Antibodies or ligands that specifically bind to the protein encoded by the PEDF gene.

In addition, the kit can also include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solution, amplification solution, hybridization solution, enzyme, control solution , Chromogenic solution, lotion, etc.

In addition, the kit may also include instructions for use and/or nucleic acid sequence analysis software and the like.

drug screening

After knowing the close correlation between PEDF and liver regeneration, substances that inhibit the expression or activity of PEDF or its coding gene can be screened based on this feature. Really useful drugs for promoting liver regeneration or preventing, alleviating or treating liver damage can be found from said substances.

Therefore, the present invention provides a method for screening potential substances (candidate substances or candidate drugs) for promoting liver regeneration or preventing, alleviating or treating liver damage, the method comprising: treating a system expressing PEDF with a candidate substance; and detecting the The expression or activity of PEDF in the above system; if the candidate substance can inhibit the expression or activity of PEDF, it indicates that the candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating or treating liver damage. The system for expressing PEDF is preferably a cell (or cell culture) system, and the cells may be cells expressing PEDF endogenously; or cells expressing PEDF recombinantly. In addition, it is also possible to evaluate whether the potential substance is useful by observing the interaction between PEDF and its upstream and downstream proteins.

In combination with the research results of the present inventors, as a preferred method of the screening method of the present invention, the potential substances (candidate substances or drug candidates) can be further determined by analyzing the proliferation ability or ring-forming ability of endothelial cells derived from liver tissue effectiveness. This can often be assayed by culturing PEDF-expressing hepatocytes, or a screening system in which they are co-cultured with endothelial cells. An observable increase in proliferative capacity or looping capacity is indicative of the effectiveness of the potential substance.

In a preferred mode of the present invention, when screening, in order to more easily observe changes in the expression or activity of PEDF, a control group (Control) can also be set, and the control group can be the expression of the candidate substance without adding The system of PEDF. The control group includes, but is not limited to: a blank control without adding candidate substances, an empty plasmid control, and the like.

As a preferred mode of the present invention, the method also includes: conducting further cell experiments and/or animal experiments on the obtained potential substances, so as to further select and determine the substances that are really useful for promoting liver regeneration or preventing, alleviating or treating liver damage. substance.

On the other hand, the present invention also provides potential substances for promoting liver regeneration or preventing, alleviating or treating liver damage obtained by the screening method. These initially screened substances can constitute a screening library, so that people can finally screen out substances that can inhibit the expression and activity of PEDF, thereby promoting liver regeneration or preventing, alleviating or treating liver damage.

pharmaceutical composition

The present invention also provides a pharmaceutical composition, which contains an effective amount (such as 0.000001-50wt%; preferably 0.00001-20wt%; more preferably 0.0001-10wt%) of the inhibitor of PEDF or its coding gene , and a pharmaceutically acceptable carrier.

As a preferred mode of the present invention, a composition for promoting liver regeneration or preventing, alleviating or treating liver damage is provided, the composition contains an effective amount of PEDF or an inhibitor of its coding gene, and a pharmaceutical acceptable carrier.

In a preferred mode of the present invention, the inhibitors include, but are not limited to: reagents for knocking out or silencing PEDF, binding molecules (such as antibodies or ligands) that specifically bind to PEDF, chemical small molecule antagonists against PEDF or inhibitors, etc. In a more specific manner, the inhibitors include, but are not limited to: interfering molecules that specifically interfere with the expression of genes encoding PEDF, CRISPR gene editing reagents for PEDF, homologous recombination reagents or site-directed mutagenesis reagents for PEDF, The above-mentioned homologous recombination reagent or site-directed mutagenesis reagent is used to carry out loss-of-function mutation of PEDF. Especially preferably, the composition contains an antibody that specifically neutralizes PEDF; or an interfering molecule that targets positions 382-402 and 323-343 in the nucleotide sequence shown in SEQ ID NO:1 or a combination thereof.

In the present invention, the term "comprising" means that various components can be used together in the mixture or composition of the present invention. Accordingly, the terms "consisting essentially of" and "consisting of" are included in the term "comprising".

As used herein, the "effective amount" refers to an amount that can produce functions or activities on humans and/or animals and that can be accepted by humans and/or animals. The "pharmaceutically acceptable carrier" refers to a carrier used for the administration of therapeutic agents, including various excipients, diluents, solvents, suspending agents, etc., which may be liquid or solid, which are suitable for human and/or Substances that have a reasonable benefit/risk ratio in animals without undue adverse side effects (such as toxicity, irritation and allergic reactions). The term refers to pharmaceutical carriers which, by themselves, are not essential active ingredients and which are not unduly toxic upon administration. Suitable vectors are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in compositions may contain liquids, such as water, saline, buffers. In addition, there may also be auxiliary substances in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, and the like. The carrier may also contain cell transfection reagents.

