Application of adeno-associated virus vector in preparation of medicine for treating hepatic fibrosis

Abstract

The invention discloses application of an adeno-associated virus vector in preparation of a medicine for treating hepatic fibrosis, human miR-29a and a flanking sequence are used as core sequences to construct an expression cassette, the expression cassette is transferred into the adeno-associated virus vector to construct a recombinant adeno-associated virus vector, and after the vector is packaged into a virus, the liver fibrosis can be improved, a target gene Col1a1 of the liver fibrosis can be reduced, and the liver fibrosis can be improved. The compound can be used for preparing drugs for treating hepatic fibrosis and drugs for inhibiting expression of hepatic fibrosis target genes and / or target proteins. The recombinant adeno-associated virus is constructed by using the human miR-29a and intercepting a part of flanking sequences, and the recombinant adeno-associated virus is more suitable for preparing the medicines for treating the hepatic fibrosis diseases, and is higher in safety coefficient and more remarkable in effect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to an application of an adeno-associated virus vector in preparing a drug for treating liver fibrosis. Background Art

[0002] Hepatic fibrosis refers to the excessive deposition of extracellular matrix (especially collagen) in the liver. It is not an independent disease, but many chronic liver diseases can cause hepatic fibrosis. Its causes can be roughly divided into infectious (chronic hepatitis B, C and D, schistosomiasis, etc.), congenital metabolic defects (hepatolenticular degeneration, hemochromatosis, α1-antitrypsin deficiency, etc.) and chemical metabolic defects (chronic alcoholic liver disease, chronic drug-induced liver disease) and autoimmune hepatitis, primary hepatic cirrhosis and primary sclerosing cholangitis, etc. In addition, various inflammatory factors and oxidative stress are also constantly promoting the process of hepatic fibrosis. If early hepatic fibrosis is not treated, it will eventually lead to cirrhosis and even liver cancer.

[0003] MicroRNA (miRNA) is a group of endogenous non-coding RNAs that regulate gene expression. MiRNA can inhibit the expression of target genes by regulating transcription or post-transcriptional translation. Changes in miRNA expression lead to changes in genes involved in a series of biological processes, which can lead to many human diseases. Therefore, miRNA has become a potential target for the treatment of many diseases. And because of its high stability in body fluids, circulating miRNA is considered a promising biomarker for diagnosis and disease prognosis.

[0004] The miR-29 family consists of miR-29a, miR-29b-1, miR-29b-2, and miR-29c, and is produced by two main transcripts: the pri-miR-29a / b1 cluster and the pri-miR-29b2 / c cluster, which are located on human chromosomes 7q32.3 and 1q32.2, respectively

[18] . The miR-29 family is often considered to be a very effective gene therapy for fibrosis because its target genes are highly overlapped with fibrosis-related genes. For example, in 2022, the research team of Maurizio Chioccioli from the Yale School of Medicine coupled BippB with a modified miR-29 mimic to treat bleomycin-induced pulmonary fibrosis mouse model to treat mouse pulmonary fibrosis. In the application of liver fibrosis treatment, the miR-29 family has shown significant therapeutic effects in different liver fibrosis models. The research team from Warren used adeno-associated virus AAV8 as a delivery tool to deliver the miR-29a overexpression plasmid into the liver of CCL4-modeled mice, and alleviated the fibrosis of mice by inhibiting the expression of Col1a1 in the extracellular matrix; Geng's research team found that TGFβ1 secretory glycoprotein Fstl1 is the target gene of miR-29a through TGFβ1 stimulation of LX-2 cells and CCL4-induced mouse liver fibrosis, and miR-29a alleviated CCL4-induced liver fibrosis by inhibiting the upregulation of Fstl1 in human and mouse fibrotic livers and activated HSCs.

[0005] With more and more research on miRNA in liver fibrosis, miRNA has been proven to be one of the important means of diagnosing and treating liver fibrosis. However, at present, when miR-29a is used to diagnose and treat liver fibrosis, mouse-derived miR-29a is generally used, and only AAV viral vectors are used to deliver simple miR-29a sequences into cells for diagnosis and treatment. Although mouse-derived miR-29a has a good effect in mouse models, there is uncertainty in the diagnosis and treatment of human liver fibrosis disease models. The expression of mouse-derived miR-29a in human cells may be limited or have other adverse effects. In addition, how to improve the tissue characteristics and safety of the delivery vector is also particularly important for the diagnosis and treatment of liver fibrosis. Summary of the invention

[0006] In response to the above technical problems, the present invention provides an application of an adeno-associated virus vector in the preparation of a drug for the treatment of liver fibrosis. Human miR-29a and flanking sequences are used as core sequences to construct an expression cassette, which is transferred into an adeno-associated virus vector to construct a recombinant adeno-associated virus vector. The vector can downregulate liver fibrosis target genes and target proteins and is used to prepare a drug for the treatment of liver fibrosis.

