CN112111520A - Adeno-associated virus expression vector of GRIM-19 gene overexpression and application thereof in inhibiting pathological process of hepatic fibrosis - Google Patents

Abstract

The invention relates to the field of biology, in particular to an adeno-associated virus expression vector of GRIM-19 gene overexpression and application thereof in inhibiting pathological processes of hepatic fibrosis. The adeno-associated virus expression vector comprises GRIM-19 gene and adeno-associated virus AAV8 linearized vector GV599, and the adeno-associated virus AAV8 linearized vector GV599 comprises a thyroglobulin TBG promoter capable of mediating the specific expression of hepatocytes. The adeno-associated virus expression vector can obviously and specifically recover the expression of GRIM-19 gene in liver cells of a mouse liver fibrosis model induced by CCl4, and can effectively reduce or reverse CCl 4-induced liver fibrosis pathological process.

Description

Adeno-associated virus expression vector overexpressing GRIM-19 gene and its effect on inhibiting liver fibrosis Application in the pathological process of dimensionality

technical field

The invention relates to the biological field, in particular to an adeno-associated virus expression vector overexpressing GRIM-19 gene and its application in inhibiting the pathological process of liver fibrosis.

Background technique

Chronic liver disease (CLD) refers to a clinicopathological syndrome of liver cell inflammation and necrosis caused by various reasons. Chronic hepatitis B virus, hepatitis C virus, nonalcoholic fatty liver disease, and alcoholic liver disease are major causes of chronic liver disease. The common pathological processes of these chronic liver diseases are: hepatitis-hepatic fibrosis-liver cirrhosis-hepatocellular carcinoma. Hepatitis is a "defensive" measure taken by the normal liver in response to external stimuli. If the accumulation of continuous damage factors will cause liver fibrosis, fibrosis, and poor blood flow, which will lead to damage to liver function and eventually develop into liver disease. hardening. At present, there is no safe and effective clinical intervention, and there is also a lack of therapeutic drugs to directly prevent or reverse fibrosis, making liver fibrosis a difficult clinical treatment for chronic liver disease for a long time.

GRIM-19 (Gene associated with retinoid-IFN-induced mortality-19) protein, also known as NDUFA13 protein, is an essential component of mitochondrial respiratory chain (MRC) complex I. Its gene is mainly located in human chromosome 19, 19p13.2, It is a protein molecule encoding 144 amino acids with a molecular weight of about 16kDa. It is mainly located in the inner membrane of mitochondria, and a small part exists in the cytoplasm and nucleus. GRIM-19 plays an important role in mitochondrial MRC complex membrane potential, maintenance of mitochondrial respiratory oxidative phosphorylation, apoptosis and regulation of various signal transduction. Studies have shown that GRIM-19, as a tumor suppressor gene, is lost in the expression of colorectal cancer, prostate cancer, liver cancer, gastric cancer, glioma and other tumors, and is related to the occurrence and development of tumors. Studies have also shown that GRIM-19 deletion is closely related to the pathological process of chronic atrophic gastritis of "inflammation-atrophy-intestinal metaplasia", suggesting that GRIM-19 is involved in the pathological progression of digestive tract inflammation.

The inventor's team found for the first time that GRIM-19 gene deficiency in mice is associated with the pathology of hepatitis and liver fibrosis. Knockout of the mouse liver can directly induce non-infectious hepatitis and further progress to spontaneous liver fibrosis. And disclosed the use of GRIM-19 gene as hepatitis or liver fibrosis drug or diagnostic target (CN111019970A). It constructs a liver-specific GRIM-19 (ie NDUFA13) heterozygous knockout mouse model, thereby inducing the liver to produce the pathological process of spontaneous liver fibrosis, and can be used as a diagnostic marker for spontaneous hepatitis-hepatic fibrosis.

However, the above findings are hepatocyte-specific knockout of the GRIM-19 gene, resulting in a mouse animal model of spontaneous liver fibrosis disease. At present, there is no evidence of GRIM-19 gene expression in human liver fibrosis clinical samples, and whether GRIM-19 overexpression can be used for the treatment of inhibiting the pathological process of liver fibrosis remains unclear.

