CN115957301A - Cell secretion factor for promoting myocardial infarction repair and application - Google Patents

Abstract

The invention provides a cell-secreted factor for promoting myocardial repair of myocardial infarction and its application. The application of insulin-like growth factor binding protein 7 (IGFBP7) in compositions or medicines for promoting myocardial repair after myocardial infarction (MI), improving cardiac function after myocardial infarction, and protecting myocardial ischemia is disclosed for the first time. Based on the above functions of IGFBP7, inhibition can be used as a drug screening target to screen substances that promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemia damage.

Description

A cell-secreted factor promoting myocardial repair in myocardial infarction and its application

technical field

The invention belongs to the field of biotechnology and pharmacy, and more specifically, the invention relates to a cell-secreted factor for promoting myocardial repair of myocardial infarction and its application.

Background technique

Cardiovascular disease is one of the most serious diseases threatening human health today. Among cardiovascular diseases, ischemic heart disease (Ischemic Heart Disease, IHD) is undoubtedly the most serious problem. Ischemic heart disease is characterized by myocardial ischemic damage caused by an imbalance between the myocardial demand for oxygen and energy substances and the supply of coronary blood flow. Acute myocardial infarction (MI) is usually due to sudden blockage of a coronary artery, resulting in cutoff of blood flow, resulting in ischemia of the myocardium. Sustained ischemia leads to the death of a large number of cardiomyocytes. Due to the lack of regeneration ability of adult cardiomyocytes, the dead cardiomyocytes cannot be effectively replenished, resulting in the proliferation of fibroblasts, which leads to excessive fibrosis and scar formation, and ventricular remodeling occurs. Decreased heart function.

How to rebuild the blood supply for the ischemic myocardium is the key problem in solving myocardial infarction. Clinically, through drug thrombolysis, bypass and other means, the blood supply to the ischemic site can be re-realized, the size of the myocardial infarction can be effectively reduced, and the long-term survival rate can be improved. Cardiac function, reduce mortality. However, studies have shown that in the process of restoring the blood supply of the ischemic heart, it will cause new damage to the heart, which together with the damage caused by ischemia is collectively referred to as ischemia/reperfusion (I/R) injury.

Myocardial infarction is a sharp reduction or interruption of coronary blood supply on the basis of coronary artery disease, which in turn leads to acute and persistent myocardial ischemia in the myocardium at the coronary artery supply site, resulting in severe cellular energy metabolism disorders, causing a large number of myocardial cells damaged and dead. Since cardiomyocytes are terminally differentiated cells, their regenerative capacity is very limited. Lost cardiomyocytes are replaced by fibrous scars, resulting in irreversible myocardial damage. In severe cases, arrhythmia, aneurysm formation, and cardiac rupture occur. Heart failure is a complex clinical symptom group of cardiac insufficiency caused by various heart diseases; in most cases, it refers to the decrease of myocardial contractility, so that the cardiac output cannot meet the metabolic needs of the body, and the blood perfusion of organs and tissues is insufficient.

The death modes of myocardial cells after myocardial ischemia mainly include autophagy, apoptosis, pyroptosis, ferroptosis, and necrosis. Among them, necrosis is considered to be the main type of myocardial cell death caused by myocardial infarction. About 30% of cardiomyocytes died from ischemic injury due to apoptosis and 70% due to necrosis. Further research found that there is a death mode in cells that is different from apoptosis and traditional necrotic cells. This process has typical necrosis-like morphological characteristics, such as membrane integrity defect and inflammation, but its process is strictly regulated by a series of molecules. It's called necrosis. Programmed necrosis cells account for about 50% of necrotic cells after myocardial infarction. Activation of death receptors is the main mechanism of necroptosis. Tumor necrosis factor receptor (Tumor Necrosis Factor Receptor, TNFR)-mediated regulation of receptor-interacting protein kinase 1 (Receptor Interacting Protein kinase 1, RIP1) and RIP3 activation is the main feature of death receptor-dependent programmed necrosis. TNFR activation promotes the dimerization and activation of RIP1, and the activated RIP1 is ubiquitinated by PELI1 to form a complex with RIP3, recruit mixed lineage kinase domain-like protein (MLKL), and execute the process of programmed necrosis.

In view of the extensiveness and multiple occurrences of myocardial infarction, there is an urgent need in the field to find more new solutions for relieving myocardial infarction and its post-occurrence symptoms.

Contents of the invention

The object of the present invention is to provide a cell-secreted factor for promoting myocardial repair of myocardial infarction and its application.

In the first aspect of the present invention, there is provided an application of insulin-like growth factor binding protein 7 (IGFBP7) or its up-regulator in the preparation of medicine or composition, and the medicine or composition has the following functions: promoting post-myocardial infarction Myocardial repair, improve cardiac function after myocardial infarction, reduce myocardial ischemia injury.

In one or more embodiments, the promotion of myocardial repair after myocardial infarction, improvement of cardiac function after myocardial infarction, and protection of myocardial ischemia include: reducing the area of myocardial infarction after myocardial infarction; Scar area; increase the number of blood vessels in the myocardial infarction area and the myocardial infarction border area after myocardial infarction; promote myocardial cell resistance to injury; inhibit the programmed necrosis after myocardial infarction, preferably by inhibiting the activation of the programmed necrosis RIP1/RIP3 signaling pathway , thereby inhibiting the programmed necrosis of injured cardiomyocytes.

In one or more embodiments, the up-regulator of insulin-like growth factor-binding protein 7 includes a substance selected from the group consisting of (a) enhancing the activity of insulin-like growth factor-binding protein 7; (b) enhancing insulin-like growth The expression, stability or effective action time of factor binding protein 7.

In one or more embodiments, the up-regulator of insulin-like growth factor-binding protein 7 includes: the expression construct (including expression vector) for recombinantly expressing insulin-like growth factor-binding protein 7, enhancing insulin-like growth factor A polypeptide or compound that binds protein 7 to inhibit the RIP1 (Receptor Interacting Protein Kinase 1)/RIP3 signaling pathway, a chemical upregulator of insulin-like growth factor binding protein 7, and promotes the promoter of insulin-like growth factor binding protein 7 gene An upregulator of driving ability, a downregulator of insulin-like growth factor binding protein 7 gene-specific microRNA, or a combination thereof.

In one or more embodiments, the insulin-like growth factor binding protein 7 is selected from the group consisting of: (a) a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1; (b) the polypeptide shown in (a) The amino acid sequence of the amino acid sequence has (a ) or (b) insulin-like growth factor binding protein 7 derivatives with polypeptide function, or active fragments thereof; (c) the sequence has a homology ≥ 80% compared with the amino acid sequence shown in SEQ ID NO: 1 (such as homologous ≥85%, ≥90%, ≥95%, ≥98% or ≥99%) insulin-like growth factor binding protein 7 derivatives, or active fragments thereof.

In one or more embodiments, the expression constructs (expression vectors) include: viral vectors, non-viral vectors; preferably, the expression vectors include: adeno-associated virus vectors, lentiviral vectors, adeno-associated virus vectors, Viral vector.