The dosage form of the pharmaceutical composition of the present invention can be various feasible dosage forms, as long as the dosage form can make the active ingredient reach the body of mammals effectively. As a preferred mode of the present invention, the dosage form of the pharmaceutical composition is a solution, wherein the PEDF antibody exists in a suitable liquid carrier or diluent in the form of a sol.

After knowing the use of the inhibitor of the PEDF or its encoding gene, various methods well known in the art can be used to administer the inhibitor or its encoding gene or its pharmaceutical composition to mammals or person.

Preferably, it can be carried out by means of gene therapy. For example, the inhibitor of PEDF can be directly administered to the subject through methods such as injection; or, the expression unit (such as an expression vector or virus, etc., or siRNA) carrying the inhibitor of PEDF can be delivered to the subject through a certain route. On the target and make it express an active PEDF inhibitor, the specific situation depends on the type of the inhibitor. Preferably, the inhibitor is introduced into the target site (focus, such as postoperative liver) through an expression construct (expression vector); the expression construct includes: viral vectors, non-viral vectors; preferably the viral vectors Including but not limited to: adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors.

The effective amount of the inhibitor of PEDF or its encoding gene described in the present invention may vary with the mode of administration and the severity of the disease to be treated. The selection of a preferred effective amount can be determined by those of ordinary skill in the art based on various factors (eg, through clinical trials). The factors include but are not limited to: pharmacokinetic parameters such as bioavailability, metabolism, half-life, etc. of the inhibitor of PEDF or its encoding gene; the severity of the disease to be treated by the patient, the patient's body weight, the patient's immune status, route of administration, etc.

In the specific examples of the present invention, some dosage regimens for animals such as mice are given. It is easy for those skilled in the art to convert the dosage of animals such as rats into the dosage suitable for humans. For example, it can be calculated according to the Meeh-Rubner formula: Meeh-Rubner formula: A=k×(W ^2/ 3)/10,000. In the formula, A is body surface area, calculated in ^m2 ; W is body weight, calculated in g; K is a constant, which varies with animal species. Generally speaking, mice and rats are 9.1, guinea pigs are 9.8, rabbits are 10.1, cats are 9.9, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It should be understood that, depending on the drug and the clinical situation, the conversion of the dosage can be changed according to the evaluation of an experienced pharmacist.

In a preferred embodiment, the MOI of the viral vector as an active ingredient in the composition may be 1-100, more preferably 5-50; further more preferably 8-20. The dosage range of the PEDF antibody or PEDF-related inhibitor is 0.1 mg-1 g/kg (more preferably 1 mg-0.5 g/kg; further preferably 10 mg-0.1 g/kg). It should be understood that, according to actual clinical needs, the dosage may also be higher or lower than these ranges.

In a preferred embodiment, the pharmaceutical composition is an oral preparation or an injection preparation.

In a preferred embodiment, effective drugs that can stimulate liver regeneration other than PEDF antibodies or PEDF-related inhibitors are added to the composition.

The present invention also provides a kit containing the pharmaceutical composition or directly containing the inhibitor of PEDF or its coding gene. In addition, the medicine box may also include instructions for explaining the method of using the medicine in the medicine box.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Experimental methods not indicating specific conditions in the following examples are usually according to conventional conditions such as edited by J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions described in the manufacturer suggested conditions.

sequence information

PEDF gene sequence (SEQ ID NO: 1; NM_001329904.2):

atgaaagggaagctcgccaggtccacaaaggaaattcccgatgagatcagcattctccttctcggtgtggcgcacttcaaggggcagtgggt

aacaaagtttgactccagaaagacttccctcgaggatttctacttggatgaagagaggaccgtgagggtccccatgatgtcggaccctaaggc

tgttttacgctatggcttggattcagatctcagctgcaagattgcccagctgcccttgaccggaagcatgagtatcatcttcttcctgcccctga

aagtgacccagaatttgaccttgatagagaggagagcctcacctccgagttcattcatgacatagaccgagaactgaagaccgtgcaggcggt

cctcactgtccccaagctgaagctgagttatgaaggcgaagtcaccaagtccctgcaggagatgaagctgcaatccttgtttgattcacca

gactttagcaagatcacaggcaaacccatcaagctgactcaggtggaacaccgggctggctttgagtggaacgaggatggggcgggaacc

accccccagcccagggctgcagcctgcccacctcaccttcccgctggactatcaccttaaccagcctttcatcttcgtactgagggacacagac

acaggggcccttctcttcattggcaagattctggacccccaggggcccctaa

PEDF amino acid sequence (SEQ ID NO: 2; NM_001329904.2):

