CN112111520A

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

Info

Publication number: CN112111520A
Application number: CN202010999942.0A
Authority: CN
Inventors: 黄轶; 曾欣; 徐小惠; 李锐; 王欣; 薛炳乾; 黄道超
Original assignee: Childrens Hospital of Chongqing Medical University
Current assignee: Childrens Hospital of Chongqing Medical University
Priority date: 2020-09-22
Filing date: 2020-09-22
Publication date: 2020-12-22

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 of GRIM-19 gene overexpression and application thereof in inhibiting pathological process of hepatic fibrosis

Technical Field

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.

Background

Chronic Liver Disease (CLD) refers to the clinical pathological syndrome of inflammation and necrosis of liver cells caused by various reasons. Chronic hepatitis b virus, hepatitis c virus, non-alcoholic fatty liver disease and alcoholic liver disease are the leading causes of chronic liver disease. The pathological processes that these chronic liver diseases follow in common are: hepatitis-hepatic fibrosis-cirrhosis-liver cancer. Hepatitis is a defensive measure of normal liver in response to external stimulation, and if persistent injury factors are accumulated, hepatic fibrosis, fibroplasia and unsmooth blood flow can be caused, so that the liver function is damaged, and finally, cirrhosis is developed. At present, no safe and effective clinical intervention means exists, and a therapeutic drug for directly preventing or reversing fibrosis is also lacked, so that the hepatic fibrosis becomes a difficult point for clinical treatment of chronic liver diseases for a long time.

GRIM-19(Gene associated with a secreted IFN-induced motility-19) protein, also known as NDUFA13 protein, is an essential component of Mitochondrial Respiratory Chain (MRC) complex I, and its Gene is mainly located on chromosome 19p13.2 of human body, and encodes 144 amino acids protein molecules with molecular weight of about 16kDa, mainly located in the inner mitochondrial membrane, and a small part of it is located in cytoplasm and nucleus. GRIM-19 plays an important role in the membrane potential of mitochondrial MRC complex, maintenance of mitochondrial respiratory oxidative phosphorylation function, apoptosis and regulation of various signal transduction. Research shows that GRIM-19 as one kind of cancer inhibiting gene is expressed and deleted in colorectal cancer, prostate cancer, liver cancer, gastric cancer, glioma and other tumors and is related to tumor development. Studies have also shown that GRIM-19 deletion is closely related to the pathological course of "inflammation-atrophy-intestinal metaplasia" chronic atrophic gastritis, suggesting that GRIM-19 is involved in the pathological progression of inflammation of the digestive tract.

The inventor group discovers for the first time that the GRIM-19 gene defect of the mouse is related to the pathology of hepatitis and hepatic fibrosis, and the knockout of the liver of the mouse can directly induce non-infectious hepatitis and further progress to spontaneous hepatic fibrosis. Also discloses the application of GRIM-19 gene as hepatitis or liver fibrosis medicine or diagnosis target (CN 111019970A). The method constructs a liver specificity GRIM-19 (namely NDUFA13) heterozygous knockout mouse model, thereby inducing the liver to generate a pathological process of spontaneous hepatic fibrosis diseases, and being used as a diagnostic marker of spontaneous hepatitis-hepatic fibrosis.

However, the finding is liver cell specific knockout of GRIM-19 gene, resulting in spontaneous liver fibrosis disease mouse animal model. At present, there is no evidence of expression of GRIM-19 gene in human liver fibrosis clinical samples, and it is not clear whether GRIM-19 overexpression can be used for inhibiting the pathological process of liver fibrosis.

Disclosure of Invention

In view of the above, the present invention aims to provide an adeno-associated virus expression vector with GRIM-19 gene over-expressed and application thereof in inhibiting pathological process of liver fibrosis.

In order to achieve the purpose, the technical scheme of the invention is as follows:

an adeno-associated virus expression vector, which comprises GRIM-19 gene and adeno-associated virus AAV8 linearized vector GV599, wherein the adeno-associated virus AAV8 linearized vector GV599 comprises a Thyroid-binding globulin (Thyroid-binding globulin) TBG promoter.

Furthermore, the linearization original element sequence of the adeno-associated virus AAV8 linearization vector GV599 is pTBG-MCS-EGFP-3Flag-SV 40-PolyA.