SUMMARY OF THE INVENTION

In view of this, the purpose of the present invention is to provide an adeno-associated virus expression vector overexpressing GRIM-19 gene and its application in inhibiting the pathological process of liver fibrosis.

For achieving the above object, the technical scheme of the present invention is:

An adeno-associated virus expression vector comprising the GRIM-19 gene and an adeno-associated virus AAV8 linearized vector GV599 comprising a Thyroid-binding globulin TBG promoter.

Further, the linearization original sequence of the adeno-associated virus AAV8 linearization vector GV599 is pTBG-MCS-EGFP-3Flag-SV40-PolyA.

During the research process, the inventors unexpectedly discovered that the GRIM-19 gene and the adeno-associated virus AAV8 linearized vector GV599 (the adeno-associated virus AAV8 linearized vector GV599 contains the thyroid-binding globulin (Thyroid-bindingglobulin) TBG promoter) adenocarcinoma. The related viral expression vector can significantly restore the expression of GRIM-19 in mouse hepatocytes induced by CCl4 treatment, and can effectively alleviate or reverse the pathological process of CCl4-induced mouse liver fibrosis.

The second purpose of the present invention is to protect the adeno-associated virus particles, which comprise the adeno-associated virus expression vector.

The third object of the present invention is to protect the adeno-associated virus expression vector or the adeno-associated virus particle composition.

The fourth object of the present invention is to protect a method for overexpressing GRIM-19 protein in hepatocytes, which comprises the following steps: contacting hepatocytes with the adeno-associated virus expression vector, adeno-associated virus particle or composition.

The fifth object of the present invention is to protect the application of the adeno-associated virus expression vector, expression particle or composition in the preparation of a medicine for treating liver fibrosis or hepatitis.

Further, the drug is used to promote the expression of GRIM-19 in the liver.

Further, the adeno-associated virus expression vector, adeno-associated virus particle or composition is administered by tail vein injection.

The beneficial effects of the present invention are:

The discovery of the present invention that the expression of GRIM-19 is down-regulated in human liver fibrosis clinical samples provides clinical evidence for liver fibrosis treatment.

The discovery of the present invention that the expression of GRIM-19 is down-regulated in the liver of mice with CCl4-induced liver fibrosis disease provides an experimental basis for the treatment of liver fibrosis.

The adeno-associated virus expression vector of the present invention can significantly and specifically restore the expression of GRIM-19 gene in mouse hepatocytes induced by CCl4 treatment, and can effectively reduce or reverse CCl4-induced mouse liver fibrosis pathological process.

Description of drawings

Figure 1 is a graph showing the results of tissue chip immunohistochemistry (IHC) analysis in Example 1, wherein expression is expression, and IHC staining score is IHC staining score;

Figure 2 shows the results of Masson staining analysis of liver fibrosis and Ishak score of liver fibrosis in Example 2, wherein A is the result of Masson staining of liver fibrosis, and B is the result of Ishak score of liver fibrosis; Masson staining is the result of detecting fibrosis Masson staining, control is the control group, CCl4 is the experimental group, and Ishak score is the Ishak score for evaluating the degree of fibrosis;

Figure 3 is the result of GRIM-19 expression analysis in Example 3, wherein A is the result of GRIM-19 gene mRNA detection in liver tissue (β-actin is the internal reference), and B is the detection of GRIM-19 protein expression in liver tissue by western blot analysis (β-actin is the internal reference) result; C is the result of immunofluorescence analysis of GRIM-19 protein expression in hepatocytes (CK-18 is the marker); MFI (Mean Fluorescence Intensity) is the quantitative analysis of mean fluorescence density;

Figure 4 is a graph showing the results of hepatic stellate cell activation and collagen expression in Example 4, wherein A is the activated hepatic stellate cell in the liver tissue (Desmin is the hepatic stellate cell surface marker, α-SMA is the hepatic stellate cell Activation marker) expression result graph, B is the expression result graph of activated hepatic stellate cells and Collagen-III, MFI (Mean Fluorescence Intensity) is the quantitative analysis of mean fluorescence density;

