CN114755415B - Method for treating liver fibrosis diseases by inducing degradation endothelial cells with combined factors - Google Patents

Abstract

The invention discloses a method for treating hepatic fibrosis diseases by combining factor-induced degradation endothelial cells, in particular to a method for screening the capacity of factors to degrade extracellular matrix, which comprises the steps of 1) inoculating cells to be detected on the surface of collagen, wherein the collagen is combined with fluorescent molecules, and 2) culturing the cells in the step 1); 3) Treating the cells with a test agent; 4) Performing fluorescence detection on the supernatant of the culture solution in the step 3), wherein the detected excitation light is 546nm, the emitted light is 575nm, and the fluorescence value obtained by detection represents the degradation capability of cells. According to this method, cells having a high degradability can be selected.

Description

Method for treating liver fibrosis by inducing degradative endothelial cells with combined factors

Technical Field

The invention relates to the field of biotechnology, to chemical factors stimulating endothelial cells and treating liver fibrosis, and in particular to a method for screening cell degradation ability and a drug for treating liver fibrosis.

Background technique

Chronic inflammation often leads to fibrosis, involving various organs or tissues, including skin, kidney, liver, lung, etc. In the development of fibrosis, different cell types can synthesize type 1 collagen, including myofibroblasts, macrophages, etc. The excessive secretion of cells leads to the deposition of type I collagen.

Fibrosis is the excessive accumulation of extracellular matrix proteins, accompanied by inflammation, and most of them occur in chronic diseases. The degradation of the excessively accumulated extracellular matrix is mainly mediated by metal matrix proteases. As the degree of liver fibrosis becomes more and more serious, the MMP13 gene of astrocytes gradually becomes low-expressed, the MMP3 gene first increases and then decreases, and the MMP9 and MMP12 genes of inflammatory macrophages, kuffer cells, and activated astrocytes are highly expressed. Myofibroblasts have also become the main cells for synthesizing extracellular matrix.

Currently, there is still a need to improve the treatment of fibrotic diseases.

Summary of the invention

The present invention proposes for the first time a method for screening cells for their ability to degrade extracellular matrix. According to this method, cells with high degradation ability can be screened out. These cells degrade the extracellular matrix in vitro or even in vivo, thereby reversing liver fibrosis. The inventors have used this endothelial cell therapy (i.e., screened cells with degradation ability are used for disease treatment) for the first time to treat liver fibrosis and related diseases.

In the first aspect of the present invention, the present invention proposes a method for screening factors for the ability to degrade extracellular matrix. According to an embodiment of the present invention, the method comprises: 1) inoculating the cells to be tested on the surface of collagen, the collagen is combined with fluorescent molecules, 2) culturing the cells in step 1); 3) treating the cells with the factors to be tested; 4) performing fluorescence detection on the supernatant of the culture solution after treatment in step 3), wherein the excitation light of the detection is 546nm, the emission light is 575nm, and the fluorescence value obtained by the detection characterizes the degradation ability of the cells. The present invention proposes for the first time a method for screening the ability of cells to degrade extracellular matrix. According to the method, factors to be tested with high degradation ability (which may be a combination of factors or a single factor) can be screened out, and these factors to be tested degrade the extracellular matrix in vitro or even in vivo, thereby reversing liver fibrosis. It should be noted that the operations such as inoculation here, unless otherwise specified, can all be carried out by conventional technical means.

According to an embodiment of the present invention, the above method further includes at least one of the following additional technical features:

According to an embodiment of the present invention, the fluorescent molecule is selected from 5(6)-TAMRA SE, FITC, CY3 and CY7, etc. It should be noted that there is no special requirement for the fluorescent molecule, and the required fluorescent molecule can be selected according to actual needs.

According to an embodiment of the present invention, the culture is carried out in the dark for two days at 37° C. It should be noted that the culture here has special requirements and can be adjusted according to the culture conditions of different cells.

According to an embodiment of the present invention, the fluorescence detection is performed by an ELISA reader.

According to an embodiment of the present invention, the fluorescence value is greater than the fluorescence value of the supernatant after incubation of pure culture medium with collagen material, indicating that the cells have the ability to degrade the extracellular matrix.