In one or more embodiments, the inhibiting the necroptosis RIP1/RIP3 signaling pathway includes inhibiting RIP3.

In one or more embodiments, the inhibition of RIP3 includes: reducing the stability of RIP3 mRNA in an IGF-independent manner, thereby inhibiting myocardial cell necrosis after myocardial infarction.

In another aspect of the present invention, the application of insulin-like growth factor binding protein 7 is provided for screening drugs or compounds (i.e. as drug screening targets), and the drugs or compounds have the following functions: promoting myocardial repair after myocardial infarction , Improve cardiac function after myocardial infarction, protect myocardial ischemia injury.

In another aspect of the present invention, there is provided a method for screening drugs or compounds (or potential drugs or compounds) that promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemia damage, including: (1) treating an expression system with a candidate substance, which expresses insulin-like growth factor-binding protein 7; and, (2) detecting the expression or activity of insulin-like growth factor-binding protein 7 in the system; if the candidate substance is in Statistically increasing the expression or activity of IGF-7 indicates that the candidate substance is a desired (interesting) drug or compound.

In one or more embodiments, step (1) includes: in the test group, adding candidate substances to the expression system; and/or, step (2) includes: detecting insulin-like growth in the system The expression or activity of factor binding protein 7, and compared with the control group, wherein the control group is an expression system without adding the candidate substance; if the candidate substance is statistically increased (such as increased by more than 20%, compared with Preferably, the expression or activity of insulin-like growth factor binding protein 7 can be increased by more than 50%; more preferably, it can be increased by more than 80%, which indicates that the candidate substance is a desired (interested) drug or compound.

In one or more embodiments, the system described in step (1) also includes the programmed necrosis RIP1/RIP3 signaling pathway or pathway protein RIP3; and, step (2) also includes: detecting The interaction between insulin-like growth factor-binding protein 7 and the programmed necrosis RIP1/RIP3 signaling pathway or pathway protein RIP3, if insulin-like growth factor-binding protein 7 is inhibited (such as inhibiting more than 20%, preferably inhibiting 50%) Above; preferably more than 80% inhibition) of the necroptosis RIP1/RIP3 (especially inhibiting RIP3) signaling pathway or pathway protein RIP3, it indicates that the candidate substance is a desired (interesting) drug or compound.

In one or more embodiments, in the system comprising the programmed necrosis RIP1/RIP3 signaling pathway, the IGFBP7 acts on RIP3.

In one or more embodiments, the system is selected from: cell system (or cell culture system), subcellular system (or subcellular culture system), solution system, tissue system, organ system or animal system .

In one or more embodiments, the candidate substances include (but are not limited to): those designed for insulin-like growth factor binding protein 7, its fragment or variant, its coding gene or its upstream and downstream molecules or signaling pathways Overexpressed molecules such as constructs, activity-promoting molecules, small chemical molecules, interacting molecules, etc.

In one or more embodiments, the method also includes: further cell experiments and/or animal experiments are carried out on the obtained drug or compound (potential drug or compound), to further select and determine from the candidate substances for promoting A composition useful for myocardial repair after myocardial infarction, improvement of cardiac function after myocardial infarction, and protection (slowdown) of myocardial ischemia damage.

In another aspect of the present invention, use of a reagent for specifically recognizing or amplifying insulin-like growth factor binding protein 7 is provided for preparing a reagent or a kit for evaluating or prognosing disease progression after myocardial infarction.

In one or more embodiments, if the insulin-like growth factor binding protein 7 is underexpressed (ie: the expression is lower than the average or conventional value of the species population), it indicates that the prognosis of the subject after myocardial infarction is poor ( relatively poor).

In one or more embodiments, the insulin-like growth factor binding protein 7 is normally expressed or highly expressed (that is: the expression is equal to, equivalent to or higher than the average or normal value of the species population; wherein the higher than e.g. higher than 5%, 10%, 15%, 20%, 30%, 50%, 60%, 80%, 90% or higher), indicating that the subject has a normal or good prognosis after myocardial infarction (relatively good) .

In one or more embodiments, the reagents include (but are not limited to): binding molecules that specifically bind to IGF-7; primers that specifically amplify the IGF-7 gene; A probe specifically recognizing the insulin-like growth factor binding protein 7 gene; or a chip specifically recognizing the insulin-like growth factor binding protein 7 gene.

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

Description of drawings

Figure 1. Human IGFBP7 domain.

Fig. 2. Distribution of IGFBP7 in human organs and expression and localization in mouse myocardium and fibroblasts.

(A) The expression of IGFBP7 in different human tissues, among which the heart is the only muscle tissue expressing IGFBP7 (data from ProteomicsDB database); (B) Immunofluorescence staining of adult mouse cardiomyocytes showed that IGFBP7 was widely distributed in cardiomyocytes, and no Obvious localization preference; in fibroblasts, IGFBP7 is distributed in a punctate manner, and partly co-localizes with vimentin-positive punctate (white arrow); (C) WB results show that the expression of IGFBP7 in adult mouse cardiomyocytes is significantly higher than that in adult mice Mouse fibroblasts. Scale bar = 50 μm.

Fig. 3. Expression of IGFBP7 in supernatant of hCVPCs and heart tissue of mice with myocardial infarction.

(A) Antibody chip results show that the content of IGFBP7 in CVPC-CdM is much higher than that in the control DMEM; (B) Elisa assay detects the content of IGFBP7 in hCVPC-CdM; (C) WB results show the protein level of IGFBP7 in heart tissue of mice with myocardial infarction Gradually increased, n=3; (D) QPCR results showed that the level of IGFBP7 mRNA in the heart tissue of mice with myocardial infarction continued to increase, n=6; (E) Elisa detected the level of IGFBP7 in the serum of mice with myocardial infarction, and it also showed the same level after myocardial infarction Uptrend, n=5. ***p<0.001 vs. normal group.

Figure 4. Myocardial-specific knockout of IGFBP7 exacerbates myocardial injury after myocardial infarction.

(A) Construction of IGFBP7 ^CM-/- mice; (B) IGFBP7 ^CM -/- does not affect the cardiac function and cardiomyocyte size of mice under physiological conditions, and myocardial injury in IGFBP7 ^CM -/- mice is aggravated after myocardial infarction, n =8. *p<0.05, **p<0.01, ***p<0.001.

Figure 5. Myocardial injection of IGFBP7 significantly improved cardiac function in mice with myocardial infarction and reduced the area of fibrous scars.

(A) Survival curve of mice undergoing myocardial infarction; (B) Cardiac function indicators of mice, IGFBP7 can significantly improve cardiac function 1 week after myocardial infarction, left ventricular ejection fraction (LVEF); (C-D) Masson staining diagram of scar area And statistical graphs, n=10; (E) IGFBP7 myocardial injection significantly reduced the number of Tunel-positive cells in the myocardial infarction area of mice, n=5; (F) IGFBP7 myocardial injection significantly increased the number of blood vessels in the myocardial infarction border area of mice , n=5*p<0.05, **p<0.01.