The inventor unexpectedly discovers in the research process that an adeno-associated virus expression vector comprising GRIM-19 gene and adeno-associated virus AAV8 linearized vector GV599 (the adeno-associated virus AAV8 linearized vector GV599 comprises a Thyroid-binding globulin (TBG) promoter) can remarkably restore the expression of GRIM-19 in mouse hepatocytes induced after CCl4 treatment, and can effectively reduce or reverse the pathological process of mouse liver fibrosis induced by CCl 4.

The invention also aims to protect adeno-associated virus particles, which comprise the adeno-associated virus expression vector.

It is a further object of the present invention to provide a composition for protecting the adeno-associated virus expression vector or adeno-associated virus particle.

The fourth object of the present invention is a method for protecting the overexpression of 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.

The fifth purpose of the invention is to protect the application of the adeno-associated virus expression vector, the expression particle or the composition in preparing the drugs for treating hepatic fibrosis or hepatitis.

Further, the medicament is used for promoting expression of GRIM-19 in liver.

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

The invention has the beneficial effects that:

the invention relates to the discovery that the expression of GRIM-19 is down-regulated in human liver fibrosis clinical samples, and provides clinical evidence for liver fibrosis treatment.

The invention relates to the discovery that GRIM-19 is expressed and down-regulated in the liver of a CCl 4-induced liver fibrosis disease mouse, and provides an experimental basis for treating liver fibrosis.

The adeno-associated virus expression vector can remarkably recover the expression of GRIM-19 gene in mouse liver cells induced after CCl4 treatment, and can effectively reduce or reverse CCl4 induced mouse liver fibrosis pathological process.

Drawings

FIG. 1 is a graph showing the results of the Immunohistochemistry (IHC) analysis of the tissue chip in example 1, wherein expression is expression and IHC staining score is IHC staining score;

FIG. 2 is a graph of the results of Masson's liver fibrosis staining analysis and Ishak's liver fibrosis score in example 2, wherein A is a graph of the results of Masson's liver fibrosis staining and B is a graph of the results of Ishak's liver fibrosis score; masson staining for detecting fibrosis, control, CCl4 and Ishak score for evaluating the fibrosis degree;

FIG. 3 is a graph showing the results of GRIM-19 expression analysis in example 3, wherein A is a graph showing the results of mRNA detection (β -actin is an internal reference) of GRIM-19 gene in liver tissue, and B is a graph showing the results of immunoblot analysis of GRIM-19 protein expression detection (β -actin is an internal reference) in liver tissue; c is a graph of the results of immunofluorescence analysis of the expression of GRIM-19 protein in liver parenchymal cells (CK-18 is a marker); MFI (mean Fluorescence intensity) is the mean Fluorescence intensity quantification;

FIG. 4 is a graph showing the results of activation of hepatic stellate cells and Collagen expression in example 4, in which A is a graph showing the results of expression of activated hepatic stellate cells (Desmin is a hepatic stellate cell surface marker and. alpha. -SMA is a hepatic stellate cell activation marker) in liver tissues, B is a graph showing the results of expression of activated hepatic stellate cells and Collagen-III, and MFI (mean Fluorescence intensity) is a quantitative analysis of mean Fluorescence density;

FIG. 5A is the schematic structural diagram of AAV8-TBG-mGRIM-19 constructed in example 5, and 5B and 5C are PCR amplification electrophoretogram and enzyme digestion electrophoretogram, respectively;

FIG. 6 is a flow chart of an experiment in example 7, wherein sacrified is a mouse sacrifice;

FIG. 7 is a graph showing the Masson staining analysis of hepatic fibrosis and the Ishak score of hepatic fibrosis in the CCl 4-induced mice in example 7 (i.e., dry prognosis), wherein A is a graph showing the Masson staining result of hepatic fibrosis and B is a graph showing the Ishak score of hepatic fibrosis;

FIG. 8 is a graph showing the results of gene expression analysis of GRIM-19 in liver tissue of mice induced liver fibrosis by CCl4 after intervention in example 8 (i.e., after intervention); wherein A is a result picture of liver tissue GRIM-19 gene mRNA detection (beta-actin is internal reference), B is a result picture of liver tissue GRIM-19 protein expression detection (beta-actin is internal reference) by immunoblot analysis; c is a graph of the results of immunofluorescence analysis of liver tissue parenchymal cells (CK-18 marker) GRIM-19 protein expression analysis; MFI (mean Fluorescence intensity) is the mean Fluorescence intensity quantification;