5A is a schematic structural diagram of the adeno-associated virus expression vector AAV8-TBG-mGRIM-19 constructed in Example 5, and 5B and 5C are PCR amplification electrophoresis images and enzyme digestion electrophoresis images, respectively;

The experimental flow chart in Fig. 6 embodiment 7, wherein, sacrificed is the mouse sacrifice;

Figure 7 is the result of Masson staining analysis and liver fibrosis Ishak score of CCl4-induced mouse liver fibrosis in Example 7 (ie, after intervention), wherein A is the Masson staining result of liver fibrosis, and B is the liver fibrosis Ishak score result graph;

Figure 8 is a graph showing the analysis results of GRIM-19 gene expression in liver tissue of mice with liver fibrosis induced by CCl4 after intervention in Example 8 (ie, after intervention); wherein, A is the detection of GRIM-19 gene mRNA in liver tissue (β-actin is an internal reference) ), B is the result of Western blot analysis of GRIM-19 protein expression in liver tissue (β-actin is the internal reference); C is the result of immunofluorescence analysis of GRIM-19 protein expression in liver parenchyma cells (labeled by CK-18). ; MFI (Mean FluorescenceIntensity) is the quantitative analysis of mean fluorescence density;

Figure 9 is a graph showing the results of hepatic stellate cell activation and collagen expression in Example 9 (ie, after intervention), wherein A is the activated hepatic stellate cell (Desmin is a hepatic stellate cell marker, and α-SMA is a hepatic stellate cell marker) in the liver tissue. Hepatic stellate cell activation marker) expression results, B is the expression results of activated hepatic stellate cells and Collagen-III, MFI (Mean Fluorescence Intensity) is the quantitative analysis of mean fluorescence density;

Figure 10 shows the results of expression of NLRP3 and inflammation-related factors in liver tissue of mice with hepatic fibrosis induced by CCl4 before and after intervention, wherein, A is the expression of GRIM-19 and NLRP3 of inflammasome in liver tissue of mice induced by CCl4 before intervention ;B is the expression of GRIM-19 and inflammasome NLRP3 in the liver tissue of mice induced by CCl4 after intervention; C is the expression of IL-1β and TNF-α related to inflammation in the liver tissue of mice induced by CCl4 before the intervention (MFI is the average Quantitative analysis of fluorescence density) results; D is the results of CCl4-induced expression of IL-1β and TNF-α in the liver tissue of mice after intervention (MFI is the quantitative analysis of mean fluorescence density).

Detailed ways

The cited embodiments are used to better illustrate the content of the present invention, but the content of the present invention is not limited to the cited embodiments. Therefore, those skilled in the art make non-essential improvements and adjustments to the embodiments according to the above-mentioned contents of the invention, which still belong to the protection scope of the present invention.

Example 1 Analysis of the expression of GRIM-19 protein in pathological tissues of different human liver diseases

The human liver disease spectrum tissue chip was used to analyze the expression of human GRIM-19 protein in the pathological tissues of different liver diseases, and the results are shown in Figure 1.

Among them, the human liver disease spectrum tissue chip was purchased from US Biomax, Inc. (LV20812). The tissue chip has a total of 104 different tissue samples, and each sample has two repeated sampling sites, including 16 normal liver tissues, 24 hepatitis tissues, and 24 liver cirrhosis tissues. And 32 cases of hepatocellular carcinoma (HCC).

As can be seen from Figure 1, compared with normal liver tissue samples, the expression of GRIM-19 protein was down-regulated in hepatitis, liver cirrhosis and liver cancer tissues, and showed a gradual down-regulation trend with the pathological process of fibrosis. The results indicate that GRIM-19 may be the target of external inducing factors, and the down-regulation of GRIM-19 expression may be an important factor in the progression of liver fibrosis induced by external stimuli.