Figure 6. Myocardial injection of IGFBP7 significantly improved cardiac function in mice with myocardial infarction and reduced the area of fibrous scars.

(A) Schematic diagram; (B) Cardiac function indicators of mice, IGFBP7 myocardial infarction 7, 14, and 28 can be detected significantly improved cardiac function, left ventricular ejection fraction (LVEF); (C) Masson staining map of the scar area And statistical graphs, n=10. *p<0.05.

Figure 7. IGFBP7 protects adult mouse cardiomyocytes against injury.

(A) Morphology of adult mouse cardiomyocytes before and after OGD injury, the proportion of rod cells in the group treated with IGFBP7 was significantly higher than that in the OGD control group, and the group added with NBI31772 was also significantly higher than that in the OGD control group, but there was no difference with the IGFBP7 group; ( B) Statistical results of the proportion of rod cells in each group, n=6; (C) Detection of LDH release, n=5; (D) Morphology of cardiomyocytes in wild-type and IGFBP7 knockout mice before and after OGD injury; (E) WT , KO rod-shaped cell proportion statistics, n=5. *p<0.05. Scale bar: 200 μm.

Figure 8. IGFBP7 inhibits the protein level of RIP3 in heart tissue.

RIP3 protein expression in heart tissue samples 3 days after myocardial infarction, n=3. ***p<0.001.

Figure 9. IGFBP7 significantly inhibited RIP3 mRNA level.

Detection of RIP3 mRNA level in heart tissue samples 3 days after myocardial infarction, n=8-10. **p<0.01, ***p<0.001.

Figure 10. IGFBP7 reduces RIP3 mRNA stability.

Adult mouse cardiomyocytes were treated with actinomycin D (10 ug/mL), and RIP3 mRNA levels were detected at 0h, 1h, 3h, 6h, and 8h, n=5. *p<0.05.

Detailed ways

On the basis of in-depth research, the present inventors revealed for the first time a composition or composition that insulin-like growth factor binding protein 7 (IGFBP7) can promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemia injury. application in medicine. Based on the above functions of IGFBP7, inhibition can also be used as a drug screening target to screen substances that promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemia damage.

IGFBP7

The IGFBPs family is a class of secreted proteins with a conserved IGFBP motif at the N-terminus, which regulates the metabolism and biological activity of IGFs by forming a complex with insulin-like growth factor I (insulin-like growth factor I, IGF-I) and IGF-II. play a key role. Currently, it is known that there are 7 members of the IGFBPs family, and their functions and biological processes are different. Among them, IGFBP1-3 and 6 are mostly involved in the regulation of obesity-related diseases metabolism and tumors. IGFBP7 is a newly discovered member of the IGFBPs family. In addition to the conserved N-terminal domain similar to IGFBP1-6, it also has a specific Kazal-type serine protease inhibitory domain and an immunoglobulin-like C2 domain. It binds to IGFs. The ability is much lower than that of other IGFBPs, suggesting that IGFBP7 has different functions and modes of action than other IGFBPs. Current studies have found that IGFBP7 not only regulates the bioavailability of IGFs and insulin, but also participates in the regulation of biological processes such as proliferation, differentiation, cell senescence and apoptosis of tumor cells such as breast cancer and lung cancer, but its exact role is still controversial. Tissues, cell types and environments may play different roles. For example, in the study of regulating aging, Wajapeyee et al. found that the activation of the proto-oncogene BRAF (B-Raf Proto-Oncogene) in human melanoma cells and fibroblasts inhibits the BRAF-MEK-ERK signaling pathway by up-regulating the synthesis and secretion of IGFBP7, and induces cell senescence and apoptosis. Scurr et al. demonstrated that BRAF signaling could neither induce IGFBP7 expression nor affect BRAF target gene expression; in terms of angiogenesis regulation, IGFBP7 was highly expressed in malignant glioma vascular endothelial cells, promoting endothelial cell proliferation and angiogenesis. Studies have found that IGFBP7 inhibits normal vascular endothelial cell proliferation and angiogenesis by inhibiting the expression of vascular endothelial growth factor. The role of IGFBP7 in myocardial infarction, myocardial ischemia/reperfusion and the effect on injured myocardial cells have not been reported.

The IGFBP7 of the present invention may be naturally occurring, for example, it may be isolated or purified from a mammal. In addition, the IGFBP7 can also be artificially prepared, for example, the recombinant IGFBP7 can be produced according to the conventional genetic engineering recombination technology, so as to be applied in experiments or clinics. Where applicable, recombinant IGFBP7 can be used. The IGFBP7 includes full-length IGFBP7 or its biologically active fragments. Preferably, the amino acid sequence of the IGFBP7 can be substantially identical to the sequence shown in SEQ ID NO:1. According to the amino acid sequence of IGFBP7, its corresponding nucleotide coding sequence can be easily obtained, for example, it can be substantially identical to the sequence shown in SEQ ID NO:1. Proteins with homology to this IGFBP7 from other species may also be included in the present invention.

The amino acid sequence of IGFBP7 formed by substitution, deletion or addition of one or more (such as 1-30) amino acid residues is also included in the present invention. IGFBP7 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. These techniques allow those skilled in the art to recognize that, in general, changes to single amino acids in non-essential regions of a polypeptide do not substantially alter biological activity.

Biologically active fragments isolated from IGFBP7 can also be used in the present invention. Here, the biologically active fragment of IGFBP7 refers to a polypeptide that can still maintain all or part of the functions of the full-length IGFBP7. Usually, the biologically active fragments retain at least 50% of the activity of the full-length IGFBP7. Under more preferred conditions, the active fragment can maintain 60%, 70%, 80%, 90%, 95%, 99% or 100% of the activity of the full-length IGFBP7.

Modified or improved IGFBP7 can also be used in the present invention, for example, IGFBP7 modified or improved to enhance its half-life, effectiveness, metabolism, and/or protein potency can be used. The modified or improved IGFBP7 may be a conjugate of IGFBP7, or it may comprise substituted or artificial amino acids. The modified or improved IGFBP7 may have little in common with the naturally occurring IGFBP7, but it can also promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and more specifically include: reducing myocardial infarction The area of fibrous scars, reducing the area of myocardial infarction after myocardial infarction, increasing the number of blood vessels in the border area of myocardial infarction after myocardial infarction, promoting the resistance of myocardial cells to damage, and inhibiting the programmed necrosis after myocardial infarction.

As used in the present invention, the "myocardial infarction" or "myocardial infarction" can be used interchangeably.

Application of IGFBP7, its involved signaling pathways and its regulators

In the research work of the present inventors, it was found that cardiovascular progenitor cells (cardiovascular progenitor cells, hCVPCs) secreted a high abundance of IGFB7 factor, and proved the role of IGFBP7 in promoting myocardial repair after myocardial infarction. Specifically, IGFBP7 can protect the cardiac function of myocardial infarction animals, reduce the fibrous scar after myocardial infarction, and promote the repair of ischemic injured myocardium. Mechanistic studies have found that its mechanism of action is at least partly through reducing the stability of RIP3 mRNA in an IGF-independent manner, thereby inhibiting the programmed necrosis of myocardial cells after myocardial infarction.