MKGKLARSTKEIPDEISILLLGVAHFKGQWVTKFDSRKTSLEDFYLDEERTVRVPMMSDPKAVLRYGLDS

DLSCKIAQLPLTGSMSIIFFLPLKVTQNLTLIEESLTSEFIHDIDRELKTVQAVLTVPKLKLSYEGEVTKSLQE

MKLQSLFDSPDFSKITGKPIKLTQVEHRAGFEWNEDGAGTTPSPGLQPAHLTFPLDYHLNQPFIFVLRDTD

TGALLFIGKILDPRGP

Example 1. The effect of targeted inhibition of PEDF on liver regeneration after ALPPS

In this example, an animal (mouse) model of stepwise hepatectomy combined with liver segmentation and portal vein ligation (ALPPS) was prepared, and PEDF antibody (purchased from R&D systems, AF1177) was administered to observe the recovery of the liver. The main operation steps are as follows:

(1) Fifty C57BL/6J male mice (8 weeks old) were randomly divided into two groups, both received the same ALPPS operation, and the second-stage operation was performed 48 hours after the first-stage operation.

(2) From 12 hours before the first-stage operation to 12 hours after the second-stage operation, the PEDF group was intraperitoneally injected with PEDF antibody 10 mg/kg (R&D Systems, AF1177) every 12 hours, and the control group was injected with the same volume of normal saline, a total of 7 times ; Record the death situation of two groups of mice.

(3) Starting from the phase I operation as planned, 5 mice were sacrificed every day, the liver weight and body weight were weighed before sacrifice, 300ul blood was taken from the tail vein, and the serum was collected after centrifugation to detect alanine aminotransferase (ALT) and Tianmen Aspartate aminotransferase (AST); half of the liver was frozen and stored in a -80°C ultra-low temperature refrigerator for later use; the other half was fixed in formalin and embedded in paraffin for later use.

The postoperative measurement results are shown in Figure 1. There was no significant difference in the survival rate of mice in the PEDF antibody group after ALPPS stage I surgery, but the survival rate was higher after stage II surgery (the second day after stage I surgery) (left); liver Body ratio recovery rate is faster (right).

The measurement results of liver function are shown in Figure 2, the serum ALT level (left) and AST level (right) of the mice in the PEDF antibody group after Phase II surgery (the second day after Phase I surgery) were lower.

The above results indicate that targeted inhibition of PEDF can accelerate the regeneration of the remnant liver after ALPPS, accelerate the recovery of liver function, and reduce the mortality related to liver failure after ALPPS.

Example 2. Immunofluorescence histochemical staining to detect the effect of targeted inhibition of PEDF on liver regeneration after ALPPS

The ALPPS operation was performed as described in Example 1, and a mouse ALPPS operation model was made, treated with PEDF antibody and a control, and the livers of mice in the PEDF antibody group and control group mice were obtained 2 days and 6 days after the first phase operation. organize. Immunofluorescence staining was performed on the specific marker of liver cells HNF4α, the specific marker of liver sinusoidal endothelial cells (LSEC) (LYVE1) and the specific nuclear antigen Ki-67 of proliferating cells after sectioning, and the counts were observed and counted by confocal microscopy. Ki-67 is a proliferating cell-associated nuclear antigen, its function is closely related to mitosis, and it is essential in cell proliferation, and its positive staining indicates active cell proliferation.

The main steps of the immunofluorescence staining method are as follows:

(1) Embedding the liver tissue in paraffin, obtaining paraffin sections, and dewaxing to water;

(2) 3% H ₂ O ₂ at room temperature for 10 minutes, washed with water;

(3) Antigen acidic repair;

(4) 1% BSA blocked for 30 minutes;

(5) Add Ki-67 antibody (1:100, Cell Signaling Technology) dropwise, overnight at 4°C;

(6) Add horseradish peroxidase-labeled secondary antibody (Shanghai Changdao Biology) dropwise, 37°C for 30min;

(7) Add TSA fluorescent staining solution (Perkin Elmer) dropwise, room temperature for 30 minutes;

(8) film washing;

(9) Add the antibodies HNF4α (1:200, Abcam) and LYVE1 (1:200, Abcam) dropwise according to steps 5-8;

(10) DAPI staining, glycerol mounting, and Leica confocal microscope observation.

As shown in the left panel of Figure 3, the proportion of proliferating hepatocytes in the PEDF antibody group and the control group was similar on the 2nd day after stage I surgery (the peak period of hepatocyte proliferation). As shown in the right panel of Figure 3, the proportion of LSEC proliferating cells in the PEDF antibody group was much higher than that in the control group on the 6th day after stage I surgery (the peak of LSEC proliferation).