[0007] In order to achieve the above object, the present invention provides an adeno-associated virus vector for use in preparing a drug for treating liver fibrosis, wherein the adeno-associated virus vector is capable of downregulating Col1a1 Adeno-associated viral vector for expression.

[0008] Preferably, the ability to downregulate Col1a1 The expression cassette of the adeno-associated virus vector contains wu-miR-01 or wu-Mir-01.

[0009] Further preferably, in the expression cassette, wu-miR-01 is human miR-29a plus flanking sequences, and the sequence is SEQ ID NO: 1; and the wu-Mir-01 is mouse miR-29a and flanking sequences, and the sequence is SEQ ID NO: 2.

[0010] Preferably, the adeno-associated virus vector is any one of AAV2, AAV5, and AAV8.

[0011] Further preferably, the adeno-associated virus vector is AAV5.

[0012] Preferably, the adeno-associated virus vector is used to deliver Col1a1 The recombinant adeno-associated virus vector constructed by the expression cassette is packaged into a virus and then injected intravenously for use in the preparation of drugs for inhibiting liver fibrosis.

[0013] Further preferably, the use of adeno-associated virus vectors to deliver Col1a1 The recombinant adeno-associated virus vector constructed by the expression cassette is packaged into a virus and then injected intravenously for use in preparing a drug for inhibiting the expression of liver fibrosis target genes and / or target proteins.

[0014] Further preferably, the target gene is Col1a1 Genes and / or Col1a4 gene, and the target protein is Col1a1 protein and / or α-SMA protein.

[0015] Furthermore, the expression cassette contains wu-miR-01 or wu-Mir-01.

[0016] Preferably, the wu-miR-01 in the expression cassette is human miR-29a plus flanking sequences, and the sequence is SEQ ID NO:1; the wu-Mir-01 is mouse miR-29a and flanking sequences, and the sequence is SEQ ID NO:2.

[0017] The beneficial effects of the present invention are: 1. Human miR-29a and flanking sequences were used as core sequences to construct an expression cassette with CMV as the promoter, and then a recombinant adeno-associated virus vector with AAV adeno-associated virus vector as the backbone was constructed. After the recombinant vector was packaged into a recombinant adeno-associated virus, it can efficiently infect the LX-2 cell line in vitro, and has high infectivity to the 293T cell line and T6 cell line.

[0018] 2. The recombinant adeno-associated virus containing human miR-29a and flanking sequences was transferred into LX-2 cells stimulated by TGFβ1, which could significantly downregulate liver fibrosis-related target genes Col1a1 and Col1a4 expression.

[0019] 3. When the recombinant adeno-associated virus containing human miR-29a and flanking sequences was injected into the mouse liver fibrosis model through the tail vein, the recombinant adeno-associated virus had liver tissue tropism, and restored the liver surface of the liver fibrosis mouse model to a normal state with smooth surface, no obvious fine granular nodules, and sharp edges. At the same time, the vacuolar degeneration and necrosis of stem cells were significantly reduced, and the fibrous intervals were reduced. In addition, the expression of ALT and AST in the mouse serum was significantly reduced. Col1a1 The expression levels of gene, Col1a1 protein and a-SMA protein.

[0020] 4. The present invention uses adeno-associated virus vector AAV5, which enhances the delivery efficiency and safety of recombinant adeno-associated virus, and also has good liver tissue tropism, which enhances the targeting of drugs to fibrotic liver tissue, and can further reduce the dosage of drugs for treating liver fibrosis and reduce costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a diagram of the construction and identification results of the recombinant adeno-associated virus vector constructed using wu-miR-01 in Example 1, in which A is a schematic diagram of the recombinant vector expression cassette, B is a gel electrophoresis diagram of wu-miR-01, C is a double enzyme digestion electrophoresis diagram of the recombinant vector, and D is a comparison diagram of the sequencing results of the recombinant vector; in Figure B, 1 is a DNA ladder, and 2-6 are PCR products of wu-miR-01; in Figure C, 1 is a DNA ladder, 2-5 are recombinant vector double enzyme digestion products, and 6 is a vector backbone double enzyme digestion product.