Based on the new discovery of the present inventors, the present invention provides the application of IGFBP7 or its upregulator for the preparation of compositions or drugs for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemia injury; Specifically include: reducing the area of fibrous scar after myocardial infarction, reducing the area of myocardial infarction after myocardial infarction, increasing the number of blood vessels in the border area of myocardial infarction after myocardial infarction, promoting myocardial cell resistance to injury, and inhibiting programmed necrosis after myocardial infarction. The present invention also provides the application of IGFBP7 as a target for screening substances that inhibit the above-mentioned diseases or symptoms.

As used herein, the up-regulator of IGFBP7 includes promoters, agonists and the like. Any substance that can improve the activity of IGFBP7, maintain the stability of IGFBP7, promote the expression of IGFBP7, promote the secretion of IGFBP7, prolong the effective time of IGFBP7, or promote the transcription and translation of IGFBP7 can be used in the present invention as an effective substance.

As a preferred mode of the present invention, the IGFBP7 upregulator includes (but not limited to): an expression vector or an expression construct capable of expressing (preferably overexpressing) IGFBP7 after being transferred into cells. Usually, the expression vector comprises a gene cassette, and the gene cassette contains the gene encoding IGFBP7 and the expression control sequence operably linked thereto. The term "operably linked" or "operably linked to" refers to the condition that certain parts of a linear DNA sequence can regulate or control the activity of other parts of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

In the present invention, the IGFBP7 polynucleotide sequence can be inserted into a recombinant expression vector, so that it can be transferred into cells and overexpressed to produce IGFBP7. Any plasmids and vectors can be used in the present invention as long as they can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements. For example, the expression vectors include: viral vectors, non-viral vectors; preferably, the expression vectors include (but not limited to): adeno-associated virus, lentiviral vectors, adenoviral vectors and the like.

Methods well known to those skilled in the art can be used to construct an expression vector containing the DNA sequence of IGFBP7 and appropriate transcription/translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like.

The present invention also provides a composition, which contains an effective amount (such as 0.000001-20wt%; preferably 0.00001-10wt%) of the IGFBP7, or its up-regulator (such as an expression vector that overexpresses the IGFBP7), or its analogue, and a pharmaceutically acceptable carrier.

The composition of the invention can be directly used to promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemia injury. In addition, it can also be used in combination with other therapeutic agents or adjuvants at the same time.

Generally, these materials can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, usually at a pH of about 5-8, preferably at a pH of about 6-8.

As used herein, the term "comprising" means that various ingredients 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 term "effective amount" or "effective dose" 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.

As used herein, a "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and/or mammals without undue adverse side effects (eg, toxicity, irritation and allergic reactions), ie, has a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.

The composition of the present invention contains a safe and effective amount of IGFBP7, or its upregulator (such as an expression vector that overexpresses the IGFBP7), or its analogue, and a pharmaceutically acceptable carrier. Such carriers include, but are not limited to: saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably produced under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparations of the present invention can also be made into sustained-release preparations.

The effective amount of IGFBP7 or its up-regulator in the present invention can 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: the pharmacokinetic parameters of the IGFBP7 or its upregulator such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the patient's body weight, and the immune status of the patient , the route of administration, etc. Usually, when the IGFBP7 of the present invention or its up-regulator is administered at a dose of about 0.00001 mg-10 mg/kg animal body weight per day, satisfactory results can be obtained

The administration method of IGFBP7 or its up-regulator, or its analogs of the present invention is not particularly limited, and may be systemic or local. For example, the IGFBP7 or its up-regulator of the present invention can be administered by local tissue injection, preferably myocardial injection. In addition, other ways of injection are also possible, for example, including but not limited to intraperitoneal injection, intravenous injection, oral administration, subcutaneous injection, intrathecal injection of spinal cord, intradermal injection, etc. to animals.

After knowing the use of the IGFBP7, various methods well known in the art can be used to administer the IGFBP7 or its coding gene, or its pharmaceutical composition to mammals. Preferably, gene therapy can be used, for example, IGFBP7 can be directly administered to the subject through methods such as injection; or, the expression unit carrying the IGFBP7 gene (such as an expression vector or virus, etc.) Delivered to the target and express active IGFBP7.

As an embodiment of the present invention, the IGFBP7 can be directly administered to mammals (such as humans), or the gene encoding IGFBP7 can be cloned into an appropriate vector (such as conventional prokaryotic or eukaryotic Expression vectors, or viral vectors such as herpes virus vectors or adenovirus vectors), the vectors are introduced into cells that can express the IGFBP7, so that the cells express IGFBP7. Expression of IGFBP7 can be achieved by introducing an appropriate amount of the cells into an appropriate part of the body of a mammal.

The mode of administration of an IGFBP7 up-regulator or analogue will largely depend on the type and identity of said up-regulator, which can be assessed by a person skilled in the art.

IGFBP7 as a drug screening target

After knowing the function and mechanism of action of IGFBP7, substances that promote the expression or activity of IGFBP7 can be screened based on this feature.

Therefore, the present invention provides a method for screening potential substances that can be used to promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemia damage. The method includes: combining the candidate substance with the expression IGFBP7 system contact; and detecting the impact of the candidate substance on IGFBP7; if the candidate substance can increase the expression or activity or secretion of IGFBP7, it indicates that the candidate substance can be used to promote myocardial repair after myocardial infarction and improve heart function after myocardial infarction , Protect myocardial ischemia injury.

In the study of the mechanism in the present invention, it is also found that the expression of RIP3, the key protein of programmed necrosis, is significantly reduced after IGFBP7 treatment, and the inhibition of RIP3 by IGFBP7 includes the regulation of transcription level. Based on this new discovery, in a preferred mode of the present invention, the system further includes: programmed necrosis RIP1/RIP3 signaling pathway or pathway protein RIP3. And the method further includes: detecting the interaction between insulin-like growth factor-binding protein 7 and the programmed necrosis RIP1/RIP3 signaling pathway or pathway protein RIP3 in the system, if insulin-like growth factor-binding protein 7 inhibits the The above-mentioned necroptosis RIP1/RIP3 (especially inhibiting RIP3) signaling pathway or pathway protein RIP3 indicates that the candidate substance is a desired (interesting) drug or compound.

In a preferred mode of the present invention, when performing screening, in order to more easily observe changes in the expression or activity of IGFBP7 and its involved signaling pathway proteins or their upstream and downstream proteins, a control group can also be set. A system expressing IGFBP7, RIP1/RIP3 signaling pathway or pathway protein RIP3 without adding the candidate substance may be used.