FIG. 9 is a graph showing the results of activation of hepatic stellate cells and Collagen expression in example 9 (i.e., dry prognosis), wherein A is a graph showing the results of expression of activated hepatic stellate cells (Desmin is a hepatic stellate cell marker and. alpha. -SMA is a hepatic stellate cell activation marker) in liver tissues, B is a graph showing the results of expression of activated hepatic stellate cells and Collagen-III, and MFI (mean Fluorescence intensity) is a quantitative analysis of mean Fluorescence density;

FIG. 10 is a graph showing the results of CCl4 inducing liver fibrosis mouse liver tissue inflammasome NLRP3 and inflammation-related factor expression before and after intervention, wherein A is a graph showing the results of CCl4 inducing GRIM-19 and inflammasome NLRP3 expression in mouse liver tissue before intervention; b is a graph of the result of CCl4 inducing GRIM-19 and inflammasome NLRP3 expression in mouse liver tissues after intervention; c is a graph of the results of the expression (MFI is mean fluorescence density quantitative analysis) of inflammation-related factor IL-1 beta and TNF-alpha in mouse liver tissues induced by CCl4 before intervention; d is a graph of the results of the induction of inflammation-related factor IL-1 beta and TNF-alpha expression (MFI is mean fluorescence density quantitative analysis) in mouse liver tissues by CCl4 after intervention.

Detailed Description

The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

EXAMPLE 1 analysis of GRIM-19 protein expression in human pathological tissues of various liver diseases

The expression of human GRIM-19 protein in different pathological tissues of liver diseases is analyzed by using a human liver disease spectrum tissue chip, and the result is shown in figure 1.

Among these, human liver disease spectrum tissue chips were purchased from US Biomax, Inc (LV 20812). The tissue chip has 104 different tissue samples, and each sample has two repeated sample application sites, wherein 16 Normal liver tissues (Normal liver tissues), 24 Hepatitis tissues (Hepatitis), 24 cirrhosis tissues (liver cirrhosis), and 32 liver cancer tissues (HCC) are included.

As shown in FIG. 1, the expression of GRIM-19 protein was down-regulated in hepatitis, liver cirrhosis and liver cancer tissues and gradually decreased with the progress of fibrosis pathology, compared with the normal liver tissue sample. The result shows that GRIM-19 may be the target of external inducing factor, and the down regulation of GRIM-19 expression may be the important factor of external stimulating factor to induce liver fibrosis to progress.

Example 2 establishment of CCl4 induced liver fibrosis mouse disease model

Taking 30 male BABL/C mice (7-8 weeks old), randomly dividing into two groups, namely an experimental group and a control group, wherein each group comprises 15 mice;

the experiment group mice are injected with 0.8ml/kg CCl4 (mixed and diluted with corn oil according to the volume ratio of 1: 4) in the abdominal cavity, and the control group mice are injected with only pure corn oil (1ml/kg) and do not exceed 300ul at most;

the injection is performed every other day, and is performed twice a week;

after 3 weeks, 5 weeks and 7 weeks, 5 mice were sacrificed from the experimental group and the control group, respectively, liver was obtained, and Masson staining analysis for hepatic fibrosis and Ishak score for fibrosis were performed, and the results are shown in fig. 2.

As can be seen from FIG. 2, the liver of the mice in the experimental group is obviously fibrotic, and the fibrotic Ishak score is obviously increased compared with the control group.

Example 3 analysis of GRIM-19 protein expression in hepatocytes in a mouse model of CCl 4-induced liver fibrosis

The real-time quantitative RT-PCR specifically comprises the following steps: using Tripure Isolation Reagent to crack mouse liver tissue, separating out chloroform, and precipitating RNA by isopropanol; cDNA was obtained by reverse transcription using QuanntiNova reverse transcription kit (2 XSSYBR Green Mix 5. mu.l, RT product 1. mu.l, Primer F (10. mu.M) 0.4. mu.l, Primer R (10. mu.M) 0.4. mu.l, RNase Free ddH2O supplemented to 10. mu.l, beta-actin was used as an in-assay control, and relative quantitation (qRT-PCR, PCR amplified primers used are shown in Table 1) was performed by comparing CT values (2-. DELTA.CT).