The results indicated that targeted inhibition of PEDF promoted liver regeneration mainly by stimulating LSEC proliferation.

Example 3. Western blot detection of the effect of targeted inhibition of PEDF on liver regeneration after ALPPS

ALPPS surgery was performed as described in Example 1, and a mouse ALPPS surgery model was made, treated with PEDF antibody and control, and LSEC-specific marker molecules in PEDF antibody group and control group were detected by Western blotting. CD146 and LYVE1 are specific marker molecules of LSEC, and their abundance in liver tissue represents the proportion of LSEC in all liver cells.

The main steps of the western blot detection method are as follows:

(1) The liver tissue was dissolved in IP lysate (Shanghai Biyuntian) by ultrasound, and centrifuged at 12000 rpm/min at 4°C.

(2) After centrifugation, the supernatant was measured by BCA (Shanghai Thermo Fisher Scientific) to measure the protein concentration of each sample, added SDS denaturing solution, and heated at 95°C for 5 minutes.

(3) The prepared denatured samples were added to the polyacrylamide gel to start electrophoresis, and the separated bands were transferred to the nitrocellulose membrane.

(4) 5% skimmed milk powder was blocked for 30 minutes, and diluted PEDF (1:200, santa cruiztechnology), CD146 (1:100, CELL SIGNALING TECHNOLOGY), LYVE1 (1:500, Abcam), internal reference molecule GAPDH (1: 5000, CELL SIGNALING TECHNOLOGY), incubated overnight at 4°C.

(5) After washing the next day, add fluorescent protein-coupled secondary antibody (1:10000, CELL SIGNALINGTECHNOLOGY), and incubate at room temperature for 2 hours.

(6) After washing, use the Odyssey membrane scanner to read the expression intensity of related molecules.

The results are shown in Figure 4. Compared with the control group, the expression of LSEC-specific markers in the PEDF antibody group did not change much at 2 days after stage I surgery, and the expression of LSEC-specific markers at 6 days after stage I increased significantly.

The results indicated that targeted inhibition of PEDF accelerated liver regeneration mainly by promoting the proliferation of LSECs in the late regeneration stage.

Example 4, the effect of targeted inhibition of PEDF on the proliferation ability of LSEC in vitro

In this example, ALPPS surgery was performed as described in Example 1, and a mouse model of ALPPS surgery was made, treated with PEDF antibody and control, and the effect of targeted inhibition of PEDF on the proliferation ability of LSEC in vitro was detected. It is analyzed by detecting EDU-positive cells. EDU is a thymidine analog that can be inserted into the DNA molecule being replicated during cell proliferation, and can effectively detect the percentage of cells in the S phase.

The main operation steps are as follows:

(1) 8-week-old C57BL/6J male mice were selected for ALPPS operation, and the liver was perfused and digested in situ after anesthesia on the second day after the second-stage operation.

(2) and through density gradient centrifugation, primary mouse hepatocytes and primary LSECs were obtained.

(3) The obtained primary hepatocytes were inoculated at a density of 4×10 ⁵ /ml in a 3D culture system based on Matrigel (Corning), the medium was changed every 48 hours, and cultured for three to four weeks.

(4) When the number of liver organoids exceeds 50/well, collect the old medium when changing the medium, and take the supernatant after centrifugation.

(5) The mouse LSECs obtained by in situ perfusion digestion were inoculated on a 6-well plate at a density of 1×10 ⁶ /well, and the cells were stabilized after adherence.

(6) Use the hepatocyte culture supernatant collected in step 4) to prepare a mixed culture medium at a ratio of 1:1, add PEDF antibody to the mixed culture medium at a concentration of 100ng/ml, and add an equal volume of antibody diluent to the control group .

(7) After culturing LSECs in the mixed medium for 48 hours, the proliferation ratio of LSECs was detected using the EDU kit (Shanghai Ribobio).

The results are shown in Figure 5, the number of EDU-positive cells in the PEDF antibody group was significantly more than that in the control group.

The results indicated that targeted inhibition of PEDF can promote the proliferation of liver sinusoidal endothelial cells (LSEC).

Example 5. The effect of targeted inhibition of PEDF on the ability of LSEC to form rings in vitro

In this example, ALPPS surgery was performed as described in Example 1, and a mouse ALPPS surgery model was made, treated with PEDF antibody and a control, and the effect of targeted inhibition of PEDF on the in vitro ring-forming ability of mouse LSECs was detected.

The main operation steps are as follows:

Steps (1)-(4) are the same as in Example 4.

(5) LSECs were seeded on u-slide angiogenesis glass slides at a density of 5×10 ³ /well, and the well plates were plated with 10 μl Matrigel before seeding.

(6) With embodiment 4 (6) step.