[0022] Figure 2This is a diagram of the construction and identification results of the recombinant adeno-associated virus vector constructed using wu-Mir-01 in Example 1, in which A is a schematic diagram of the recombinant vector expression cassette, B is a gel electrophoresis diagram of wu-Mir-01, C is a double enzyme digestion electrophoresis diagram of the recombinant vector, and D is a comparison diagram of the sequencing results of the recombinant vector; in Figure B, 1 is a DNA ladder, and 2 is the PCR product of wu-Mir-01; in Figure C, 1 is a DNA ladder, 2 is a double enzyme digestion product of the recombinant vector, and 3 is a double enzyme digestion product of the vector backbone.

[0023] Figure 3 The figures are the functional verification results of the recombinant adeno-associated virus vector in Example 2, wherein A is a fluorescent photograph of the recombinant vector after transfection into 293T cells, B is a bar graph of the expression levels of miR-29a 3p and 5p; C is a bar graph of the expression level of the liver fibrosis-related target gene Col1a1 after cells were infected with AAV-wu-miR-01 containing GFP, and D is a diagram of the mechanism of miR-29a targeting Col1a1; in the figure, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.005, and **** indicates P<0.001.

[0024] Figure 4 The results of the effect of the flanking sequence on the expression of miR-29a in Example 3 are shown in Figure A, which is a bar graph of the expression level in T6 cells, and Figure B is a bar graph of the expression level in LX-2 cells.

[0025] Figure 5 These are fluorescent photographs of different cell lines infected in vitro with the AAV-GFP-wu-miR-01 recombinant adeno-associated virus in Example 4; the scale bar in the figure is 200 μm.

[0026] Figure 6 These are fluorescent photographs of the LX-2 cell line at different infection times in vitro with the AAV-GFP-wu-miR-01 recombinant adeno-associated virus in Example 4; the scale bar in the figure is 200 μm.

[0027] Figure 7 The infection efficiency of different vector skeletons on LX-2 in Example 4.

[0028] Figure 8 The expression of miR-29a mature target genes after LX-2 cells were infected with recombinant adeno-associated virus under TGFβ1 stimulation in Example 5, A is a bar graph of miR-29a 3p expression, B is a bar graph of miR-29a 5p expression, C is a bar graph of Col1a1 expression, and D is a bar graph of Col1a4 expression; in the figure, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.005, and **** indicates P<0.001.

[0029] Fig. 9 These are tissue photographs of mouse organs under different treatment conditions in Example 6. Figure A is a tissue photograph of mouse organs under different treatment conditions, and Figure B is a fluorescent photograph of mouse organs under different treatment conditions. From left to right in the figure, the organs are heart, liver, spleen, lung, and kidney.

[0030] Fig.10 These are gross photographs of the liver tissues of mice under different treatment conditions in Example 7. The scale bar in the figure is 0.5 cm.

[0031] Fig.11 HE staining photos of liver tissues of mice under different treatment conditions in Example 7. The scale bar in the figure is 100 μm Fig.12 Masson staining photos of liver tissue of mice under different treatment conditions in Example 7. The scale bar in the figure is 500 μm Fig.13 The changes in serological indicators of mouse ocular venous blood under different treatment conditions in Example 8, in which A is a bar graph of ALT expression, B is a bar graph of AST expression, and C is a bar graph of HA expression; ** in the figure indicates P<0.01, and *** indicates P<0.005.

[0032] Fig.14 The expression of mature mouse miR-29a and target genes under different treatment conditions in Example 9, A is a bar graph of miR-29a 3p expression, B is a bar graph of miR-29a 5p expression, and C is a bar graph of target gene Col1a1 expression; in the figure, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.005, and **** indicates P < 0.001.

[0033] Fig.15 The expression of mouse target protein under different treatment conditions in Example 9, A is the Western blot electrophoresis of the target protein, B is the quantitative analysis bar graph of Western Blotting electrophoresis by imageJ, C is the expression bar graph of Col1a1 protein, and D is the expression bar graph of a-SMA protein; in the figure, * indicates P<0.05, ** indicates P<0.01, *** indicates P<0.005, and **** indicates P<0.001. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. It is worth noting that the following embodiments are only preferred embodiments of the present invention and should not be understood as limiting the present invention. The protection scope of the present invention shall be based on the contents recorded in the claims. Any modification or replacement made by those skilled in the art to the technical solution of the present invention without creative work shall fall within the protection scope of the present invention.