According to an embodiment of the present invention, the cells to be tested are selected from at least one of endothelial cells, immune cells and astrocytes. It should be noted that there is no particular limitation on the cells to be tested, and the inventors can select different cells as needed. The inventors used liver sinusoidal endothelial cells, human umbilical vein endothelial cells and other cells for corresponding tests.

In another aspect of the present invention, the present invention also proposes the use of a pharmaceutical composition in the preparation of a drug for treating fibrotic diseases, inflammation, sclerotic diseases or cancer. According to an embodiment of the present invention, the pharmaceutical composition includes a PMA factor. The inventors have found that the PMA factor can stimulate cells such as hepatic sinusoidal endothelial cells and human umbilical vein endothelial cells, giving them a strong ability to degrade extracellular matrix, greatly slowing down and improving the process of inflammation, fibrosis, sclerosis and canceration of organs such as the liver, lungs and kidneys.

According to an embodiment of the present invention, the use further includes at least one of the following technical features:

According to an embodiment of the present invention, the pharmaceutical composition further includes at least one selected from Accutase, type I immune factor and type II immune factor. The inventors found that the combination of at least one of Accutase type I immune factor and type II immune factor and PMA factor (referred to as P) (such as IL4+P, IL13+P, IL1β+P, IL6+P, IFNγ+P, TNFα+P, etc.) can stimulate cells such as hepatic sinusoidal endothelial cells and human umbilical vein endothelial cells, so that they have a strong ability to degrade extracellular matrix, greatly alleviating and improving inflammation, fibrosis, sclerosis, and cancerous diseases of organs such as the liver, lungs, and kidneys.

According to an embodiment of the present invention, the Accutase is 100X-1000X Accutase (A1110501), and the PMA factor is 50 ng/ml. The inventors found that under this combination, the degradation ability is stronger.

According to an embodiment of the present invention, the fibrotic disease is selected from liver fibrosis, kidney fibrosis and lung fibrosis.

According to an embodiment of the present invention, the fibrotic disease is induced by carbon tetrachloride.

According to an embodiment of the present invention, the cirrhotic disease is selected from liver cirrhosis.

According to an embodiment of the present invention, the cancer is selected from liver cancer.

According to an embodiment of the present invention, the inflammation is selected from hepatitis.

According to an embodiment of the present invention, the pharmaceutical composition is obtained by screening according to the aforementioned method. Accutase and PMA factors are obtained by the inventors by screening according to the aforementioned method, and the fluorescence value of the combination of these two factors is 550.

In another aspect of the present invention, the present invention also proposes a method for treating fibrosis, inflammation, sclerosis or cancer. According to an embodiment of the present invention, the method comprises: administering a pharmaceutical composition to a patient suffering from fibrosis, inflammation, sclerosis or cancer, wherein the pharmaceutical composition comprises a PMA factor. The inventors have found that the combination of PMA factors can stimulate hepatic sinusoidal endothelial cells, giving them a strong ability to degrade extracellular matrix, greatly slowing down and improving the progression of organ inflammation, fibrosis, sclerosis and cancer.

According to an embodiment of the present invention, the method further includes at least one of the following technical features:

According to an embodiment of the present invention, the pharmaceutical composition further comprises at least one selected from Accutase, type I immune factor and type II immune factor. The inventors have found that the combination of at least one of type I immune factor and type II immune factor of Accutase and PMA factor can stimulate cells such as hepatic sinusoidal endothelial cells and human umbilical vein endothelial cells, making them have a strong ability to degrade extracellular matrix, greatly alleviating and improving inflammation, fibrosis, sclerosis, and cancerous diseases of organs such as liver, lung, and kidney.

According to an embodiment of the present invention, the Accutase is 100X-1000X Accutase (A1110501), and the PMA factor is 50 ng/ml. The inventors found that under this combination, the degradation ability is stronger.

According to an embodiment of the present invention, the fibrotic disease is selected from liver fibrosis, kidney fibrosis and lung fibrosis.

According to an embodiment of the present invention, the fibrotic disease is induced by carbon tetrachloride.

According to an embodiment of the present invention, the pharmaceutical composition is obtained by screening according to the aforementioned method. For example, Accutase and PMA factors are obtained by the inventors by screening according to the aforementioned method, and the fluorescence value of the combination of these two factors is 550.