Example 2 Establishment of CCl4-induced liver fibrosis mouse disease model

30 male BABL/C mice (7-8 weeks old) were randomly divided into two groups, the experimental group and the control group, with 15 mice in each group;

The mice in the experimental group were intraperitoneally injected with 0.8ml/kg CCl4 (mixed and diluted with corn oil at a volume ratio of 1:4), while the mice in the control group were injected with only pure corn oil (1ml/kg), with a maximum of 300ul;

Injections every two days, twice a week;

After 3 weeks, 5 weeks and 7 weeks respectively, 5 mice in the experimental group and the control group were sacrificed, and the livers were collected for Masson staining analysis of liver fibrosis and Ishak score for fibrosis. The results are shown in Figure 2.

As can be seen from Figure 2, compared with the control group, the mice in the experimental group developed significant fibrosis in the liver, and the Ishak score of fibrosis was significantly increased.

Example 3 Analysis of the expression of GRIM-19 protein in hepatocytes in the CCl4-induced liver fibrosis mouse model

Real-time quantitative RT-PCR, specifically: using Tripure Isolation Reagent to lyse mouse liver tissue, chloroform precipitation, isopropanol precipitation for RNA; using QuantiNova reverse transcription kit for reverse transcription to obtain cDNA, 2×SYBRGreen Mix 5μl, RT product 1μl, Primer F (10μM) 0.4μl, Primer R (10μM) 0.4μl, RNaseFree ddH2O was added to 10μl, β-actin was used as an internal control for detection, and relative quantitative analysis (qRT-PCR) was performed by comparing CT values (2-ΔΔCT). , the primers used for PCR amplification are shown in Table 1).

Western blotting, specifically: extracting mouse liver tissue protein, lysing with Western and IP cell lysis buffer (Biyuntian), homogenizing, lysing on ice for at least 30min, centrifuging at 12000g for 3min at 4°C, collecting the supernatant into an Ep tube, Take part to measure the protein concentration using BCA protein concentration assay kit (Biyuntian), add the corresponding volume of SDS-PAGE protein loading buffer (5×), mix well, heat at 100°C for 10min, use immediately after centrifugation or store at -80 °C refrigerator. SDS-PAGE electrophoresis, gel preparation and sample loading, using 12.5% SDS-PAGE separating gel and 5% SDS-PAGE stacking gel, 80V constant pressure running stacking gel, 120V constant pressure running separating gel, until bromophenol blue electrophoresis reaches Gel bottom. Transfer the membrane (wet transfer method), use PVDF as the solid phase carrier, transfer the membrane at 230mA for 50min, take out the membrane and see that the marker has been transferred, put the membrane in TBST and wash twice, 5min each time. Blocking and antibody incubation, blocking with 5% BSA (TBST dilution), shaking at room temperature for about 1 h. Add the corresponding concentration of primary antibodies: β-actin (sigma, 1:10000), GRIM-19 (Santa Cruz, 1:300), shake at 4°C overnight. The primary antibody was recovered and washed three times with TBST for 10 min, 5 min and 5 min respectively. The corresponding secondary antibody diluted in TBST (1:5000) was incubated on a shaker at room temperature for 1.5 h, rinsed three times with TBST as before, and exposed.

Immunofluorescence technique, specifically: using OCT embedding medium to embed mouse liver tissue and store it in -80°C refrigerator, take out the frozen tissue, slice the tissue into 8um slices, fix it in 4% PFA for 30min, and use PBS Wash three times, 5min each time. For blocking, use goat serum:PBS=1:1 for dilution, block at room temperature for 1 hour, incubate with primary antibody, and use immunofluorescence blocking solution dilution (Biyuntian) to dilute the primary antibody, overnight at 4 degrees. Wash three times with PBS, 5 min each time, incubate the secondary antibody, select the appropriate secondary antibody according to the source of the primary antibody, dilute the secondary antibody with immunofluorescence blocking solution at a ratio of 1:200, incubate at room temperature for 1 h in the dark, wash 3 times with PBS, each time 5min times. DAPI was counterstained at room temperature for 10 min (final concentration 1 ug/ml), and the slides were mounted with anti-fluorescence quenching mounting solution (purchased from Soleibo Company). Before detection, they were stored in a humid box at 4°C and protected from light, and images were taken by confocal laser.

Real-time quantitative RT-PCR, Western blotting and immunofluorescence techniques were used to analyze the expression of GRIM-19 gene in the liver of the experimental group and control mice after 3 weeks, 5 weeks and 7 weeks of the model constructed in Example 2. The results are shown in Figure 3 Show.