[0035] Experimental animals, cells and plasmids Specific pathogen free (SPF) grade male C57BL / 6 mice were purchased from the Experimental Animal Center of Three Gorges University and kept in cages in the Experimental Animal Center of Three Gorges University. The initial weight of the experimental mice was 18-20 g. The animal breeding room maintained a constant temperature of (22±2)ºC and a constant humidity of (60±5)%. During the feeding period, the experimental mice had a normal free diet. All animal feeding was approved by the Animal Experiment Ethics Committee, and the experimental mouse feeding was carried out in accordance with the "Guidelines for the Care and Use of Laboratory Animals of Three Gorges University". HEK-293T cell line: donated by Professor Zha Yunhong of Yichang Central People's Hospital.

[0036] HSC-T6 cell line (rat hepatic stellate cells): donated by Professor Song Yuhu of Tongji Hospital, Huazhong University of Science and Technology.

[0037] LX-2 cell line (human hepatic stellate cells): gifted by Professor Liu Changbai of the Basic Medical College of China Three Gorges University.

[0038] AAV-CMV-GFP plasmid: purchased from Addgene official website.

[0039] pAAV5 plasmid and pAAV8 plasmid were purchased from Addgene official website.

[0040] pAdDeltaF6 plasmid: donated by Professor Li Botao from the School of Health Medicine.

[0041] Example 1 Construction of recombinant adeno-associated virus vector (1) Obtain mouse and human miR-29a and its flanking sequences from the NCBI website, respectively, and design primers for PCR amplification. The human miR-29a and flanking sequence (denoted as wu-miR-01) is SEQ ID NO: 1, and the mouse miR-29a and flanking sequence (denoted as wu-Mir-01) is SEQ ID NO: 2. Amplification primers are designed. The primer sequences are shown in Table 1. (2) AAV5-GFP and AAV5 were double-digested using HindⅢ, XolⅠ and 10×Tango buffer to obtain linearized vectors; (3) The miR-29a sequence was mixed with the linearized vector at a molar ratio of 7:1 by homologous recombination, and then transformed, screened, and sequenced for detection ( Figure 1-2 ) The successfully constructed recombinant adeno-associated virus vectors were obtained: AAV5-GFP-Mir-29a, AAV5-GFP-wu-miR-01, AAV5-Mir-29a, AAV5-wu-miR-01, and the recombinant adeno-associated plasmid vector plasmid was extracted for later use.

[0042] Table 1 Amplification primers for miR-29a with flanking sequences

[0043] Example 2 Functional verification of recombinant adeno-associated virus vector Taking AAV5-GFP-wu-miR-01 and AAV5-wu-miR-01 recombinant adeno-associated virus vectors as examples, after transfection, observe whether miR-29a can be successfully expressed and whether GFP will affect the expression of wu-miR-01. The specific methods are as follows: (1) Experimental group (AAV5-GFP-wu-miR-01): AAV5-GFP-wu-miR-01-1 recombinant vector was transfected into 293T cells using a transfection reagent; Control group (AAV5-wu-miR-01): AAV5-wu-miR-01 recombinant vector was transfected into 293T cells using transfection reagent; Blank control group (MOCK): 293T cells were treated with an equal amount of transfection reagent; (2) Observe the fluorescence intensity of transfected cells by fluorescence microscopy 48 hours after transfection; (3) Total RNA was extracted from each group of cells, and the expression of miR-29a 3p, miR-29a 5p, and liver fibrosis-related target gene Col1a1 was detected by qPCR; The results showed that the AAV5-GFP-wu-miR-01 group had stronger fluorescence expression, while the AAV5-wu-miR-01 group had no fluorescence expression ( Figure 3 A); qPCR detection showed that the expression of mature miR-29a 3p and 5p in AAV5-GFP-wu-miR-01 group and AAV5-wu-miR-01 group was significantly higher than that in MOCK group ( Figure 3 B), indicating that the constructed plasmid can overexpress the mature miR-29a and the removal of the GFP protein on the plasmid has no effect on the expression of the inserted wu-miR-01 target sequence; compared with the MOCK group, the expression level of Col1a1 in the AAV5-GFP-wu-miR-01 group was significantly decreased ( Figure 3C).