According to an embodiment of the present invention, the cirrhotic disease is selected from liver cirrhosis.

According to an embodiment of the present invention, the cancer is selected from liver cancer.

According to an embodiment of the present invention, the inflammation is selected from hepatitis.

Definitions and general terms

Certain embodiments of the present invention are now described in detail, examples of which are illustrated by the accompanying structural formulas and chemical formulae. The present invention is intended to encompass all substitutions, modifications, and equivalent technical solutions, which are all included within the scope of the present invention as defined in the claims. It should be appreciated by those skilled in the art that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the combined documents, patents, and similar materials differ from or contradict the present application (including but not limited to defined terms, term applications, described technologies, etc.), the present application shall prevail.

It should be further appreciated that certain features of the invention, which for clarity are described in the context of multiple separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Unless otherwise specified, all technical terms used in the present invention have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All patents and publications related to the present invention are incorporated herein by reference in their entirety.

Unless otherwise indicated, the following definitions used herein shall apply. For purposes of the present invention, chemical elements are consistent with the Periodic Table of the Elements, CAS version, and Handbook of Chemistry and Physics, 75th edition, 1994. In addition, general principles of organic chemistry can be found in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry" by Michael B. Smith and Jerry March, John Wiley & Sons, New York: 2007, the entire contents of which are incorporated herein by reference.

Unless otherwise specified or there is a clear conflict in context, the articles "a", "an", and "the" as used herein are intended to include "at least one" or "one or more". Therefore, these articles as used herein refer to articles that refer to one or more than one (i.e., at least one) of the objects. For example, "a component" refers to one or more components, i.e., there may be more than one component contemplated for use or use in the implementation of the described embodiment.

The term "subject" as used in the present invention refers to an animal. Typically, the animal is a mammal. Subjects, for example, also refer to primates (e.g., humans, male or female), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In certain embodiments, the subject is a primate. In other embodiments, the subject is a human.

The term "patient" used in the present invention refers to humans (including adults and children) or other animals. In some embodiments, "patient" refers to humans.

The term "comprising" is an open expression, that is, including the contents specified in the present invention but not excluding other contents.

As used herein, the term "treating" any disease or condition, in some embodiments, refers to ameliorating the disease or condition (i.e., slowing or preventing or alleviating the development of the disease or at least one clinical symptom thereof). In other embodiments, "treating" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treating" refers to regulating the disease or condition physically (e.g., stabilizing perceptible symptoms) or physiologically (e.g., stabilizing physical parameters), or both. In other embodiments, "treating" refers to preventing or delaying the onset, occurrence, or worsening of a disease or condition.

The pharmaceutical compositions provided by the present invention can be formulated into immediate or modified release dosage forms, including delayed-, sustained-, pulsed-, controlled-, targeted-, and programmed release forms.

The pharmaceutical composition provided by the present invention can be co-formulated with other active ingredients that do not impair the intended therapeutic effect, or co-formulated with substances that supplement the intended effect.

Pharmaceutical composition provided by the invention can be administered parenterally by injection, infusion or implantation, for local or systemic administration. Parenteral administration as used in the present invention includes intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial and subcutaneous administration.

Pharmaceutical composition provided by the invention can be formulated into any dosage form suitable for parenteral administration, including solution, suspension, emulsion, micelle, liposome, microsphere, nano system and the solid form suitable for making solution or suspension in liquid before injection. Such dosage form can be prepared according to the conventional method known to the technicians in the field of pharmaceutical science (referring to Remington:The Science and Practice of Pharmacy, the same).

The endothelial cell therapy described in the present invention refers to the use of hepatic sinusoidal endothelial cells to treat liver fibrosis diseases and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

FIG1 is a high-throughput screening result according to an embodiment of the present invention;

FIG2 is a cell morphology diagram of dLSEC and nLSEC according to an embodiment of the present invention and a morphology diagram after being degraded on collagen for two days;

FIG3 is a diagram showing the ability of dLSEC and nLSEC to degrade substrates according to an embodiment of the present invention;

FIG4 is a SEM diagram showing the degradation of collagen by dLSEC and nLSEC according to an embodiment of the present invention;

FIG5 is a qPCR characterization of MMPs genes according to an embodiment of the present invention;

6 is a diagram showing the in vivo distribution of dLSEC and nLSEC injected via the spleen according to an embodiment of the present invention;

FIG7 is a comparison diagram of Sirius red staining, immunofluorescence staining, and in situ gelatin zymography staining according to an embodiment of the present invention;

FIG8 is a diagram of in vivo ACTA2 gene detection according to an embodiment of the present invention;

FIG. 9 is a diagram showing the in vivo MMPs gene expression according to an embodiment of the present invention.

