CN115927322A

CN115927322A - Application of inhibitor of Thbs2 gene or THBS2 protein in preparation of anti-hepatic fibrosis medicine

Info

Publication number: CN115927322A
Application number: CN202211061437.7A
Authority: CN
Inventors: 陈巍; 张宁; 尤红; 吴晓宁; 张文
Original assignee: Beijing Friendship Hospital
Current assignee: Beijing Friendship Hospital
Priority date: 2022-08-31
Filing date: 2022-08-31
Publication date: 2023-04-07

Abstract

The invention discloses application of an inhibitor of Thbs2 gene or THBS2 protein in preparing a medicament for resisting hepatic fibrosis. The invention discovers for the first time that the targeted silent mouse hepatic stellate cell THBS2 has the effect of resisting hepatic fibrosis, and the anti-fibrotic drugs obtained based on the research comprise siRNA and shRNA transcribed by the specific targeted mouse Thbs2 gene, can effectively inhibit the expression of THBS2 protein and have better effect of resisting hepatic fibrosis.

Description

Application of inhibitor of Thbs2 gene or THBS2 protein in preparation of anti-hepatic fibrosis drug

Technical Field

The invention relates to the field of biomedicine. More particularly, relates to the application of inhibitors of Thbs2 genes or THBS2 proteins in preparing anti-hepatic fibrosis drugs.

Background

Thrombospondin 2 (THBS 2) knock-out mouse connective tissue abnormalities, including collagen fibroblastic abnormalities, increased vessel wall thickness and hemorrhagic disease, uneven, disorganized, discrete or even disrupted bundles of fibrils in the tissue (kyniakides TR ZY, smith LT, bain SD, yang Z, lin MT, danielson KG, iozzo RV, laMarca M, mcnney CE, ginns EI, born protein p.rice lack porous 2 displacement connected tissue abnormality, and staining diagnosis, j 419-biological extracellular matrix 1998), indicating the involvement of the cells in ECM 2 (ECM 2). Recent studies have found that THBS2 can inhibit angiogenesis and participate in cancer progression and metastasis: THBS2 in the tumor microenvironment reduces angiogenesis and promotes tumor-associated lymphangiogenesis, promotes intrahepatic bile duct cancer progression (Carpino G, cardiale V, di Giamberardino A, overi D, donfantant S, colasanti T, amato G, mennini G, franchitto M, conti F, rossi M, riminicci M, gaudio E, alvaro D, mancone C.Thrombospondin 1 and 2 along with PEDF inhibitor angiogenisis and promoter lyhangiogenie in intrahepatic cholestenocardial 20275 (6): 1377-1386.); THBS2 ⁺ Fibroblasts are an important cell population for promoting early lung adenocarcinoma metastasis (Yang H, sun B, fan L, ma W, xu K, hall SRR, wang Z, schmid RA, peng RW, marti TM, gao W, xu J, yang W, yao F. Multi-scale integration analysis THBS2+ cancer-associated fibrates as a key organic promoter aggregating growth in early-stage adoptive administration. Therapeutics 20212 (7): 3104-3130.). In addition, THBS2 is also a serum marker of nonalcoholic steatohepatitis (NASH) and its associated Liver fibrosis (Kozumi K, kodama T, murai H, sakane S, govaere O, cockell S, motooka D, kakita N, yamada Y, kondo Y, tahata Y, yamada R, hikita H, sakamori R, kamada Y, daly, anstee QM, tatsumi T, morii E, takehara T. Transcriptomics identity Thrombosondin-2 as a Biomarker for NASH and Advanced Liver fibrosis. Hepatology 2021 AK 74 (5 2452-2466.).