Western blot, specifically: extracting mouse liver tissue protein, cracking with Western and IP cell lysate (Biyun day), homogenizing, cracking on ice for at least 30min, centrifuging at 12000g at 4 ℃ for 3min, collecting supernatant to Ep tube, taking part, determining protein concentration with BCA protein concentration determination kit (Biyun day), adding SDS-PAGE protein sample buffer (5X) with corresponding volume, mixing, heating at 100 ℃ for 10min, centrifuging, and immediately using or storing in refrigerator at-80 ℃. SDS-PAGE electrophoresis, gel preparation and sample loading, adopting 12.5% SDS-PAGE separating gel and 5% SDS-PAGE concentrating gel, running the concentrating gel at 80V constant pressure, and running the separating gel at 120V constant pressure until the bromophenol blue electrophoresis reaches the bottom of the gel. And (4) transferring the membrane (wet transfer method), using PVDF as a solid phase carrier, transferring the membrane for 50min at 230mA, taking out the membrane, transferring the marker, and washing the membrane in TBST for 2 times, 5min each time. Blocking and antibody incubation, blocking with 5% BSA (TBST dilution), and shaking at room temperature for about 1 h. Add the corresponding concentration primary antibody: beta-actin (sigma,1:10000), GRIM-19(Santa Cruz,1:300), shaking overnight at 4 ℃. Recovering primary antibody, and rinsing with TBST for 3 times at 10min, 5min and 5min respectively. TBST dilution the corresponding secondary antibody (1:5000) was incubated for 1.5h at room temperature in a shaker, rinsed 3 times with TBST as before, and exposed.

The immunofluorescence technique specifically comprises the following steps: embedding mouse liver tissue with OCT embedding medium, storing in a refrigerator at-80 deg.C, taking out frozen tissue, continuously slicing into 8um slices, fixing with 4% PFA for 30min, washing with PBS for three times, and 5min each time. Blocking, dilution with goat serum PBS 1:1, blocking for 1h at room temperature, incubation of primary antibody, dilution with immunofluorescence blocking solution (petun sky), 4 ° overnight. Washing with PBS for three times, each time for 5min, incubating secondary antibody, selecting appropriate secondary antibody according to the species of the primary antibody, diluting the secondary antibody with immunofluorescence blocking solution at a ratio of 1:200, incubating for 1h at room temperature in a dark place, and washing with PBS for 3 times, each time for 5 min. DAPI was counter-stained at room temperature for 10min (final concentration 1ug/ml), mounted with an anti-fluorescence quenching mounting solution (from Solibao) and stored in a wet box at 4 ℃ in the dark before detection, and the images were obtained by confocal laser scanning.

The expression of GRIM-19 gene in the livers of experimental and control mice after 3, 5 and 7 weeks of the model constructed in example 2 was analyzed by real-time quantitative RT-PCR, Western blotting and immunofluorescence techniques, and the results are shown in FIG. 3.

TABLE 1 primers for qRT-PCR detection of GRIM-19mRNA expression

Upstream amplification primer of GRIM-19	5’-GGTGGGCGAGTCTGTGTTC-3’
		Downstream amplification primer of GRIM-19	5’-ATTGCTCATCTCCTCCTTGGTG-3’

As can be seen from FIG. 3, the liver tissues of the treated mice showed a significant decrease in GRIM-19mRNA and protein expression compared to the control group. Thus, CCl4 was shown to cause down-regulation of mouse liver GRIM-19 expression.

Example 4 analysis of inflammatory factors and degree of fibrosis in liver in CCl 4-induced liver fibrosis mouse model group

The expression conditions of factors such as inflammatory factors NLRP3, IL-1 beta, TNF-alpha, hepatic stellate cell activation markers Desmin, alpha-SMA, fibrosis markers colage III and the like in mice of a control group and an experimental group are analyzed and detected by using an immunofluorescence technique, and the results are shown in fig. 4 and 10, wherein fig. 4 is a diagram of the results of hepatic stellate cell activation and collagen expression, and fig. 10 is a diagram of the results of CCl4 induced hepatic fibrosis mouse hepatic tissue inflammasome NLRP3 and inflammation related factor expression before and after intervention.