Detailed ways

Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

The raw materials used in the present invention are all purchased from commercial sources, Accutase: A1110501, Gibco. PMA: plant-derived recombinant human serum albumin OsrHSA (culture medium grade), HYC002M01, oryzogen/Wuhan Heyuan. 5(6)-TAMRASE: HY-D0723, MCE. Rat tail type I collagen 100 mg: 354236, BD. PMA: V1171, Promega.

General approach

1. Preparation of fluorescent glue:

1 mL of 3.17 mg/mL type I collagen (3.17 mg/mL) was added to a 10 kDa dialysis bag, placed in 1 L of pre-cooled labeling buffer (0.25 M NaHCO3, 0.4 M NaCl, solvent: deionized water, PH = 9.5), and dialyzed at 4°C overnight; 1 mg of fluorescent molecule (5(6)-TAMRA SE) was dissolved in 100 μL of DMSO at room temperature, and then 900 μL of labeling buffer (filtered with 0.22 μm filter membrane) was added and mixed, and pre-cooled on ice (protected from light); the dialyzed collagen was squeezed out, added to the above fluorescent molecule solution and mixed, and 500 μL was dispensed into each tube, and mixed overnight at 4°C in the dark; the mixed collagen was added to the dialysis bag, placed in 1 L of pre-cooled labeling buffer, and dialyzed at 4°C in the dark overnight; the dialysis bag was placed in 1 L of pre-cooled 0.2% acetic acid solution with a pH of 4, and dialyzed at 4°C in the dark overnight.

2. Fluorescent glue forming:

Prepare the reagents according to the following dosages: 27.55μl of type I collagen, 2.75μl of fluorescent gel, 19μl of DMEM basal medium, and 0.6969μl of 1M sodium hydroxide. Mix the DMEM medium with the NaOH solution and pre-cool it on ice; mix the collagen with the fluorescent gel; add PBS to the wells around the gel on the 96-well plate to keep it moist, and place the 96-well plate on ice; after blowing and aspirating evenly, quickly add it to the wells of the 96-well plate, 50μL per well; move the 96-well plate to a 37℃ incubator away from light; take it out after 1h 30min for use.

3. Fluorescent glue degradation experiment

2×10 ⁴ cells were added into the wells in a volume of 150 μL and moved into a 37°C incubator. After 2 days of degradation, the wells were taken out in the dark. The supernatant in the wells was transferred to a black well plate. The fluorescence value of the supernatant was detected with an excitation light of 546 nm and an emission light of 575 nm. The fluorescence value can quantitatively characterize the degradation ability of the cells.

4. Immunofluorescence Staining

Remove the culture medium and wash twice with PBS. Fix with PFA for 30 min and wash twice with PBS. Block with membrane blocking solution for 1 h. Dilute primary antibody with primary antibody diluent and incubate at 4°C overnight. Wash three times with PBS. Secondary antibody (1:400, dissolved in PBS), RT, 1 h. Wash three times with PBS. DAPI (1:2000), F-actin (1:200), 15 min, RT. Wash three times with PBS.

5. Sirius Red Staining

Frozen sections were allowed to recover at room temperature for 5-10 min. PFA fixation was performed for 30 min. Rinse with running water for 1 min. Staining was performed with Sirius red stain for 3 min. After 3 min, staining was stopped with deionized water. Dehydration was performed with 70% ethanol for 5 min, 90% ethanol for 5 min, 100% ethanol for 5 min, xylene I, xylene II. After air drying, the sections were sealed with neutral resin.