At present, no specific target and medicine for effectively interfering hepatic fibrosis are available clinically except for removing causes of diseases. Early studies found that THBS2 is significantly up-regulated in fibrotic liver, mainly in the areas of the funnels and the fibrous septa; and THBS2 is obviously up-regulated in Liver tissues of Liver fibrosis mouse models induced by different etiology and different modeling modes, which indicates that THBS2 expression disorder is not disturbed by the etiology and is specific to Liver fibrosis (Chen W, wu X, yan X, xu A, yang A, you H. Polytranscripto animal analysis genetic specific with Liver fibrosis progression index of ecology. Am J Physiol tissue physiology physiological physiology physical 2019 (6): G744-G754.).

However, the THBS2 has an anti-fibrosis effect or a fibrosis promotion effect by obviously up-regulating the expression in the fibrotic liver, and no relevant report exists at home and abroad at present.

Disclosure of Invention

The invention aims to provide application of an inhibitor of Thbs2 gene or THBS2 protein in preparation of a medicament for resisting hepatic fibrosis.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention firstly provides the application of the inhibitor of the Thbs2 gene or the THBS2 protein in the preparation of the anti-hepatic fibrosis medicine.

Further, the inhibitor is siRNA, dsRNA, shRNA or miRNA which takes THBS2 protein or a transcript thereof as a target sequence and can inhibit the expression of the THBS2 protein or the transcription of the Thbs2 gene.

Further, the inhibitor is any one of the following:

c) The target sequence of the siRNA is shown as SEQ ID NO. 1;

d) shRNA, wherein the sequence of the shRNA is shown as SEQ ID NO. 2;

c) A vector capable of expressing the siRNA of a) or the shRNA of b);

d) A virus comprising the vector of c).

The invention constructs an adeno-associated virus AAV6 coated whole-body promoter (CMV) -shRNA (Thbs 2) vector (namely AAV6-CMV-shRNA (Thbs 2)) mouse tail intravenous injection minimMouse, can obviously inhibit CCl ₄ The liver fibrosis caused by intraperitoneal injection progresses, which indicates that the Thbs2 gene or the THBS2 protein is an effective target for resisting the liver fibrosis.

Further, the target sequence guide strand of the siRNA is shown as SEQ ID NO. 3.

The invention also provides an siRNA molecule, and the target sequence of the siRNA molecule is shown in SEQ ID NO. 1.

Further, the target sequence guide strand of the siRNA molecule is shown as SEQ ID NO. 3.

The invention also provides a shRNA molecule, and the sequence of the shRNA molecule is shown in SEQ ID NO. 2.

The invention also provides an expression vector which can express the siRNA molecule or the shRNA molecule.

The invention also provides a virus comprising the expression vector.

In a specific embodiment of the invention, the virus is adeno-associated virus type 6 (AAV 6).

In a specific embodiment of the invention, the expression vector capable of expressing the siRNA molecule or the shRNA molecule is an adeno-associated virus type 6 vector, specifically AAV6-CMV-shRNA (Thbs 2).

The invention also provides an anti-hepatic fibrosis drug which comprises the siRNA molecule, the shRNA molecule, the expression vector and/or the virus and a pharmaceutically acceptable vector.

In the present invention, the Thbs2 gene or Thbs2 protein is a hepatic stellate cell Thbs2 gene or Thbs2 protein.

The invention has the following beneficial effects:

the invention discovers for the first time that the targeted silent mouse hepatic stellate cell Thbs2 has the effect of resisting hepatic fibrosis, and the anti-fibrosis medicine obtained based on the research comprises siRNA and shRNA transcribed by the specific targeted mouse Thbs2 gene, can effectively inhibit the expression of the THBS2 protein and has better effect of resisting hepatic fibrosis.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a map of the empty GV143 plasmid and the GV143-Thbs2 plasmid; wherein, A: GV143 empty plasmid; b: GV143-Thbs2 plasmid.

FIG. 2 is an electrophoretogram before and after the cleavage of vector GV 143; wherein, the 1#:10kb Marker (the bands are 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb,1.5kb,1kb,750bp,500bp and 250bp from top to bottom), the 2#: GV143 vector after enzyme digestion, and the 3#: GV143 vector without enzyme digestion.