As can be seen from FIGS. 4 and 10, compared with the control group, the expression of the inflammatory factor NLRP3 and the fibrosis factors such as Desmin, alpha-SMA, collage III and the like of the mouse in the experimental group is obviously increased, which indicates that the CCl4 induced liver fibrosis model of the mouse is successfully constructed.

Example 5 hepatocyte-specific GRIM-19 overexpression adeno-associated viral vector construction

The GV599 vector (the linearization original sequence is pTBG-MCS-EGFP-3Flag-SV40-PolyA, and contains a hepatocyte specific thyroid binding globulin TBG promoter) is purchased from Shanghai Jikai GeneBiochemical technology Co., Ltd, and entrusts the Shanghai Jikai GeneBiochemical technology Co., Ltd to construct the adeno-associated virus expression vector GV 599-TBG-mGRIM-19.

Entrusted Shanghai Jikai Genencochemistry technology Limited company to carry out positive clone sequencing result analysis on the obtained adeno-associated virus expression vector GV599-TBG-mGRIM-19, and the comparison result is as follows:

in this case, the italic part indicates that the sequencing result is identical to the target sequence, and the underlined part indicates the cleavage site.

As can be seen from the sequencing result, the adeno-associated virus expression vector GV599-TBG-mGRIM-19 is successfully constructed.

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

The obtained adeno-associated virus expression vector GV599-TBG-mGRIM-19 was subjected to PCR amplification electrophoresis and enzyme digestion electrophoresis by Shanghai Jikai Genenco Chemicals Co., Ltd. to obtain results shown in FIGS. 5B and 5C, respectively, where M is a DNA marker.

As shown in FIGS. 5B and 5C, the plasmid after single digestion shows a uniform electrophoretic band 5B, which can be compared with Marker to determine its molecular weight of 478 bp.

Example 6 adeno-associated Virus titre Q-PCR assay

1. Preparing materials: 2X SYBR Green Master mix, Q-Zfcas9 quantitative PCR primers, Zfcas9 plasmid standard, DNase/RNase-Free Water, 96-hole quantitative PCR plate, plastic sealing film, DNase/RNase-Free gun head and Ep tube;

2. preparing an instrument: a quantitative PCR instrument;

3. the experimental steps are as follows: establishing a standard curve by using plasmids as standard substances, and comparing a sample to be detected with the standard curve to obtain the titer of the sample to be detected, wherein the method specifically comprises the following steps:

1) for each sample, 4 aliquots of 90. mu.l water were prepared and placed in Ep tubes; adding 10uL of sample into the first part of water, uniformly mixing and naming as-1, then taking 10 mu l of sample out of the first part of water and adding the sample into the second part of water, naming as-2, and the like; finally obtaining 8 diluted samples, taking the obtained 5 diluted samples as a template and performing quantitative PCR;

2) adding 10uL of each sample into 40 mu l of water, uniformly mixing and naming as-2, preparing 4 parts of 90 mu l of water, filling the water into an Ep tube, taking 10 mu l of the water out of the Ep tube, adding the water into 90 mu l of water, naming as-3, and the like; the difference is that only 4 dilutions are obtained, and the obtained later 3 diluted samples are taken to enter quantitative PCR;

3) measuring the number of required reaction holes, wherein 2X SYBR Green Mix in each reaction is 10 mul, the upstream and downstream primers in the table 1 are respectively 0.5 mul, Rox reference dye is 0.2 mul, and water is added to supplement the amount to 15 mul; preparing a reaction system for every 20 reactions;

4) adding the prepared reaction system into a 96-well reaction plate, wherein each well is 15 mu l; adding 5 mul of each sample, and arranging multiple holes;

5) PCR was carried out according to the SYBR Premix Ex Taq II (TAKARA) kit;

6) the Ct value data obtained 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

Constructing according to the logarithm value of the concentration of the standard substance and the Ct average value to obtain a standard curve; wherein, the standard curve copy number is Conc/(MW per bp Vector Size) Avogadro constant/Dilution Factor; in the formula, Conc represents concentration, MW per bp represents MW bp value, Vector Size represents carrier Size, Avogadro constant represents Avogadaro constant, and Dilution Factor represents Dilution Factor; original concentration of plasmid: 2.58E +13Copies/mL, E denoting the ten index, Copies denoting copy number; plasmid size: 5354 bp;

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 a standard curve;

7) the final value of the concentration of the sample to be tested is obtained by dividing the measurement by the dilution and multiplying by 2, where 2 is multiplied because the standard is double stranded and the AAV viral particles are single stranded.