The expression system can be, for example, a cell (or cell culture) system, and the cell can be a cell endogenously expressing IGFBP7, RIP1/RIP3 signaling pathway or pathway protein RIP3; or can be a recombinant expression IGFBP7, RIP1/RIP3 RIP3 signaling pathway or pathway protein RIP3 in cells. The system for expressing IGFBP7, RIP1/RIP3 signaling pathway or pathway protein RIP3 can also be (but not limited to) subcellular system, solution system, tissue system, organ system or animal system (such as animal model).

As a preferred mode of the present invention, the method further includes: conducting further cell experiments and/or animal experiments on the obtained potential substances, so as to further select and determine substances that are really useful for exerting the functions claimed by the present invention.

The method for detecting the expression, activity, amount or secretion of IGFBP7 is not particularly limited in the present invention. Conventional protein quantitative or semi-quantitative detection techniques can be used, such as (but not limited to): SDS-PAGE method, Western-Blot method, ELISA and the like.

On the other hand, the present invention also provides the compound, composition or drug obtained by the screening method, or some potential substances. Some initially screened substances can constitute a screening library, so that people can finally screen out substances that can promote myocardial repair after myocardial infarction, improve cardiac function after myocardial infarction, and protect myocardial ischemia damage. For clinical use.

IGFBP7 as a diagnostic target

Based on the above new findings of the present inventors, IGFBP7 can be used as a marker for evaluating/prognosing disease progression after myocardial infarction: (i) performing classification, differential diagnosis, and/or susceptibility analysis of diseases after myocardial infarction; (ii) Evaluate the treatment drugs, drug efficacy, and prognosis of relevant populations after myocardial infarction, and select appropriate treatment methods; (iii) Early assessment of disease risks in relevant populations, early monitoring and early prevention and treatment. For example, high-risk groups due to abnormal expression of the IGFBP7 gene can be isolated, so that more targeted prevention or treatment can be carried out.

Therefore, the present invention provides the use of IGFBP7 for preparing a reagent or kit for evaluating or predicting disease progression after myocardial infarction.

Various techniques known in the art can be used to detect the presence, expression, expression level or activity of the IGFBP7 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 present invention also provides reagents for detecting the presence or absence and expression of the IGFBP7 gene in the analyte. Preferably, when performing detection at the gene level, primers that specifically amplify IGFBP7 can be used; or probes that specifically recognize IGFBP7 can be used to determine the presence or absence of the IGFBP7 gene; when performing detection at the protein level, specificity can be used The expression of IGFBP7 protein is determined by an antibody or ligand that binds to the protein encoded by IGFBP7. As a preferred mode of the present invention, the reagent is a primer, which can specifically amplify the IGFBP7 gene or gene fragment. The design of specific probes for the IGFBP7 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 IGFBP7 gene, but not specific to other genes other than the IGFBP7 gene Sexual binding, and the probe has a detectable signal. In addition, the method of detecting the expression of IGFBP7 protein in an analyte using an antibody that specifically binds to IGFBP7 protein is also well known to those skilled in the art.

The present invention also provides a kit for detecting the presence or absence and expression of the IGFBP7 gene in an analyte, the kit comprising: a primer for specifically amplifying the IGFBP7 gene; a probe for specifically recognizing the IGFBP7 gene; or a specific Antibodies or ligands that specifically bind to the IGFBP7 protein.

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, color development solution, etc. Color liquid, lotion, etc. In addition, the kit may also include instructions for use and/or nucleic acid sequence analysis software and the like.

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

Human IGFBP7 CDS sequence (SEQ ID NO: 2):

atggagcggccgtcgctgcgcgccctgctcctcggcgccgctgggctgctgctcctgctcctgcccctctcctcttcctcctcttcggacacctgcggcccctgcgagccggcctcctgcccgcccctgcccccgctgggctgcctgctgggcgagacccgcgacgcgtgcggctgctg ccctatgtgcgcccgcggcgagggcgagccgtgcgggggtggcggcgccggcaggggggtactgcgcgccggggcatggagtgcgtgaagagccgcaagaggcggaagggtaaagccggggcagcagccggcggtccgggtgtaagcggcgtgtgcgtgtgcaagagccgctacccggtgtgcgg cagcgacggcaccacctacccgagcggctgccagctgcgcgccgccagccagagggccgagagccgcggggagaaggccatcacccaggtcagcaagggcacctgcgagcaaggtccttccatagtgacgccccccaaggacatctggaatgtcactggtgcccaggtgtacttgagctgtgaggtcatcggaatcccgac acctgtcctcatctggaacaaggtaaaaaggggtcactatggagttcaaaggacagaactcctgcctggtgaccgggacaacctggccattcagacccggggtggcccagaaaagcatgaagtaactggctgggtgctggtatctcctctaagtaaggaagatgctggagaatatgagtgccatgcatccaattcccaaggacaggcttcagcatc agcaaaaattacagtggttgatgccttacatgaaataccagtgaaaaaaggtacacaataa

Human IGFBP7 amino acid sequence (SEQ ID NO: 1):

MERPSLRALLLGAAGLLLLLLPLSSSSSSDTCGPCEPASCPPLPLCLLGETRDACGCCPMCARGEGEPCGGGGAGRGYCAPGMECVKSRKRRKGKAGAAAGGPGVSGVCVCKSRYPVCGSDGTTYPSGCQLRAASQRAESRGEKAITQVSKGTCEQGPSIVTPPKDIWNVTGAQVYLSCEVIGIPTPVLIWNKVKR GHYGVQRTELLPGDRDNLAIQTRGGPEKHEVTGWVLVSPLSKEDAGEYECHASNSQGQASASAKITVVDALHEIPVKKGTQ

The structure domain of human IGFBP7 is shown in Fig. 1 .

Embodiment 1, the acquisition of IGFBP7

The present inventors analyzed the secretion profile of cardiovascular progenitor cells (hCVPCs) by using a cytokine chip, and found that hCVPCs secreted at least 500 factors, including cell growth factors, chemokines, and inflammatory response factors.

After large-scale research and analysis, the inventors found that among the factors secreted by hCVPCs, the presence of insulin-like growth factor binding protein 7 (Insulin-like Growth Factor Binding Protein 7, IGFBP7) in the hCVPCs conditioned medium was higher than that of the control group. The increase range was more than 500 times. Preliminary studies suggested that it may be an important effect factor for hCVPCs to play the role of myocardial repair.

Example 2. IGFBP7 inhibits programmed necrosis of myocardial cells in ischemic injury

(1) Animal model

Preparation of IGFBP7 myocardial specific knockout (IGFBP7 ^CM-/- ) mice:

Using the Cas9/RNA system gene targeting technology, construct a gRNA targeting the mouse Igfbp7 gene to guide the Cas9 protein to cut DNA double strands at the specific site of the crRNA guide sequence target; at the same time, make a homology arm carrying the target site and conditional knockout The Donor vector except for the element was co-injected with the Cas9 system, and the loxp element was recombined to both sides of exon 3 of the target site through homologous recombination after the Cas9 system cut the DNA chain to obtain igfbp7 flox mice. After mating this mouse with myh6-Cre mice, knockout of IGFBP7 can be induced by tamoxifen induction.