FIG. 3 shows the result of 1% agarose gel electrophoresis of colony PCR products; wherein, 1#: negative control (ddH) ₂ O), 2#: self-coupled control, 3#: positive control, 4#: marker (bands: 5kb,3kb,2kb,1.5kb,1Kb,750bp,500bp,250bp,100bp from top to bottom), no. 5-12: 1-8 transformants.

FIG. 4 shows EGFP green fluorescent protein expression after transfection of 293T cells with GV143-Thbs2 plasmid.

FIG. 5 shows Western Blot analysis of Thbs2 protein expression, where M: protein Marker,1: positive control, 2: self-ligation control, 3: GV143-Thbs2 plasmid.

FIG. 6 is a map of the GV687-CMV-NC vector and the GV687-CMV-shRNA (Thbs 2) vector; wherein, A: GV687-CMV-NC vector; b: GV687-CMV-shRNA (Thbs 2) vector.

FIG. 7 shows the confirmation of the silencing efficiency of GV687-CMV-shRNA (Thbs 2) plasmid by Western Blot; wherein, 1: CON,2: OE,3: OE + KD low, 4: OE + KD is high.

FIG. 8 shows the caudal vein injection of AAV6-CMV-NC vector and AAV6-CMV-shRNA (Thbs 2) vector with CCl ₄ The HE staining and sirius red staining results (A) and the positive area ratio (B) of the sirius red staining of the liver tissues of the mice after the abdominal cavity is continuously stimulated for 6 weeks.

FIG. 9 shows the caudal vein injection of AAV6-CMV-NC vector and AAV6-CMV-shRNA (Thbs 2) vector with CCl ₄ After the abdominal cavity is stimulated continuously for 6 weeks, the Western Blot experiment detects the expression of Thbs2, collagen I and alpha SMA protein of the liver tissues of the mice.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

Example 1 siRNA specifically targeting mouse Thbs2 Gene and validation of silencing efficiency thereof

1. Construction of Thbs2 Gene overexpression vector

1. Acquisition of target Gene

Taking a mouse gene library provided by Haijiki Gene medicine science and technology Co., ltd as a template, carrying out PCR amplification by using primers Thbs2-p1 and Thbs2-p2 (shown in table 1) to obtain a PCR product, carrying out agarose gel electrophoresis detection, carrying out gel recovery, and purifying to obtain a Thbs2 gene containing Xhol and Kpnl enzyme cutting sites; the PCR reaction system and the PCR reaction conditions are shown in Table 2 and Table 3, respectively.

TABLE 1 primer sequences

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TABLE 2PCR reaction System

Reagent	Volume (μ L)
		ddH ₂ O	32.5
5x PS Buffer	10
		dNTP Mix(2.5mM each)	4
Upstream amplification primer Thbs2-p1 (10. Mu.M)	1
		Downstream amplification primer Thbs2-p2 (10. Mu.M)	1
Stencil (10 ng/. Mu.L)	1
		PrimeSTAR HS DNA polymerase	0.5
Total of	50

TABLE 3PCR reaction conditions

2. Enzyme-cleaved GV143 vector

Preparing 50 mu l of enzyme digestion system according to table 4, sequentially adding reagents in sequence, gently blowing and uniformly mixing by using a pipette, centrifuging for a short time, reacting at 37 ℃ for 3h or overnight, carrying out enzyme digestion on the GV143 vector (namely, the GV143 empty plasmid, the structural schematic diagram of which is shown in A in figure 1) to obtain an enzyme digestion vector product, carrying out electrophoresis detection on 1% agarose gel (the electrophoresis diagram is shown in figure 2), recovering the gel, and purifying to obtain the GV143 vector after enzyme digestion.

TABLE 4 enzyme digestion System

3. Exchanging PCR product with carrier

Preparing an exchange system shown in the table 5 in an ice water bath, gently blowing and uniformly mixing by using a pipette, centrifuging for a short time to avoid generating bubbles, reacting at 37 ℃ for 30min, and then placing in the ice water bath for cooling for 5min to obtain an exchange reaction product for immediate conversion.