(7) After culturing LSECs with the mixed medium for 6 hours, observe the ringing of LSECs under a microscope in bright field.

The results are shown in Figure 6. The number of LSEC rings in the PEDF antibody group was significantly more than that in the control group, indicating that the PEDF antibody has a very significant promotion effect on the ring-forming ability of liver sinusoidal endothelial cells.

Example 6. Correlation between PEDF transcription level in liver tissue of patients undergoing hepatectomy and postoperative ALT level

In this example, the PEDF transcription level in the liver tissue of patients undergoing major hepatectomy and the postoperative ALT level were analyzed.

The main operation steps are as follows:

(1) After obtaining the approval of the Ethics Committee of our hospital and signing the informed consent form with the patient, fresh paracancerous tissue from the patient during the operation was taken, part of which was lysed with Trizol, and RNA was extracted by ethanol extraction; the other part was stored at 4°C Medium, used in Examples 7, 8, and 9 within 8 hours.

(2) cDNA was obtained by reverse transcription system of M-MLV (Invitrogen, 28025013).

(3) The transcription level of PEDF in liver tissue was detected by qRT-PCR system.

(4) Correlation analysis was carried out between PEDF transcription level and serum ALT level 3 days after operation.

The results are shown in Figure 7. According to the level of PEDF transcription in liver tissue, the patients can be divided into three groups (high: No.1-7; medium: No.8-15; low: No.16-22). ), combined with the analysis of ALT level 3 days after operation, it was found that the postoperative ALT level of the PEDF transcription level group was higher than that of the PEDF transcription level group.

Therefore, the PEDF transcription level in the liver tissue of patients undergoing major hepatectomy is related to the postoperative ALT level. The higher the PEDF transcription level, the higher the postoperative ALT level.

Example 7, the promotion effect of the culture of hepatocytes in the PEDF low transcription level group on the ring-forming ability of HUVEC cells

In this example, the effect of culture of hepatocytes in the PEDF low transcript level group on the ring-forming ability of HUVEC cells was analyzed.

The main operation steps are as follows:

(1) Take the fresh paracancerous tissue of the patient during the operation in Example 6, cut the tissue into about 1-2 mm, digest it with 1 mg/ml collagenase at 37 °C for 1-1.5 hours, and add it when no tissue fragments are visible to the naked eye. Equal volume of complete medium to stop digestion.

(2) After filtering the cell suspension with a 70 μm filter, centrifuge twice at 50 g for 3 min to collect hepatocytes at the bottom of the tube.

(3) After the medium was resuspended, the hepatocytes were planted in a 6-well plate coated with type I collagen at a planting density of 1.2-1.5×10 ⁶ per well, and 5 wells of each patient's hepatocytes were planted.

(4) Change the medium after 6 hours to remove cells that are not attached.

(5) Culture for 48 hours after changing the medium, and collect the conditioned medium of the hepatocytes for use.

(6) HUVECs were seeded on u-slide angiogenesis slides at a density of 5×10 ³ /well, and the well plates were plated with 10 μl Matrigel before seeding.

(7) Hepatocyte culture medium was collected, and the conditioned medium was mixed with complete medium 1:1 to cultivate HUVEC cells.

(8) After culturing HUVEC with the mixed medium for 24 hours, the ring formation was observed under a microscope under bright field.

The results are shown in FIG. 8 , the conditioned medium of hepatocytes in the PEDF low transcription level group stimulated the ringing ability of HUVEC cells more significantly. It is suggested that PEDF in hepatocytes can affect the recovery of liver function after hepatectomy by regulating the function of vascular endothelium.

Therefore, the conditioned medium of hepatocytes in the PEDF low transcript level group can promote the ring-forming ability of HUVEC cells.

Example 8. The effect of targeted interference of PEDF in hepatic cells of patients undergoing hepatectomy on the proliferation of HUVEC cells

In this example, adenovirus-mediated targeted interference with PEDF in hepatocytes of patients undergoing hepatectomy was used to analyze the effect on the proliferation of HUVEC cells.

The adenovirus vector is: H17692 (obtained from Shanghai Heyuan Biological Co., Ltd.).

Sequence 1: cccaagctgaagctgagttat (SEQ ID NO:3; corresponding to No. 382-402 in SEQ ID NO:1);

Sequence 2: ccgagttcattcatgacatag (SEQ ID NO: 4; corresponding to No. 323-343 in SEQ ID NO: 1);

Sequence 3: cggaagcatgagtatcatctt (SEQ ID NO:5; corresponding to No. 246-266 in SEQ ID NO:1);

Sequence 4: cttgtttgattcaccagactt (SEQ ID NO: 6; corresponding to positions 447-467 in SEQ ID NO: 1).

The above sequence was introduced into the adenoviral vector.