Table 1 Primers used to detect GRIM-19 mRNA expression by qRT-PCR

Upstream amplification primers for GRIM-19 5’-GGTGGGGCGAGTCTGTGTTC-3’ Downstream Amplification Primers for GRIM-19 5'-ATTGCTCATCTCCTCCTTGGTG-3'

As can be seen from Figure 3, compared with the control group, the mRNA and protein expressions of GRIM-19 in the liver tissue of the mice in the treatment group were significantly decreased. This proves that CCl4 can lead to down-regulation of GRIM-19 expression in mouse liver.

Example 4 Analysis of inflammatory factors and fibrosis degree in liver in CCl4-induced liver fibrosis mouse model group

The expression of inflammatory factors such as NLRP3, IL-1β, TNF-α, hepatic stellate cell activation markers Desmin, α-SMA and fibrosis marker collageⅢ in mice in the control group and experimental group was analyzed by immunofluorescence technique. The results are shown in Figure 4 and Figure 10, in which Figure 4 shows the results of hepatic stellate cell activation and collagen expression, and Figure 10 shows the liver tissue inflammasome NLRP3 and inflammation-related factors in mice with hepatic fibrosis induced by CCl4 before and after intervention Expression result graph.

It can be seen from Figure 4 and Figure 10 that compared with the control group, the expression of NLRP3 inflammatory factors and fibrosis factors such as Desmin, α-SMA, and collage III in the experimental group mice was significantly increased, indicating that the CCl4-induced mouse liver fibrosis model has been successfully established.

Example 5 Construction of hepatocyte-specific GRIM-19 overexpression adeno-associated virus vector

GV599 vector (the linearized original sequence is pTBG-MCS-EGFP-3Flag-SV40-PolyA, and contains hepatocyte-specific thyroid-binding globulin TBG promoter) was purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd. and commissioned by Shanghai Jikai Gene Chemical Technology Co., Ltd. constructed the adeno-associated virus expression vector GV599-TBG-mGRIM-19.

Shanghai Jikai Gene Chemical Technology Co., Ltd. was entrusted to analyze the positive clone sequencing results of the obtained adeno-associated virus expression vector GV599-TBG-mGRIM-19. The comparison results are as follows:

其中，斜体部分表示测序结果与目标序列一致，下划线表示酶切位点。

Among them, the italic part indicates that the sequencing result is consistent with the target sequence, and the underline indicates the restriction site.

The sequencing results showed that the adeno-associated virus expression vector GV599-TBG-mGRIM-19 was successfully constructed.

The resulting adeno-associated virus expression vector GV599-TBG-mGRIM-19 is shown in Figure 5A.

Shanghai Jikai Gene Chemical Technology Co., Ltd. was entrusted to carry out PCR amplification electrophoresis and restriction electrophoresis on the obtained adeno-associated virus expression vector GV599-TBG-mGRIM-19. The results are shown in Figure 5B and 5C, respectively, M is DNA marker (marker thing).

It can be seen from Figures 5B and 5C that after the plasmid is digested with a single enzyme, a uniform electrophoresis band will be shown in Figure 5B. At this time, it can be compared with the Marker to determine the molecular weight of 478bp.

Example 6 Adeno-associated virus titer Q-PCR detection

1. Material preparation: 2X SYBR Green Master Mixture, Q-Zfcas9 quantitative PCR primer, Zfcas9 plasmid standard, DNase/RNase-Free Water, 96-well quantitative PCR plate, plastic sealing film, DNase/RNase-Free pipette tip and Ep tube ;

2. Instrument preparation: quantitative PCR instrument;

3. Experimental steps: use the plasmid as a standard to establish a standard curve, and compare the sample to be tested with the standard curve to obtain the titer of the sample to be tested, specifically:

1) Prepare 4 parts of 90μl water for each sample and put them in Ep tubes; take 10uL sample and add it to the first part of water, mix well and name it -1, then take 10μl from it and add it to the second part of water, name it -2 , and so on; finally, 8 dilution samples are obtained, and the last 5 dilution samples obtained are used as templates to enter quantitative PCR;