TABLE 5 exchange System

Note: positive control, in order to exclude the failure of transformation due to the competent cell efficiency of the final step and the transformation process; self-ligation control, GV143 vector self-ligation control without added target fragment, was used to assess ligation efficiency.

4. Transformation of

Add 10. Mu.L of the exchange reaction product to 100. Mu.L of competent cells, flick the tube wall and mix well, put on ice for 30min, heat shock at 42 ℃ for 90s, incubate in ice water bath for 2min. Adding 500. Mu.L LB medium, and shaking-culturing at 37 deg.C for 1h. Taking a proper amount of the bacterial liquid, uniformly coating the bacterial liquid on a plate containing kanamycin (100 mu g/ml), and carrying out inverted culture in an incubator for 12-16h.

5. Colony PCR identification

Using Thbs2-p3 and Thbs2-p4 as identification primers (Table 6), preparing an identification system shown in Table 7, shaking, uniformly mixing, centrifuging for a short time, picking a single colony to a 20 mu L identification system by using a sterile gun head in an ultra-clean workbench, blowing, uniformly mixing, and placing in a PCR instrument for PCR amplification, wherein the PCR reaction conditions are shown in Table 8. The results of 1% agarose gel electrophoresis of the PCR products are shown in FIG. 3.

TABLE 6 identifying primers

TABLE 7 identification System

Reagent	Volume (μ L)
		ddH ₂ O	9.2
2x Taq Plus Master Mix	10
		Upstream primer Thbs2-p3 (10. Mu.M)	0.4
Downstream primer Thbs2-p4 (10. Mu.M)	0.4
		Single colony of bacteria	-
Total of	20

TABLE 8PCR reaction conditions

6.Sanger sequencing assay

Picking a monoclonal colony, extracting colony DNA (the kit is purchased from Tiangen Biochemical technology (Beijing) Co., ltd., the product number is DP 302-02), and then carrying out Sanger sequencing, and the comparison of experimental groups indicates that: and (6) detecting.

7. Plasmid extraction

The colony with correct sequencing is transferred to 10ml LB liquid culture medium containing kanamycin (100 mug/ml), cultured overnight at 37 ℃, and subjected to plasmid extraction by using a small-medium-volume plasmid kit (Tiangen Biochemical technology (Beijing) Co., ltd., product number DP 118) without endotoxin to obtain the Thbs2 gene overexpression vector, which is named as GV143-Thbs2 plasmid, and the structural schematic diagram of the plasmid is shown as B in figure 1.

The specific operation steps are as follows:

1. collecting overnight cultured bacteria liquid in a marked 5ml centrifuge tube, centrifuging at 12000rpm for 2min, and collecting bacteria;

2. discarding the supernatant, adding 250 μ l of cell resuspension, and fully oscillating to make the bacterial mass suspend uniformly;

3. adding 250 μ l cell lysate, adding 10 μ l proteinase K, reversing the mixture from top to bottom for 5-6 times, and mixing gently; standing for 1-2min to make thallus cracking and clarifying;

4. adding 350 μ l of neutralizing solution, turning upside down, mixing to completely separate out protein, and standing in ice bath for 5min;

centrifuging at 5.10000rpm for 10min, discarding protein, and collecting supernatant in another clean sterile 1.5ml EP tube;

centrifuging at 6.12000rpm for 5min while preparing the marked recovery column, transferring the supernatant to the recovery column, centrifuging at 12000rpm for 1min, and discarding the lower layer of waste liquid;

7. adding 600 μ l of pre-prepared rinsing liquid, centrifuging at 12000rpm for 1min, discarding the lower layer waste liquid, repeating once, and allowing to idle at 12000rpm for 2min to further remove the residual rinsing liquid;

8. transferring the recovery column to a new 1.5ml EP tube in a super clean bench, standing for 10-20min, and naturally drying;

9. adding 95 μ l of nucleic-Free Water into the recovery column, standing for 2min, centrifuging at 12000rpm for 2min, collecting sample, numbering, electrophoresing, measuring concentration, and performing quality inspection.