[0044] Example 3 Effect of flanking sequences on miR-29a expression (1) Constructing a recombinant adeno-associated virus vector without flanking sequences: AAV5-GFP-miR-29a according to the method described in Example 1, wherein the primer sequence is shown in Table 2, and its sequence is SEQ ID NO: 3; (2) The recombinant adeno-associated virus vector AAV5-GFP-wu-miR-01 containing the flanking sequence in Example 1 was transfected into T6 cells and LX-2 cells respectively; AAV5-GFP was transfected into T6 cells and LX-2 cells as a control group; (3) 48 h after transfection, RNA of transfected cells was extracted, and the expression of miR-29a 3p and miR-29a 5p was detected by qPCR; The results are as follows Figure 4 As shown in the figure: after adding flanking sequences to miR-29a, the expression levels of miR-29a 3p and miR-29a 5p increased significantly. Therefore, the addition of flanking sequences can increase the expression level of miR-29a, and after being made into lentivirus, the infection efficiency can be improved, thereby reducing the dosage and cost.

[0045] Table 2 Primers for amplification of miR-29a without flanking sequences

[0046] Example 4 Investigation of the infectivity of recombinant adeno-associated virus (1) According to the method described in Example 1, a recombinant adeno-associated virus vector with an AAV8 backbone was constructed: AAV8-GFP-wu-miR-01, and the primer sequences are shown in Table 3; (2) AAV5-GFP-wu-miR-01 constructed in Example 1 and the vector prepared in step (1) were transfected into 293T cells respectively, packaged using a viral packaging system (Table 4), and then purified with iodixanol to obtain AAV virus; (3) using the AAV virus obtained in step (2) to infect LX-2 cell line, T6 cell line, and 293T cell line in vitro; (4) The fluorescence intensity was observed under an inverted fluorescence microscope at 1, 3, 5, and 7 days after infection to determine the virus infection efficiency.

[0047] The results showed that the recombinant adeno-associated virus had different infection intensities on different cell lines. On day 5 of infection, the fluorescence intensity of the AAV5-GFP-wu-miR-01 group on the LX-2 cell line was the highest, followed by the 293T cell line, and the fluorescence of the T6 cell line was the weakest, indicating that the AAV5-GFP-wu-miR-01 group had the highest infection efficiency on the LX-2 cell line and the lowest infection efficiency on the T6 cell line ( Figure 5 ). As the infection time prolonged, the infection intensity of the AAV5-GFP-wu-miR-01 group on the LX-2 cell line was also different. The fluorescence intensity of the AAV5-GFP-wu-miR-01 group reached a peak on the 5th day of infection of the LX-2 cell line ( Figure 6 ).Depend on Figure 7 It can be seen that the infection efficiency of LX-2 cells when AAV5 is used as the vector backbone is significantly higher than that when AAV8 is used as the vector backbone Table 3 AAV8 vector wu-miR-01 amplification primers

[0048] Table 4 Virus packaging system

[0049] Example 5 Effect of TGFβ1 stimulation on the function of recombinant adeno-associated virus TGFβ1 is a transforming growth factor and one of the most effective factors that promote liver fibrosis. Therefore, TGFβ1 can be used to stimulate cells to construct a liver fibrosis cell activation model, and then the liver fibrosis cell activation model can be infected with a recombinant adeno-associated virus to detect the expression of wu-miR-01 and the expression of liver fibrosis-related target genes. The specific method is as follows: (1) LX-2 cells were stimulated with TGFβ1 for 24 h to construct a liver fibrosis activation model; (2) Experimental group (TGFβ1-rAAV5-GFP-wu-miR-01): The liver fibrosis activation model constructed in step (1) was infected with the AAV5-GFP-wu-miR-01 recombinant AAV virus constructed and packaged in Example 4; Control group 1 (TGFβ1): an equal volume of 1× PBS solution with recombinant AAV virus was added to the liver fibrosis activation model of uninfected cells; Blank control group (MOCK): unstimulated LX-2 cells were added with an equal volume of 1× PBS solution as the recombinant AAV virus; (3) 48 hours after viral infection, total RNA was extracted from each group of cells, and the expression of mature miR-29a 3p and 5p was detected by qPCR. U6 and GAPDH were also used to detect liver fibrosis-related target genes. Col1a1 and Col1a4 expression of.