Mouse myocardial infarction model: male C57BL/6 mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium solution (45 mg/kg), the left front chest of the mice was depilated after fixation, and the ventilator was intubated. The thoracotomy was performed on the left side of the chest at 1 cm above the xiphoid process, 2-3 intercostal spaces were opened, and a chest expander was placed horizontally to fully expand the intercostal spaces and expose the heart. The left anterior descending coronary artery was ligated with a slipknot. After successful ligation, the left ventricle of the heart turned white and the ST segment of the electrocardiogram was elevated.

Oxygen sugar deprivation (OGD) injury model: Oxygen sugar deprivation (OGD) injury model was established on neonatal rat cardiomyocytes using hypoxic/anaerobic workstations. Workstation parameter setting: hypoxia mode oxygen concentration is less than 0.1%, carbon dioxide 5%. During the hypoxic treatment, the cell culture medium was hypoxic and normoxic. The control group was always in normal culture medium and cultured in the incubator. For cell injury assays, cells were treated anaerobically for 1 hr.

(2) Cell experiment design

cell preparation

Isolate cardiomyocytes from adult wild-type and IGFBP7 myocardial-specific knockout (IGFBP7 ^CM-/- ) mice, and simulate myocardial ischemia injury using an oxygen-glucose deprivation (OGD) injury model. Hypoxia box parameter settings: oxygen concentration in hypoxia mode is less than 0.1 %, carbon dioxide 5%. During hypoxic treatment, the cell culture medium was blood-deficient (125mM NaCl, 8mM KCl, 1.2mM KH ₂ PO ₄ , 1.25mMMgSO ₄ , 6.25mM NaHCO ₃ , 20mM HEPES-Na Salt, 5mM lactate-Na, 1.2mM CaCl ₂ and pH 6.6), the normoxic control group was always in the normal medium (M199 1% penicillin-streptomycin, 10mM 2,3-butanedione monoxime, 1% Insulin-transferrin-selenium, 1% chemically definedlipid concentrate and 5% Bovine serum albumin) And cultivated in normal incubator.

Preparation of IGFBP7 overexpressing adenovirus (Ad-IGFBP7): Using Lipofectamine 2000 as a transfection reagent, pAd-igfbp7 plasmid and adenovirus backbone plasmid were co-expressed in 293A cells. After culturing for 5-7 days, observe the cells to confirm the formation of virus clones. About 12 days later, the cells in the center of the clone were lysed to form lesions, and the virus liquid in the lesions was sucked up, and then added to 500 μL DMEM. This virus is a primary virus. The 293A cells were spread in a 100mm culture dish, and 100 μL of primary virus was added to infect the cells. Visible cell lysis is usually seen within 2-3 days after infection. Virus was harvested when about one-third to one-half of the cells were floating (usually 3-5 days post-infection). Gently scrape the cells off, transfer to a 15mL centrifuge tube, freeze the cells in liquid nitrogen, then thaw in a 37°C water bath, and shake vigorously. This step was repeated 3 times. After centrifugation at 6000 rpm at 4°C for 5 minutes, the supernatant was collected and stored. Be careful not to repeatedly freeze and thaw when storing, and store it at -20°C; in order to further amplify the adenovirus, re-infect the cells in a 100mm culture dish with the virus supernatant obtained in the previous step, and perform the same operation to collect the virus and further infect 150mm cells 293A cells in a petri dish to obtain a sufficient amount of virus.

The aforementioned recombinant adenovirus infects cardiomyocytes to obtain recombinant cardiomyocytes overexpressing IGFBP7.

Detection Indicator

For the cell injury assay, cells were treated with hypoxia for 1 hr, and cell morphology, lactate dehydrogenase (LDH) release, ATP synthesis, and mitochondrial membrane potential were measured to evaluate cell injury.

(2) Design of animal experiments

1. Experimental animals

8-12 week old C57BL6 WT mice;

8-12 week old IGFBP7 ^CM-/- mice.

Experimental group:

a: Sham operation + solvent group;

b: operation group + solvent group;

c: operation group +IGFBP7 (1ng/kg);

d: Operation group + IGFBP7 (30 ng/kg).

2. Detection indicators:

(1) Survival rate: detect the survival rate of mice in groups a, b, c, and d;

(2) Cardiac function ultrasound: Left ventricular ejection fraction (LVEF) was detected by cardiac function ultrasound at 2, 7, 14 and 28 days after operation;

(3) Scar area: Myocardium tissue was embedded with OCT and sectioned with Masson staining to evaluate the fibrous scar area;

(4) Apoptosis of cardiomyocytes: Myocardium tissue was embedded with OCT and sections were stained with TUNEL to evaluate the apoptosis of injured cardiomyocytes;

(5) Angiogenesis: Myocardial tissue was embedded with OCT and stained with a-SMA to evaluate the angiogenesis in the myocardial infarction area and marginal area;

(6) Collect myocardial tissue samples to extract RNA, protein, gene expression detection and follow-up experiments.

Embodiment 3, the expression of IGFBP7 in cardiomyocytes

In order to reveal the cardioprotective effect of IGFBP7, it is first necessary to clarify the expression of IGFBP7 in the heart. The high-throughput analysis of the database (ProteomicsDB) shows that IGFBP7 has a relatively stable expression in many tissues of the normal human body (Figure 2A, the yellow is the heart), muscle Tissues are only expressed in the heart. Immunofluorescent staining of adult mouse cardiomyocytes and fibroblasts revealed that IGFBP7 was ubiquitously distributed in cardiomyocytes (Fig. 2B), while in fibroblasts, it was distributed in a dot-like manner and partly co-localized with vimentin ( Figure 2B).

Western blot results showed that the expression of IGFBP7 was significantly higher in cardiomyocytes than in fibroblasts (Fig. 2C).

Example 4, IGFBP7 is up-regulated in myocardial infarction

The results of the antibody chip showed that IGFBP7 was a factor highly secreted by hCVPCs (Figure 3A), and the content of IGFBP7 in hCVPCs conditioned medium (CdM) was detected by Elisa (Figure 3B).

The mouse model of myocardial infarction was used to collect time-course samples of heart tissue and serum from mice with myocardial infarction, and to detect the changes of IGFBP7 expression after myocardial infarction. The results showed that within one week after myocardial infarction, the expression of IGFBP7 showed a continuous upward trend (Figure 3C-Figure 3E), suggesting that it plays an important role in the occurrence and subsequent development of myocardial infarction.

Example 5. IGFBP7 Myocardial Specific Knockout Exacerbates Myocardial Injury After Myocardial Infarction

In the induced knockout IGFBP7 ^CM-/- mice cardiomyocytes, IGFBP7 was knocked out (Figure 4A), the surface area of IGFBP7 ^CM-/- mice cardiomyocytes was not different from that of wild-type mice, the ratio of heart weight to body weight, heart function No difference.

However, after myocardial infarction surgery, the cardiac function of IGFBP7 ^CM-/- mice (KO) was significantly decreased compared with that of wild-type mice (Fig. 4B).