2. Expression detection of Thbs2 Gene overexpression vector (GV 143-Thbs2 plasmid)

1. Cell transfection

(1) Recovering 293T cells:

taking out the 293T cell freezing tube from the liquid nitrogen tank, quickly putting the tube into a water bath at 37 ℃, and unfreezing the tube as soon as possible by shaking the tube when not needed; after completely unfreezing, centrifuging at 1000rpm for 2min, wiping the frozen tube with 75% alcohol, sterilizing, and moving to an ultra-clean bench; removing supernatant from the frozen stock solution by aspiration, adding 1ml of fresh complete medium to suspend the cells, inoculating the resulting cell suspension into a 6cm petri dish containing 3ml of complete medium, shaking up gently, and culturing in a 5% CO2 incubator at 37 ℃; the culture medium was replaced the next day (i.e., complete medium) and the culture was continued.

(2) Passage of 293T cells:

subculturing the cells growing to 90% confluence, discarding the old culture solution, adding 2ml of sterilized D-Hank's solution, washing the cell growth surface, and discarding the solution; adding 1ml of pancreatin digestive juice, digesting for about 1-2min at 37 ℃ until the cells are completely digested, adding 2ml of complete culture medium, blowing and beating for a plurality of times by using a graduated pipette, washing the cells on the wall, mixing the cells uniformly, dividing the mixture into two new 6cm culture dishes, supplementing the complete culture medium to 4ml, and continuously culturing until 293T cells are in a logarithmic phase.

(3) Plasmid transfection of 293T cells:

carrying out pancreatin digestion on 293T cells in a logarithmic growth phase to prepare a cell suspension; cell suspension (cell count about 4X 10) ⁵ ) Inoculated into 24-well cell culture plates, 37 ℃ and 5% CO ₂ The incubator is used for culturing until the cell fusion degree reaches 80 percent. Transfection was performed according to Invitrogen lipofectamine 2000 transfection reagent (purchased from Invitrogen, cat # 11668) using instructions:

a) Changing the culture solution to 400ul of opti-MEM culture medium;

b) Transfecting 1 mu g of GV143-Thbs2 plasmid and 2 mu l of lipofectamine 2000 in each hole, respectively dissolving the GV143-Thbs2 plasmid and the lipofectamine 2000 in opti-MEM culture medium according to the proportion, uniformly mixing, and standing at room temperature for 5min to obtain diluted GV143-Thbs2 and lipofectamine 2000;

c) Mixing the GV143-Thbs2 and lipofectamine 2000 diluted in the step b) uniformly, and standing for 20min at room temperature to obtain a mixed solution of the GV143-Thbs2 plasmid and the lipofectamine 2000;

d) Adding the mixture of GV143-Thbs2 plasmid and lipofectamine 2000 to 293T cells having a degree of cell fusion of 80%, 5% at 37% ₂ After 5h of culture in the incubator, the medium was replaced with fresh complete medium containing 10% serum and cultured for 24h.

After 24h of transfection, the expression of the fluorescence labeling gene on the plasmid was observed to judge the transfection efficiency, as shown in FIG. 4, the fluorescence expression result indicates that: after transfection, obvious green fluorescence can be observed in the cells, which indicates that the GV143-Thbs2 plasmid fluorescence labeling gene is normally expressed, and the GV143-Thbs2 plasmid successfully transfects 293T cells.

2.Western Blot analysis of Thbs2 protein expression

Positive control, 293T cells from either control or experimental group (i.e., GV143-Thbs2 plasmid) were transfected, washed twice with PBS, and lysed with appropriate amount of pre-cooled 2 × Lysis Buffer (formulation as in table 9).