8) And averaging the titers obtained by measuring the adeno-associated viruses with different dilutions to obtain the final concentration of the adeno-associated viruses.

TABLE 4 results of titer determination

Note that the red diagonal part is data with larger 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, the titer is 1.82E +13V.G/ml, and the empty vector AAV8-TBG-NC (namely only containing GV599 vector) is packaged by the same method, and the titer is 7.03E +12 V.G/ml.

The adeno-associated virus expression vector and the empty vector are stored at-80 ℃ and are thawed and stored at 4 ℃ when being used.

Example 7 establishment of GRIM-19 overexpression adeno-associated virus animal model for treatment of hepatic fibrosis

Taking 30 male BABL/C mice (7-8 weeks old), randomly dividing the mice into two groups, namely an experimental group and a control group, wherein each group comprises 15 mice, and weighing and marking the mice;

taking out AAV8-TBG-GRIM-19(1.82E +13v.g/ml) and AAV8-TBG-NC (7.03E +12v.g/ml) from-80 deg.C to 4 deg.C in advance, and thawing;

the injection amount was prepared in an EP tube and diluted with PBS to a final concentration of 1E +11v.g/100ul adeno-associated virus/mouse;

injecting the carrier in one week in advance, and injecting AAV8-TBG-GRIM-19 as the adeno-associated virus expression vector and AAV8-TBG-NC as the empty vector plasmid into the mice as the experimental group and the control group in 1E +11v.g/100ul vector/mouse;

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

at three time points 3 weeks, 5 weeks and 7 weeks after CCl4 injection, 5 mice in each of the control and experimental groups were sacrificed (the implementation procedure is shown in FIG. 6), and liver sampling and Masson staining for liver fibrosis were performed, and the results are shown in FIG. 7.

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

Example 8 analysis of GRIM-19 protein expression in an animal model for the treatment of liver fibrosis by overexpression of adeno-associated Virus

After 3 weeks, 5 weeks and 7 weeks of the model control group constructed in example 7, GRIM-19 gene expression in mice of the experimental group and the negative control group was detected by qRT-PCR, western blot and indirect immunofluorescence (as described above), as shown in fig. 8.

As can be seen from FIG. 8, the GRIM-19 expression was significantly increased in the mice of the experimental group as compared with the control group. Thus, the GRIM-19 adeno-associated virus was successfully expressed in mice.

Example 9 analysis of liver inflammatory factors and fibrosis degree in animal model for treating hepatic fibrosis by overexpression of adeno-associated virus

The expression conditions of factors such as inflammatory factors NLRP3, IL-1 beta, TNF-alpha, hepatic stellate cell activation markers Desmin, alpha-SMA, fibrosis markers collage III and the like in mice of a control group and an experimental group are analyzed and detected by using a conventional indirect immunofluorescence technique (as described above), and the results are shown in fig. 9 and 10, wherein fig. 9 is a diagram of the results of hepatic stellate cell activation and collagen expression, and fig. 10 is a diagram of the results of CCl4 induced hepatic fibrosis mouse hepatic tissue inflammasome NLRP3 and inflammation related factor expression before and after intervention.

As can be seen from FIGS. 9 and 10, compared with the control group, the AAV8-TBG-GRIM-19 experiment injection mice NLRP3, IL-1 beta, TNF-alpha, Desmin, alpha-SMA, Collage III, F4/80, TGF-beta and other factors obviously weaken the expression, which shows that the GRIM-19 adeno-associated virus overexpression can inhibit CCl 4-induced liver fibrosis pathological process.

In summary, the following steps: the GRIM-19 overexpression adeno-associated virus constructed by the invention can obviously reduce or even reverse CCl 4-induced liver fibrosis.

Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.

<110> GRIM-19 gene over-expressed adeno-associated virus expression vector and application thereof in inhibiting hepatic fibrosis pathological process

<120> Chongqing medical university affiliated children hospital

<160>2

<210>1

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> upstream amplification primer

<400>1

ggtgggcgag tctgtgttc 19

<210>2

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> downstream amplification primer

<400>2

attgctcatc tcctccttgg tg 22

Claims

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.