6. In situ gelatin zymography

Dissolve GENMED colloid solution (Reagent A) in a 60℃ water bath. Carefully transfer 40 microliters of GENMED colloid solution (Reagent A) to a 1.5 ml centrifuge tube. Incubate in a 37℃ constant temperature water bath for 10 minutes. At the same time, preheat GENMED staining solution (Reagent B) at 37℃ instantly, and do not keep it at 37 degrees for a long time. Add 5 microliters of 37℃ preheated GENMED staining solution (Reagent B) and mix well. Add 0.1 microliter of hochest to the A+B mixture. Mix well again, and immediately add 40 microliters of the above liquid to the unfixed frozen section samples. Cover with a coverslip (note: avoid bubbles). Incubate in a 4℃ refrigerator for 10 minutes, avoid light. Incubate in a 37℃ incubator for 60 minutes, avoid light. Observe immediately under a fluorescence microscope: excitation wavelength 480nm, emission wavelength 530nm.

7. Animal Model of Liver Fibrosis

Male Balb/c nu mice, 6 weeks old, induced with 10% CCL4, 1.5 μl/g, twice a week for 4 weeks.

8. In vivo bioluminescence monitoring of LSEC-Akaluc

Mesenchymal stem cells for intrasplenic injection were engineered to stably express luciferase by lentiviral-mediated gene transfer (LSEC-Akaluc). To visualize LSEC Akaluc, mice were injected intraperitoneally with 40 μL of 15 mg/mL TokeOni (808350, Sigma) 5 minutes before imaging. Mice with liver fibrosis were injected intrasplenicly with 100 μL PBS or 4 × 10 ⁵ LSECs suspended in 100 μL PBS. Mice were imaged using the IVIS Lumina II imaging system (Caliper Life Sciences) to observe the distribution of LSECs in vivo.

9. qPCR Analysis

Cells or tissues (e.g., liver) were homogenized in RNA ISOLATORTRIZOL reagent (R401-01, Vazyme) before RNA extraction. 1 μg of DNA-free total RNA was synthesized into cDNA using reverse transcriptase (R211-01, Vazyme). Gene-specific transcriptional analysis was performed in triplicate on a CFX96 instrument (Bio-Rad) using AceQ qPCR SYBR Green Master Mix (Q121-02, Vazyme). All genes were normalized to GAPDH. Relative expression was calculated using the 2 ^ΔΔCT method.

10. SEM characterization of the interaction between cells and materials

The collagen material with dLSEC and nLSEC on the surface was fixed with 2.5% glutaraldehyde for 2 hours. After fixation, it was washed 3 times with PBS and then dehydrated with a series of 70%, 80%, 90% and 100% ethanol solutions, each step lasting 10 minutes. The samples were freeze-dried using critical point drying (Tousimis samdrii-795). The dried samples were gold-plated for 90 seconds before imaging with a scanning electron microscope (FEIQuanta 200) for microstructural evaluation.

Embodiment 1:

Type I collagen combined with fluorescent molecules is uniformly gelled in a 96-well plate, and then cells are planted on the surface of the collagen. Two days later, the cell culture supernatant in the well plate is aspirated, and then the fluorescence value of the cell culture supernatant is detected by an ELISA instrument and recorded as sampling (the excitation light is 546nm, the emission light is 575nm, and the fluorescence value represents the degradation ability of the cells). At the same time, the inventor will use the fluorescence value of the pure culture medium and collagen material after two days of incubation to record it as standard 1, and the fluorescence value of 0.5mg/ml MMP1 (the solvent is pure culture medium) and collagen material after two days of incubation (that is, the fluorescence value of completely degraded collagen material) is recorded as standard 2. The formula for calculating the proportion of cell-degraded collagen material is: Percentage of degraded collagen material = (sampling-standard 1)/(standard 2-standard 1)*100%

Example 2

Using the method in Example 1, the inventors screened endothelial cells, immune cells, astrocytes, etc. The specific results are shown in Figure 1, where A represents Accutase and P represents PMA.

The inventors have found that the combination of Accutase and PMA factor can stimulate hepatic sinusoidal endothelial cells, making them have a strong ability to degrade extracellular matrix.

In the table below, the cell types are Raw264.7 (mouse mononuclear macrophage leukemia cells), PM is primary macrophage extracted from mouse bone marrow, THP1 is human myeloid leukemia mononuclear cells, ESC-LSEC, ESC-MSC are liver sinusoidal endothelial cells differentiated from human embryonic stem cells, mesenchymal stem cells differentiated from human embryonic stem cells, LX2 is human hepatic stellate cells, C166 is yolk sac endothelial cells, 3T3 is mouse fibroblasts, HL60 is human promyelocytic acute leukemia cells. Serumfree is serum-free culture (basic medium, 100x glutamax, 0.5mg/ml albumin).