2) Add 10uL of each sample to 40μl of water, mix well and name it -2, prepare 4 parts of 90μl of water, put them in Ep tubes, then take 10μl from it and add it to 90μl of water, named -3, and so on; The difference is that only 4 dilutions are obtained, and the last 3 diluted samples obtained enter quantitative PCR;

3) Calculate the required number of reaction wells. In each reaction, 2X SYBR Green Mix is 10 μl, upstream and downstream primers in Table 1 are 0.5 μl each, Rox reference dye is 0.2 μl, and water is added to make up to 15 μl; prepare one more for every 20 reactions reaction system;

4) Add the prepared reaction system to a 96-well reaction plate, 15 μl per well; add 5 μl to each sample, and set up duplicate wells;

5) The PCR reaction was carried out according to the instructions of the SYBR Premix Ex Taq II (TAKARA Company) kit;

6) The obtained Ct value data are shown in Table 2.

Table 2 Ct value data

Standard-3 Standard-4 Standard-5 Standard-6 Standard-7 Ct1 14.83 18.08 21.46 25.14 28.38 Sample-2 Sample-3 Sample-4 Sample-5 Ct1 12.02 15.99 19.58 23.70

The standard curve was constructed according to the log value of the standard concentration and the average value of Ct; wherein, the copy number of the standard curve=Conc/(MW per bp*Vector Size)*Avogadro constant/Dilution Factor; in the formula, Conc represents the concentration, and MW per bp Represents MW bp value, Vector Size represents vector size, Avogadro constant represents Avogadro constant, Dilution Factor represents dilution factor; original plasmid concentration: 2.58E+13Copies/mL, E represents the index of ten, copies represents copy number; plasmid size: 5354bp;

Table 3 Copy number

Standard-3 Standard-4 Standard-5 Standard-6 Standard-7 Copies/ml 2.58E+10 2.58E+09 2.58E+08 2.58E+07 2.58E+06

The concentration of other samples can be calculated according to the standard curve;

7) The final value of the concentration of the sample to be tested is obtained by dividing the measured value by the dilution and then multiplying by 2, where the multiplication by 2 is because the standard is double-stranded, while the AAV virus particle is single-stranded.

8) The titers obtained by different dilutions of adeno-associated virus are averaged, which is the final concentration of adeno-associated virus.

Table 4 titer determination results

Remarks: The red slashed part is the data with large experimental error and does not participate in the calculation of the average titer.

5. The packaged adeno-associated virus expression vector is named AAV8-TBG-mGRIM-19, and the titer is 1.82E+13V.G/ml. The same method is used to package the empty vector AAV8-TBG-NC (that is, only the GV599 vector is contained). ), the titer was 7.03E+12V.G/ml.

Adeno-associated virus expression vector and empty vector were stored at -80°C, and thawed at 4°C when used.

Example 7 Establishment of GRIM-19 overexpression adeno-associated virus to treat liver fibrosis animal model

30 male BABL/C mice (7-8 weeks old) were taken and randomly divided into two groups, namely the experimental group and the control group, with 15 mice in each group, weighed and marked;

The adeno-associated virus expression vector AAV8-TBG-GRIM-19 (1.82E+13v.g/ml) and the empty vector AAV8-TBG-NC (7.03E+12v.g/ml) were taken out from -80°C to 4°C in advance melt;

Prepare the injection volume in an EP tube and dilute it with PBS to a final concentration of 1E+11v.g/100ul adeno-associated virus/mouse;

One week in advance, the mice in the experimental group and the control group were injected with the adeno-associated virus expression vector AAV8-TBG-GRIM-19 (the experimental group) and the empty vector plasmid AAV8-TBG-NC (the control group), respectively, with an injection volume of 1E +11v.g/100ul carrier/mouse;

After 10 days of injection, according to the fibrosis induction method, intraperitoneal injection of 0.8ml/kg CCl4 mice was carried out every week, 200ul/mice, twice a week, for 6-8 weeks;

At three time points after CCl4 injection 3 weeks, 5 weeks and 7 weeks, 5 mice in the control group and 5 mice in the experimental group were sacrificed (the implementation process is shown in Figure 6), and liver samples and Masson staining analysis of liver fibrosis were carried out. The results are shown in Figure 7.