The main operation steps are as follows:

(1) Take the fresh paracancerous tissue of the patient during the operation in Example 6, cut the tissue into about 1-2 mm, digest it with 1 mg/ml collagenase at 37 °C for 1-1.5 hours, and add it when no tissue fragments are visible to the naked eye. Equal volume of complete medium to stop digestion.

(2) After filtering the cell suspension with a 70 μm filter, centrifuge twice at 50 g for 3 min to collect hepatocytes at the bottom of the tube.

(3) After the medium was resuspended, the hepatocytes were planted in a 6-well plate coated with type I collagen at a planting density of 1.2-1.5×10 ⁶ per well, and 5 wells of each patient's hepatocytes were planted.

(4) Change the medium after 6 hours to remove cells that are not attached.

(5) Infect hepatocytes (MOI=8) with empty vector and adenovirus carrying different sequences, and change the medium after 48 hours.

(6) Culture for 48 hours after changing the medium, collect the hepatocyte medium, mix the conditioned medium with -complete medium 1:1, cultivate HUVEC cells, and perform CCK8 detection.

The results are shown in FIG. 9 , after infection of hepatocytes with adenovirus targeting different sequences that interfere with PEDF, the conditioned medium stimulates the proliferation ability of HUVEC cells differently. Among them, sequences 1 and 2 can significantly increase the proliferation level of HUVEC cells, and the effect is significantly better than that of other groups.

Therefore, after using adenovirus-mediated targeted interference with different sequences of PEDF in the liver cells of patients with high levels of PEDF transcription, the conditioned medium of the primary liver cells can significantly promote the proliferation of HUVEC cells compared with the empty vector control group.

Example 9. Targeted interference with PEDF in hepatocytes of patients undergoing hepatectomy can promote the ring-forming ability of HUVEC cell lines

In this example, adenovirus-mediated targeted interference with PEDF in hepatic cells of patients undergoing hepatectomy was used to analyze the effect on the ring-forming ability of HUVEC cells.

The main operation steps are as follows:

(1)-(5) are the same as embodiment 8.

(6) HUVECs were seeded on u-slide angiogenesis slides at a density of 5×10 ³ /well, and the well plates were plated with 10 μl Matrigel before seeding.

(7) Hepatocyte culture medium was collected, and the conditioned medium was mixed with complete medium 1:1 to cultivate HUVEC cells.

(8) After culturing HUVEC with the mixed medium for 24 hours, the ring formation was observed under a microscope under bright field.

The results are shown in FIG. 10 , after infection of hepatocytes with different adeno-associated viruses targeting the interfering PEDF sequence, the conditioned medium stimulated the ring-forming ability of HUVEC cells differently. Among them, the number of loops in sequence 1 and 2 groups was significantly more than that in the control group, indicating that targeting interference sequences 1 and 2 can significantly improve the looping ability of HUVEC cells. The inventors observed that it was even superior to the AF1177 antibody.

Therefore, after adenovirus-mediated targeted interference with PEDF high transcription level group with different sequences of PEDF in hepatocytes of patients undergoing hepatectomy, the conditioned medium of primary hepatocytes can promote the growth of HUVEC cells compared with the empty vector control group. ring capability.

Embodiment 10, screening method

(1) Screening based on PEDF expression or activity

Screening system: Human liver cell line (L-02) overexpressing PEDF.

Test group: culture the liver cell line overexpressing PEDF, and administer the candidate substance;

Control group: culture the liver cell line overexpressing PEDF without administering the candidate substance.

The expression or activity of PEDF in the culture medium of the test group and the control group were respectively detected and compared. If the expression or activity of PEDF in the test group is statistically lower (eg, 30% or lower) than that in the control group, it indicates that the candidate is a potential substance for promoting liver regeneration, alleviating or treating liver damage.

(2) Screening based on endothelial cell culture

Screening system: human liver cell line (L-02) overexpressing PEDF and human umbilical vein endothelial cell line (HUVEC).

Test group: use PEDF-overexpressed L-02 conditioned medium to stimulate HUVEC cells, and give candidate substances;

Control group: HUVEC cells were stimulated with PEDF-overexpressed L-02 conditioned medium, and no candidate substance was given.