Example 6. Myocardial injection of human recombinant protein IGFBP7 factor improves cardiac function and reduces scar area in mice with myocardial infarction

Using a mouse model of myocardial infarction (MI), a myocardial injection experiment of human recombinant protein IGFBP7 was carried out to evaluate its effect on cardiac function. Two doses of 1ng/kg and 30ng/kg were used to inject IGFBP7 into the myocardium at the same time as the myocardial infarction operation, and then continue to inject IGFBP7 subcutaneously at 2.4ug/kg/day within one week, and detect it on days 2, 7, 14 and 28 The heart function of the mice was evaluated for myocardial infarct size at 4 weeks (Fig. 5A, B).

The results showed that myocardial injection of IGFBP7 did not affect the survival rate of the operated mice (Fig. 5B), and the cardiac function of the mice could be significantly improved 1 week after the injection (Fig. 5C), and the area of fibrous scar was significantly reduced (Fig. 5D).

Evaluation of cardiomyocyte apoptosis by TUNEL staining found that myocardial injection of IGFBP7 significantly reduced the number of Tunel-positive cells in the myocardial infarction area of mice (Fig. 5E).

The results of a-SMA staining showed that the number of blood vessels in the border area of the myocardial infarction area in the IGFBP7 myocardial injection group increased (Fig. 5F).

Example 7, IGFBP7 antibody neutralization weakens the myocardial repair effect of hCVPCs

Using the mouse model of myocardial infarction, IGFBP7 antibody neutralization experiments were carried out (Figure 6A), the cardiac function of the mice was detected at 2, 14 and 28 days after operation, and the size of the myocardial infarction was evaluated at 28 days.

The results showed that IGFBP7 antibody could weaken the effect of hCVPCs on protecting cardiac function and limiting fibrous scar (Fig. 6B, Fig. 6C).

Example 8, IGFBP7 protects adult mouse cardiomyocytes against OGD injury

In order to further clarify that the myocardial protection of IGFBP7 is closely related to the protective effect on cardiomyocytes, adult mouse cardiomyocytes were isolated, an in vitro OGD injury model was established, and the ability of IGFBP7 treatment group and control group to resist injury was compared. The results showed that the proportion of rod cells in the IGFBP7-treated group was significantly higher than that in the OGD control group (Fig. 7A-B).

At the same time, it was found that the addition of NBI31772, an inhibitor of the combination of IGFBP7 and IGF, did not affect the protective effect of IGFBP7 on cardiomyocytes, suggesting that IGFBP7 protects the myocardium through an IGF receptor-independent pathway.

Further evaluation of the ability of cardiomyocytes of WT and KO mice to resist OGD injury revealed that the release of LDH in cardiomyocytes of KO mice was significantly increased after injury, and the proportion of rod cells was significantly lower than that of the wild type (Fig. 7C-E).

The above results indicated that IGFBP7 has the function of protecting cardiomyocytes against injury.

Example 9, IGFBP7 has inhibitory effect on RIP3 protein level in myocardial tissue after myocardial infarction

Based on the above research results, the role of IGFBP7 in protecting cardiomyocytes was clarified, and it was further revealed that its protective effect was achieved through an IGF receptor-independent pathway. Necrosis is the main way of myocardial cell death after myocardial infarction, in which programmed necrosis occupies an important proportion. In cardiac tissue samples 3 days after myocardial infarction, the classic RIP1/RIP3 pathway of programmed necrosis was detected.

The results showed that the expression of RIP3, a key protein of necroptosis in the IGFBP7 treatment group, was significantly reduced (Figure 8), suggesting that IGFBP7 has an inhibitory effect on the occurrence of necroptosis after myocardial infarction.

Embodiment 10, IGFBP7 suppresses RIP3 mRNA level

Further, QPCR detection was performed to analyze the expression of RIP3 mRNA after myocardial injection of IGFBP7.

The results showed that myocardial injection of IGFBP7 significantly suppressed RIP3 mRNA levels. This result suggested that the inhibition of RIP3 by IGFBP7 included regulation at the transcriptional level ( FIG. 9 ).

Embodiment 11, overexpression IGFBP7 reduces RIP3 mRNA stability

In order to further clarify how IGFBP7 regulates the RIP3 mRNA level, whether it reduces its transcription or promotes its degradation, actinomycin D (ActD, 10ug/mL) was used to inhibit the transcription of adult mouse cardiomyocytes, and the degradation of RIP3 mRNA by IGFBP7 was evaluated regulation.

The results showed that after adenovirus (Ad) expressed IGFBP7, it reduced the stability of RIP3 mRNA and promoted its degradation (Figure 10), indicating that IGFBP7 inhibited the expression of RIP3 by reducing the stability of RIP3 mRNA, thereby inhibiting the programmed necrosis of injured cardiomyocytes.

Embodiment 12, screening method

(1) Screening based on IGFBP7 expression or activity

Cells: cardiomyocytes overexpressing IGFBP7.

Test group: culturing the cardiomyocytes overexpressing IGFBP7 and administering candidate substances;

Control group: culture the cardiomyocytes overexpressing IGFBP7 without administering the candidate substance.

The expression or activity of IGFBP7 in the test group and the control group were respectively detected and compared. If the expression or activity of IGFBP7 in the test group is statistically higher (e.g., 30% higher or lower) than the control group, it indicates that the candidate is capable of promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, protecting Potential substance for myocardial ischemic injury.

Further, the RIP1/RIP3 signaling pathway is co-expressed in the cells, and the activity can be judged by observing the following conditions: observing the situation of IGFBP7 reducing the stability of RIP3 mRNA in the test group. If the test group can significantly promote the reduction of RIP3 mRNA stability after adding the candidate substance, it indicates that the candidate is a potential substance for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemia injury .

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims. Also, all documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference.

sequence listing

<110> Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences

<120> A cell-secreted factor that promotes the repair of myocardial infarction and its application

<130> 216701

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<170> SIPOSequenceListing 1.0

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Met Glu Arg Pro Ser Leu Arg Ala Leu Leu Leu Gly Ala Ala Gly Leu

1 5 10 15

Leu Leu Leu Leu Leu Pro Leu Ser Ser Ser Ser Ser Ser Ser Asp Thr Cys

20 25 30

Gly Pro Cys Glu Pro Ala Ser Cys Pro Pro Leu Pro Pro Leu Gly Cys

35 40 45

Leu Leu Gly Glu Thr Arg Asp Ala Cys Gly Cys Cys Pro Met Cys Ala

50 55 60

Arg Gly Glu Gly Glu Pro Cys Gly Gly Gly Gly Ala Gly Arg Gly Tyr

65 70 75 80

Cys Ala Pro Gly Met Glu Cys Val Lys Ser Arg Lys Arg Arg Lys Gly

85 90 95

Lys Ala Gly Ala Ala Ala Gly Gly Pro Gly Val Ser Gly Val Cys Val

100 105 110

Cys Lys Ser Arg Tyr Pro Val Cys Gly Ser Asp Gly Thr Thr Tyr Pro

115 120 125

Ser Gly Cys Gln Leu Arg Ala Ala Ser Gln Arg Ala Glu Ser Arg Gly

130 135 140

Glu Lys Ala Ile Thr Gln Val Ser Lys Gly Thr Cys Glu Gln Gly Pro

145 150 155 160

Ser Ile Val Thr Pro Pro Lys Asp Ile Trp Asn Val Thr Gly Ala Gln

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Val Tyr Leu Ser Cys Glu Val Ile Gly Ile Pro Thr Pro Val Leu Ile