[0050] The results are as follows Figure 8 As shown in the figure, compared with the control group, the expression of mature miR-29a 3p and 5p in the experimental group infected with AAV5-GFP-wu-miR-01 virus was significantly increased, and the liver fibrosis-related target genes in the experimental group Col1a1 and Col1a4 The expression was significantly downregulated.

[0051] Example 6 Distribution of recombinant adeno-associated virus in mice Inject recombinant adeno-associated virus into the mouse liver fibrosis model to explore the tissue distribution of the recombinant adeno-associated virus in mice and observe the tissue tropism of the recombinant adeno-associated virus (1) Experimental group (CCL4+rAAV5-GFP-wu-miR-01): Mice were injected with AAV5-GFP-wu-miR-01 recombinant virus through the tail vein after 6 weeks of 20% CCl4 modeling. The injection volume was 6×10^11 vp / mouse. Control group 3 (CCL4): mice 6 weeks after modeling with 20% CCl4 were injected with an equal volume of 1× PBS solution (the virus solvent) through the tail vein; Blank control group (MOCK): normal 6-week-old mice, injected with an equal volume of 1× PBS solution into the tail vein; (2) Four weeks after viral infection, the mice were killed and their hearts, livers, spleens, lungs, and kidneys were removed. Small animal tissue imaging was performed under white light and at an excitation wavelength of 475 nm.

[0052] The results are as follows Fig. 9 As shown: Compared with the control group of liver fibrosis mice without virus particle injection, AAV5-wu-miR-01 labeled with GFP green fluorescent protein was mainly expressed in the liver, kidney, and lung, while no fluorescence appeared in the heart and spleen, and the expression intensity in the liver was stronger, indicating that AAV5 has liver tissue tropism.

[0053] Example 7 Effect of recombinant adeno-associated virus on liver fibrosis in mice (1) Experimental group (CCL4+rAAV5-GFP-wu-miR-01): mice that had been modeled with 20% CCl4 for 2 weeks were injected with AAV5-GFP-wu-miR-01 virus through the tail vein at a volume of 6×10^11 vp / mouse; Control group 1 (CCL4+rAAV5-GFP): mice that had been modeled with 20% CCl4 for 2 weeks were injected with an equal amount of AAV5-GFP through the tail vein; Control group 2 (CCL4): mice modeled with 20% CCl4 for 2 weeks were injected with an equal volume of 1× PBS solution through the tail vein; Blank control group (MOCK): normal mice were injected with an equal volume of 1× PBS solution through the tail vein; (2) Eight weeks after viral infection, the liver tissues of mice were perfused with saline and then taken for gross liver photography to observe liver fibrosis. (3) The liver tissues of mice in each group were collected and made into paraffin sections, which were then stained with HE and Masson to observe the fibrosis of the mouse liver tissues.

[0054] The results are as follows Fig.10 As shown, the liver tissue surface of mice in the MOCK group was smooth, with sharp edges and no nodules; the liver surface of mice in the CCl4 group and CCl4+AAV5 group was relatively rough and covered with granular nodules with blunt edges; while the liver surface of mice in the CCl4+AAV5-wu-miR-01 group was smoother, without obvious fine granular nodules, and with sharper edges.

[0055] The results showed that the liver tissues of the CCl4 group and the CCl4+AAV5 group showed a large number of hepatocyte ballooning and necrosis, inflammatory cells infiltrated in the fibrous scar, and the liver fibrous septa increased significantly. Correspondingly, the hepatocyte vacuolar degeneration and necrosis in the CCl4+AAV5-wu-miR-01 group were significantly alleviated, and the fibrous septa decreased ( Fig.11 ); No blue collagen was observed in the MOCK group, but obvious blue collagen was observed in the CCl4 and CCl4+AAV5 groups. The collagen thickening in the portal area was obvious, and multiple collagen bundles were cross-linked and divided into leaflets. Compared with the empty control group, the blue collagen in the CCl4+AAV5-wu-miR-01 group was significantly reduced ( Fig.12 ).

[0056] Example 8 Effects of recombinant adeno-associated virus on mouse serological parameters The four groups of mice infected for 8 weeks in Example 7 were taken, and ocular venous blood was collected for serological detection. The test results showed that among the three serological detection indicators ALT, AST, and HA, the expression levels of the MOCK group were the lowest, and the expression levels of the CCL4 group and the CCL4+AAV5-GFP group were relatively high; the expression of ALT and AST in the serum of the mice in the CCl4+AAV5-wu-miR-01 group was significantly reduced, and the hyaluronic acid HA was also reduced to a certain extent ( Fig.13 ).