The proliferative ability or ring-forming ability of the HUVEC cells in the test group and the control group were respectively detected and compared. If the proliferative ability or ring-forming ability of HUVEC cells in the test group is significantly higher (eg, 10% or higher) than the control group, it indicates that the candidate is a potential substance for promoting liver regeneration, alleviating or treating liver damage.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

sequence listing

<110> The Third Affiliated Hospital of Naval Medical University of the Chinese People's Liberation Army

<120> Application of targeted inhibition of pigment epithelium-derived factor in promoting liver regeneration and improving liver injury

<130> 212216

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 696

<212>DNA

<213> Homo sapiens

<400> 1

atgaaaggga agctcgccag gtccacaaag gaaattcccg atgagatcag cattctcctt 60

ctcggtgtgg cgcacttcaa ggggcagtgg gtaacaaagt ttgactccag aaagacttcc 120

ctcgaggatt tctacttgga tgaagagagg accgtgaggg tccccatgat gtcggaccct 180

aaggctgttt tacgctatgg cttggattca gatctcagct gcaagattgc ccagctgccc 240

ttgaccggaa gcatgagtat catcttcttc ctgcccctga aagtgaccca gaatttgacc 300

ttgatagagg agagcctcac ctccgagttc attcatgaca tagaccgaga actgaagacc 360

gtgcaggcgg tcctcactgt ccccaagctg aagctgagtt atgaaggcga agtcaccaag 420

tccctgcagg agatgaagct gcaatccttg tttgattcac cagactttag caagatcaca 480

ggcaaaccca tcaagctgac tcaggtggaa caccgggctg gctttgagtg gaacgaggat 540

ggggcgggaa ccaccccccag cccagggctg cagcctgccc acctcacctt cccgctggac 600

tatcacctta accagccttt catcttcgta ctgagggaca cagacacagg ggcccttctc 660

ttcattggca agattctgga ccccaggggc ccctaa 696

<210> 2

<211> 231

<212> PRT

<213> Homo sapiens

<400> 2

Met Lys Gly Lys Leu Ala Arg Ser Thr Lys Glu Ile Pro Asp Glu Ile

1 5 10 15

Ser Ile Leu Leu Leu Gly Val Ala His Phe Lys Gly Gln Trp Val Thr

20 25 30

Lys Phe Asp Ser Arg Lys Thr Ser Leu Glu Asp Phe Tyr Leu Asp Glu

35 40 45

Glu Arg Thr Val Arg Val Pro Met Met Ser Asp Pro Lys Ala Val Leu

50 55 60

Arg Tyr Gly Leu Asp Ser Asp Leu Ser Cys Lys Ile Ala Gln Leu Pro

65 70 75 80

Leu Thr Gly Ser Met Ser Ile Ile Phe Phe Leu Pro Leu Lys Val Thr

85 90 95

Gln Asn Leu Thr Leu Ile Glu Glu Ser Leu Thr Ser Ser Glu Phe Ile His

100 105 110

Asp Ile Asp Arg Glu Leu Lys Thr Val Gln Ala Val Leu Thr Val Pro

115 120 125

Lys Leu Lys Leu Ser Tyr Glu Gly Glu Val Thr Lys Ser Leu Gln Glu

130 135 140

Met Lys Leu Gln Ser Leu Phe Asp Ser Pro Asp Phe Ser Lys Ile Thr

145 150 155 160

Gly Lys Pro Ile Lys Leu Thr Gln Val Glu His Arg Ala Gly Phe Glu

165 170 175

Trp Asn Glu Asp Gly Ala Gly Thr Thr Pro Ser Pro Gly Leu Gln Pro

180 185 190

Ala His Leu Thr Phe Pro Leu Asp Tyr His Leu Asn Gln Pro Phe Ile

195 200 205

Phe Val Leu Arg Asp Thr Asp Thr Gly Ala Leu Leu Phe Ile Gly Lys

210 215 220

Ile Leu Asp Pro Arg Gly Pro

225 230

<210> 3

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 3

cccaagctga agctgagtta t 21

<210> 4

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 4

ccgagttcat tcatgacata g 21

<210> 5

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 5

cggaagcatg agtatcatct t 21

<210> 6

<211> 21

<212>DNA

<213> Artificial Sequence

<400> 6

cttgtttgat tcaccagact t 21

Claims (10)

1. Use of an inhibitor of a pigment epithelium-derived factor for:

preparing a composition for promoting liver regeneration; or

Preparing a composition for preventing, alleviating and/or treating liver injury.

2. The use of claim 1, wherein said composition is further used for:

promoting proliferation of endothelial cells;

promoting the cyclization ability of endothelial cells;

stimulating the production of new blood vessels in the residual liver or damaged liver; and/or

Accelerate the recovery of liver body ratio and liver function after hepatectomy.

3. The use of claim 1, wherein the inhibitor of a pigment epithelium-derived factor comprises an inhibitor selected from the group consisting of: an agent that knocks out or silences a pigment epithelium-derived factor; binding molecules such as antibodies that specifically bind to pigment epithelium-derived factors; a chemical small molecule antagonist or inhibitor against pigment epithelium derived factor; or agents that interfere with the interaction of the pigment epithelium-derived factor with an effector molecule or its receptor.