A+P can stimulate LSEC to show the strongest degradation ability, A+P can also stimulate ESC-LSEC to show strong degradation ability, and the other combinations are weak.

Factors include IL4 (50 ng/ml), IL13 (50 ng/ml), LPS (20 ng/ml), A (200X), fMLP (2 μM), TGFβ (3 ng/ml), and P (50 ng/ml).

The results are shown in Table 1 below:

Table 1

Example 3

dLSEC is LSEC stimulated by Accutase and PMA. nLSEC is normal LSEC.

After the inventors induced the cells with Accutase+PMA on a culture dish for one day, they observed under a microscope that dLSEC had an elongated cell morphology and nLSEC had an oval cell morphology. After the cells were digested and seeded on type I collagen, dLSEC could degrade them into a mass of cells and collagen after two days, while nLSEC spread dispersedly on the collagen, as shown in Figure 2.

Through the detection of the degradation ability of fluorescent colloid cells, within two days, dLSEC can degrade 80% of type I collagen and nLSEC can degrade 5% of type I collagen, as shown in Figure 3.

Scanning electron microscopy showed that dLSEC could degrade surrounding type I collagen, resulting in large cavities, while no large cavities appeared around nLSEC (see Figure 4).

By qPCR, the expression of MMPs related genes in dLSEC and nLSEC was detected. The inventors found that the expression of MMP2, 8, 9, 10, and 14 in dLSEC was higher than that in nLSEC, among which MMP9 was as high as 200 times, as shown in FIG5 .

dLSEC-akaluc was injected into the liver fibrosis model induced for 4 weeks via the spleen, and imaging was performed on the first, third, and fifth days. The results showed that dLSEC-akaluc and nLSEC-akaluc accumulated in the liver on the first and third days; on the fifth day, almost no signal was detected in the cells injected into the body, as shown in Figure 6.

In Sirius red staining, the darker color indicates the deposition of collagen in the body. The more collagen is deposited, the more severe the degree of liver fibrosis. From Figure 7, it can be seen that the dLSEC group significantly improved the degree of liver fibrosis. According to the results of immunofluorescence staining of aSMA and col1a1 (liver fibrosis marker protein) in tissue sections, the inventors found that compared with the nLSEC group and the fibrotic mice group, the expression of aSMA and col1a1 in the dLSEC group was lower, indicating that liver fibrosis was alleviated. According to the results of in situ gelatin zymography, the dLSEC group had high expression of green fluorescence, indicating that the content of matrix metalloproteinases in the body was high, indicating that dLSEC improved the progression of liver fibrosis in vivo by degrading the extracellular matrix.

The ACTA2 gene is a key gene that characterizes the severity of liver fibrosis. Generally speaking, the higher the expression of ACTA2, the more severe the liver fibrosis. From Figure 8 (Normal mice are normal mice, Fibrotic mice are 4-week liver fibrosis mice without treatment, dLSEC is the dLSEC treatment group injected through the spleen, and nLSEC is the nLSEC treatment group injected through the spleen.) It can be seen that the expression of the ACTA2 gene in the dLSEC group is significantly lower than that in the Fibrotic mice group, indicating that dLSEC has a certain therapeutic effect on liver fibrosis.

The inventors also characterized the expression of MMPs genes in vivo after treatment of liver fibrosis. As can be seen from Figure 9, the expression of MMP2, 8, 9, 10, and 14 genes in the dLSEC group was higher than that in the nLSEC group and the fibrotic mice group. From the gene level, it was shown that dLSEC alleviated the progression of liver fibrosis by matrix degradation mediated by MMPs.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (3)

1. Use of a pharmaceutical composition comprising PMA factor and Accutase in the manufacture of a medicament for the treatment of liver fibrosis disease.

2. The use according to claim 1, wherein the pharmaceutical composition is capable of increasing the ability of endothelial cells to degrade extracellular matrix.

3. The use according to claim 2, wherein the endothelial cells are liver sinus endothelial cells and human umbilical vein endothelial cells.