[0057] Example 9 Expression of recombinant adeno-associated virus in mouse liver and regulation of target genes The four groups of mice infected for 8 weeks in Example 7 were killed and the total RNA and protein in the liver were extracted. The expression levels of the mature forms 3p and 5p of miR-29a were detected, and the expression level of the liver fibrosis target gene Col1a1 was detected at the same time. In addition, the β-actin protein was used as an internal reference, and the expression of the target protein Col1a1 protein and a-SMA protein was detected by Western Blot experiment.

[0058] The results showed that compared with the MOCK group, the expression levels of mature miR-29a 3p and 5p in the CCl4 group were significantly decreased, and the expression levels of 3p and 5p in the CCl4+AAV5 group were also significantly decreased, while the expression levels of 3p and 5p in the CCl4+AAV5-miR-29a group were significantly increased ( Fig.14 a, b); The gene level of the target gene Col1a1 was detected and it was found that the expression level in the CCL4 group and the CCL4+AAV5-GFP group was the highest, while the expression level in the CCl4+AAV5-miR-29a group was significantly reduced, and the expression level in the MOCK group was the lowest ( Fig.14 c).

[0059] Western Blot results showed that the expression levels of target proteins Col1a1 and α-SMA were the highest in the CCL4 group and CCL4+AAV5-GFP group, significantly decreased in the CCl4+AAV5-wu-miR-01 group, and the lowest in the MOCK group ( Fig.15 ).

Claims

1. Use of an adeno-associated virus vector in the preparation of a drug for treating liver fibrosis, characterized in that: The adeno-associated virus vector is capable of down-regulating Col1a1 Adeno-associated virus vector for expression.

2. Use of an adeno-associated virus vector according to claim 1 in the preparation of a drug for treating liver fibrosis, characterized in that: The energy can be downregulated Col1a1 The expression cassette of the adeno-associated virus vector contains wu-miR-01 or wu-Mir-01.

3. Use of an adeno-associated virus vector according to claim 2 in the preparation of a drug for treating liver fibrosis, characterized in that: The wu-miR-01 in the expression cassette is human miR-29a and flanking sequences, and the sequence is SEQ ID NO:1; the wu-Mir-01 is mouse miR-29a and flanking sequences, and the sequence is SEQ ID NO:

2.

4. Use of an adeno-associated virus vector according to claim 1 or 2 in the preparation of a drug for treating liver fibrosis, characterized in that: The adeno-associated virus vector is any one of AAV2, AAV5, and AAV8.

5. Use of an adeno-associated virus vector according to claim 4 in the preparation of a drug for treating liver fibrosis, characterized in that: The adeno-associated virus vector is AAV5.

6. Use of an adeno-associated virus vector according to claim 2 in the preparation of a drug for treating liver fibrosis, characterized in that: Adeno-associated viral vector delivery can downregulate Col1a1 The recombinant adeno-associated virus vector constructed by the expression cassette is packaged into a virus and then injected intravenously for use in the preparation of drugs for inhibiting liver fibrosis.

7. Use of an adeno-associated virus vector according to claim 6 in preparing a drug for treating liver fibrosis, characterized in that: Adeno-associated viral vector delivery can downregulate Col1a1 The recombinant adeno-associated virus vector constructed by the expression cassette is packaged into a virus and then injected intravenously for use in preparing a drug for inhibiting the expression of liver fibrosis target genes and / or target proteins.

8. Use of an adeno-associated virus vector according to claim 7 in the preparation of a drug for treating liver fibrosis, characterized in that: The target gene is Col1a1 Genes and / or Col1a4 gene, and the target protein is Col1a1 protein and / or α-SMA protein.

9. Use of an adeno-associated virus vector according to any one of claims 6 to 8 in the preparation of a drug for treating liver fibrosis, characterized in that: The expression cassette contains wu-miR-01 or wu-Mir-01.

10. Use of an adeno-associated virus vector according to claim 9 in preparing a drug for treating liver fibrosis, characterized in that: The wu-miR-01 in the expression cassette is human miR-29a and flanking sequences, and the sequence is SEQ ID NO:1; the wu-Mir-01 is mouse miR-29a and flanking sequences, and the sequence is SEQ ID NO:2.

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