4. The use of claim 3, wherein the agent that knocks out or silences a pigment epithelium-derived factor comprises: interference molecules specifically interfering the expression of coding genes of the pigment epithelium derived factor, CRISPR gene editing reagent aiming at the pigment epithelium derived factor, homologous recombination reagent or site-directed mutation reagent aiming at the pigment epithelium derived factor, wherein the homologous recombination reagent or the site-directed mutation reagent performs loss-of-function mutation on the pigment epithelium derived factor;

preferably, the interfering molecule comprises an shRNA, siRNA, miRNA, antisense nucleic acid, or a construct capable of forming the shRNA, siRNA, miRNA, antisense nucleic acid;

more preferably, the agent for knocking out or silencing pigment epithelium derived factor is an interference molecule, and targets 382-402 th, 323-343 th, 246-266 th, 447-467 th or the combination thereof in the nucleotide sequence shown in SEQ ID NO. 1; preferably at positions 382 to 402, 323 to 343 or combinations thereof in the nucleotide sequence shown in SEQ ID NO. 1.

5. The use of claim 4, wherein the inhibitor is introduced to the target site via an expression construct; the expression construct comprises: viral vectors, non-viral vectors; preferably the viral vector comprises: adenovirus vectors, adeno-associated virus vectors, lentivirus vectors, retroviral vectors.

6. The use of claim 1, wherein the liver injury comprises liver dysfunction following liver surgery, liver damage resulting from hepatitis, liver fibrosis, cirrhosis, end-stage liver disease, liver cancer, alcoholic liver disease, metabolic liver disease, or liver failure;

preferably, the liver operation comprises a treatment method based on the regeneration capacity of the normal liver, which is used for preserving the normal liver tissue and enabling the normal liver tissue to compensate and proliferate to play the normal liver function by destroying the liver tissue of the lesion part; more preferably, the method comprises the following steps: traditional hepatectomy, secondary hepatectomy by portal vein ligation, secondary hepatectomy by combining liver segmentation and portal vein ligation, radiofrequency ablation, microwave ablation, cryoablation, hepatic artery interventional embolization chemotherapy, hepatic artery interventional embolization radiotherapy and stereotactic radiotherapy.

7. The application of a reagent for specifically identifying or amplifying pigment epithelium derived factors, which is used for preparing a diagnostic reagent or a kit for diagnosing or prognostically evaluating the liver regeneration capability or liver injury; preferably, the reagent comprises: a binding molecule that specifically binds to a pigment epithelium-derived factor protein; primers for specifically amplifying pigment epithelium derived factor genes; a probe that specifically recognizes a pigment epithelium-derived factor gene; or a chip which specifically recognizes the pigment epithelium derived factor gene.

8. A pharmaceutical composition or kit for promoting liver regeneration or preventing, ameliorating and/or treating liver injury comprising an inhibitor of pigment epithelium-derived factor, said inhibitor comprising a compound selected from the group consisting of: interference molecules specifically interfering the expression of coding genes of the pigment epithelium derived factor, CRISPR gene editing reagent aiming at the pigment epithelium derived factor, homologous recombination reagent or site-directed mutation reagent aiming at the pigment epithelium derived factor, wherein the homologous recombination reagent or the site-directed mutation reagent performs loss-of-function mutation on the pigment epithelium derived factor; preferably, the interfering molecule comprises an shRNA, siRNA, miRNA, antisense nucleic acid, or a construct capable of forming the shRNA, siRNA, miRNA, antisense nucleic acid; more preferably, the interfering molecule targets positions 382-402, 323-343, 246-266, 447-467 or a combination thereof in the nucleotide sequence shown in SEQ ID NO 1; preferably at positions 382 to 402, 323 to 343 or combinations thereof in the nucleotide sequence shown in SEQ ID NO. 1.

9. A method of screening for potential agents for promoting liver regeneration or preventing, ameliorating and/or treating liver damage, the method comprising:

(1) Treating an expression system with a candidate substance, the expression system expressing pigment epithelium-derived factor; and the combination of (a) and (b),

(2) Detecting the expression or activity of pigment epithelium derived factor in said system; the candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating and/or treating liver damage if the candidate substance statistically modulates the expression or activity of the pigment epithelium-derived factor.

10. The method of claim 9, wherein the system of step (1) is an endothelial cell system; preferably, the endothelial cells comprise: hepatic sinus endothelial cells, vascular endothelial cells, lymphatic endothelial cells;

the step (2) further comprises: detecting the proliferation capacity or the cyclization capacity of endothelial cells in the system; if the proliferative capacity or the cyclization capacity is promoted, the candidate substance is a potential substance for promoting liver regeneration or preventing, alleviating and/or treating liver injury.

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