As can be seen from Figure 7, compared with the control group, the degree of liver fibrosis of the mice in the experimental group was significantly reduced.

Example 8 Analysis of GRIM-19 protein expression in animal models of liver fibrosis treated by overexpression of adeno-associated virus

Using qRT-PCR, Western blotting and indirect immunofluorescence techniques (as described above), the GRIM- The expression of 19 genes is shown in Figure 8.

As can be seen from Figure 8, compared with the control group, the expression of GRIM-19 in the mice in the experimental group was significantly increased. This proves that GRIM-19 adeno-associated virus is successfully expressed in mice.

Example 9 Analysis of liver inflammatory factors and fibrosis degree in animal models of liver fibrosis treated by overexpression of adeno-associated virus

Using conventional indirect immunofluorescence technique (same as above), the inflammatory factors NLRP3, IL-1β, TNF-α, hepatic stellate cell activation markers Desmin, α-SMA and fibrosis marker collageⅢ and other factors were analyzed and detected in the control group. The results are shown in Figure 9 and Figure 10, in which Figure 9 shows the results of hepatic stellate cell activation and collagen expression, and Figure 10 shows the liver tissue of mice with liver fibrosis induced by CCl4 before and after the intervention The expression results of inflammasome NLRP3 and inflammation-related factors.

As can be seen from Figures 9 and 10, compared with the control group, AAV8-TBG-GRIM-19 experimentally injected mice with NLRP3, IL-1β, TNF-α, Desmin, α-SMA, CollageⅢ, F4/80, TGF-β, etc. The factor expression was significantly attenuated, indicating that GRIM-19 adeno-associated virus overexpression could inhibit the pathological process of CCl4-induced liver fibrosis.

To sum up: the GRIM-19 overexpression adeno-associated virus constructed in the present invention can significantly reduce or even reverse CCl4-induced liver fibrosis.

In addition, it should be understood that although this specification is described in terms of embodiments, not each embodiment only includes an independent technical solution, and this description in the specification is only for the sake of clarity, and those skilled in the art should take the specification as a whole , the technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art.

<110> Adeno-associated virus expression vector overexpressing GRIM-19 gene and its application in inhibiting the pathological process of liver fibrosis

<120> Children's Hospital Affiliated to Chongqing Medical University

<160>2

<210>1

<211>19

<212> DNA

<213> Artificial sequences

<220>

<223> Upstream Amplification Primer

<400>1

ggtgggcgag tctgtgttc 19

<210>2

<211>22

<212> DNA

<213> Artificial sequences

<220>

<223> Downstream Amplification Primers

<400>2

attgctcatc tcctccttgg tg 22

Claims (8)

1. An adeno-associated virus expression vector, which is characterized by comprising a GRIM-19 gene and an adeno-associated virus AAV8 linearized vector GV599, wherein the adeno-associated virus AAV8 linearized vector GV599 comprises a thyroid-associated globulin TBG promoter.

2. The adeno-associated virus expression vector according to claim 1 wherein the linearized element sequence of adeno-associated virus AAV8 linearized vector GV599 is pTBG-MCS-EGFP-3Flag-SV 40-PolyA.

3. The adeno-associated viral particle comprising the adeno-associated viral expression vector according to claim 1 or 2.

4. The adeno-associated virus expression vector or adeno-associated virus particle composition according to any one of claims 1 to 3.

5. A method of expressing GRIM-19 protein in hepatocytes comprising the steps of: contacting a hepatocyte with the adeno-associated virus expression vector, adeno-associated virus particle, or composition of any one of claims 1 to 4.

6. Use of the adeno-associated virus expression vector, expression particle or composition according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of liver fibrosis or hepatitis.

7. The use of claim 6, wherein the medicament is for promoting expression of GRIM-19 in the liver.

8. The use of claim 6 or 7, wherein the adeno-associated virus expression vector, adeno-associated virus particle, or composition is administered by tail vein injection.

Images