180 185 190

Trp Asn Lys Val Lys Arg Gly His Tyr Gly Val Gln Arg Thr Glu Leu

195 200 205

Leu Pro Gly Asp Arg Asp Asn Leu Ala Ile Gln Thr Arg Gly Gly Pro

210 215 220

Glu Lys His Glu Val Thr Gly Trp Val Leu Val Ser Pro Leu Ser Lys

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Glu Asp Ala Gly Glu Tyr Glu Cys His Ala Ser Asn Ser Gln Gly Gln

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atggagcggc cgtcgctgcg cgccctgctc ctcggcgccg ctgggctgct gctcctgctc 60

ctgcccctct cctcttcctc ctcttcggac acctgcggcc cctgcgagcc ggcctcctgc 120

ccgcccctgc ccccgctggg ctgcctgctg ggcgagaccc gcgacgcgtg cggctgctgc 180

cctatgtgcg cccgcggcga gggcgagccg tgcgggggtg gcggcgccgg caggggggtac 240

tgcgcgccgg gcatggagtg cgtgaagagc cgcaagaggc ggaagggtaa agccggggca 300

gcagccggcg gtccgggtgt aagcggcgtg tgcgtgtgca agagccgcta cccggtgtgc 360

ggcagcgacg gcaccaccta cccgagcggc tgccagctgc gcgccgccag ccagagggcc 420

gagagccgcg gggagaaggc catcacccag gtcagcaagg gcacctgcga gcaaggtcct 480

tccatagtga cgccccccaa ggacatctgg aatgtcactg gtgcccaggt gtacttgagc 540

tgtgaggtca tcggaatccc gacacctgtc ctcatctgga acaaggtaaa aaggggtcac 600

tatggagttc aaaggacaga actcctgcct ggtgaccggg acaacctggc cattcagacc 660

cggggtggcc cagaaaagca tgaagtaact ggctgggtgc tggtatctcc tctaagtaag 720

gaagatgctg gagaatatga gtgccatgca tccaattccc aaggacaggc ttcagcatca 780

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Claims (11)

1. Use of insulin-like growth factor binding protein 7 or a upregulating agent thereof in the manufacture of a medicament or composition for the following function: promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and relieving myocardial ischemia injury.

2. The use of claim 1, wherein promoting myocardial repair following myocardial infarction, improving cardiac function following myocardial infarction, and protecting against myocardial ischemic injury comprises:

reducing the area of the myocardial infarction area after myocardial infarction;

the area of a fibrous scar after myocardial infarction is reduced;

increasing the number of blood vessels in the myocardial infarction area and the myocardial infarction edge area after myocardial infarction;

promoting myocardial cells to resist damage;

the method can inhibit programmed necrosis of myocardial infarction, preferably inhibit activation of RIP1/RIP3 signal pathway of programmed necrosis, and further inhibit programmed necrosis of injured myocardial cell.

3. The use of claim 1, wherein the up-regulator of insulin-like growth factor binding protein 7 comprises an up-regulator selected from the group consisting of: (a) A substance that enhances the activity of insulin-like growth factor binding protein 7; (b) A substance that enhances the expression, stability or effective duration of action of insulin-like growth factor binding protein 7.

4. The use of claim 1, wherein the up-regulator of insulin-like growth factor binding protein 7 comprises a peptide selected from the group consisting of: an expression construct for recombinant expression of insulin-like growth factor binding protein 7, a polypeptide or compound that enhances the inhibitory effect of insulin-like growth factor binding protein 7 on the cellular programmed necrosis RIP1/RIP3 signal pathway, a chemical up-regulator of insulin-like growth factor binding protein 7, an up-regulator that promotes the driving ability of the insulin-like growth factor binding protein 7 gene promoter, a down-regulator of insulin-like growth factor binding protein 7 gene-specific microRNA, or a combination thereof; preferably, the expression construct comprises: viral vectors, non-viral vectors; more preferably, the expression vector comprises: adeno-associated virus vectors, lentiviral vectors, and adenoviral vectors.

5. Use of insulin-like growth factor binding protein 7 for screening a drug or compound having the following functions: promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemia injury.

6. A method for screening a drug or a compound for promoting myocardial repair after myocardial infarction, improving cardiac function after myocardial infarction, and protecting myocardial ischemic injury, which comprises:

(1) Treating an expression system expressing insulin-like growth factor binding protein 7 with a candidate substance; and the combination of (a) and (b),

(2) Detecting the expression or activity of insulin-like growth factor binding protein 7 in said system; if the candidate substance statistically increases the expression or activity of insulin-like growth factor binding protein 7, it is indicative that the candidate substance is the desired drug or compound.

7. The method of claim 6, wherein step (1) comprises: in the test group, adding a candidate substance to the expression system; and/or the presence of a gas in the atmosphere,

the step (2) comprises the following steps: detecting the expression or activity of insulin-like growth factor binding protein 7 in said system and comparing said expression or activity to a control, wherein said control is an expression system without said candidate substance; if the candidate substance statistically increases the expression or activity of insulin-like growth factor binding protein 7, it is indicative that the candidate substance is the desired drug or compound.

8. The method of claim 6 or 7, wherein the system of step (1) further comprises a apoptosis RIP1/RIP3 signal pathway or pathway protein RIP3; and, step (2) also includes: and detecting the interaction condition of the insulin-like growth factor binding protein 7 and the programmed necrosis RIP1/RIP3 signal pathway or pathway protein RIP3 in the system, and if the insulin-like growth factor binding protein 7 inhibits the programmed necrosis RIP1/RIP3 signal pathway or pathway protein RIP3, indicating that the candidate substance is the required medicine or compound.

9. Use of a reagent specifically recognizing or amplifying insulin-like growth factor binding protein 7 for the preparation of a reagent or kit for assessing or prognosing disease progression after myocardial infarction.

10. The use according to claim 9, wherein a low expression of insulin-like growth factor binding protein 7 is indicative of a poor prognosis of the subject after myocardial infarction; if insulin-like growth factor binding protein 7 is normally expressed or highly expressed, it indicates that the prognosis of the subject after myocardial infarction is normal or good.

11. The use of claim 9, wherein said agent comprises:

a binding molecule that specifically binds to insulin-like growth factor binding protein 7;

primers for specifically amplifying the insulin-like growth factor binding protein 7 gene;

a probe that specifically recognizes insulin-like growth factor binding protein 7 gene; or

A chip for specifically recognizing the 7 gene of insulin-like growth factor.

Images