TABLE 9 formulation of 2 × lysine Buffer

Reagent	Concentration of
		1M Tris-HCl(pH 6.8)	100mM
Mercaptoethanol
		2％
Glycerol			20％
	SDS
		4％

Scraping the cells, transferring into 1.5ml EP tube, cracking on ice for 10-15min, and ultrasonically breaking the cells (200W for 4 times, each time for 5s, and interval for 2 s); centrifuging at 12000g for 15min at 4 deg.C, and collecting supernatant by BCA method to determine protein concentration (BCA kit, purchased from Biyuntian biology, cat. No. P0012S); each sample was adjusted to a protein concentration of 2. Mu.g/. Mu.L and stored at-80 ℃ for further use. The loading of protein was 20. Mu.g, primary antibody was FLAG (purchased from Sigma, cat # F1804, diluted 1). The secondary antibody was goat-anti-mouse secondary antibody (purchased from Santa-Cruz, cat # sc-2005, dilution ratio 1: 4000) and incubated at room temperature for 1h. Use of Pierce ^TM ECL Western Blotting Substrate kit (purchased from Thermo, cat # 32106). The results are shown in FIG. 5, where the Thbs2 protein is normally expressed.

3. RNAi adeno-associated virus vector construction (GV 687-CMV-shRNA (Thbs 2))

1. Design of synthetic siRNA specifically targeting Thbs2 Gene

For the mouse Thbs2 gene, siRNA targeting specifically the target sequence of the Thbs2 gene was designed as shown in table 10 (shanghai jekken genescience and technology gmbh).

TABLE 10 effective siRNA target sequences targeting Thbs2 gene

2. Design of second-generation shRNA (shRNAmir) specifically targeting mouse Thbs2 gene

The sequence of the second-generation shRNA (Thbs 2-shRNA) of the specific targeting mouse Thbs2 gene is shown in SEQ ID NO.2, and the specific information is shown in Table 11; the sequence of the negative control second generation shRNA (NC-shRNA) is shown in SEQ ID NO.8, and the specific information is shown in Table 12.

TABLE 11 specific information of the second generation shRNA sequence of the specific targeting mouse Thbs2 gene

TABLE 12 negative control second generation shRNA sequence details

3. Synthesis of specific targeting mouse Thbs2 gene and negative control second-generation shRNA

The sequence (shown as SEQ ID NO. 2) of the second-generation shRNA (Thbs 2-shRNA) of the specific target mouse Thbs2 gene is obtained by design and whole gene synthesis of Shanghai Jikai gene chemistry technology GmbH.

In addition, the sequence of the negative control second generation shRNA (NC-shRNA) (shown as SEQ ID NO. 8) is obtained by design and whole gene synthesis of Shanghai Kjeka GeneChemie GmbH.

Ligation of Thbs2-shRNA to CMV promoter-EGFP-MIR155 (MCS) -SV40 PolyA (vector number GV 687)

Utilizing NheI and XhoI to enzyme-cleave the CMV promoter-EGFP-MIR155 (MCS) -SV40 PolyA vector (vector number GV687, abbreviated as GV687 vector) to obtain a linearized GV687 vector which is then configured with a linking reaction system (as shown in Table 13) with the Thbs2-shRNA, linking the linearized GV687 vector with the Thbs2-shRNA overnight at 16 ℃, directly linking the Thbs2-shRNA into the enzyme-cleaved CMV promoter-EGFP-MIR155 (MCS) -SV40 PolyA to obtain the GV687-CMV-shRNA (Thbs 2); the ligation of the negative control NC-shRNA to CMV promoter-EGFP-MIR155 (MCS) -SV40 PolyA (vector number GV 687) was performed as described above to obtain GV687-CMV-NC. Wherein, the map of the GV687-CMV-NC plasmid is shown as A in figure 6, and the map of the GV687-CMV-shRNA (Thbs 2) plasmid is shown as B in figure 6.

TABLE 13 ligation reaction System

Reagent	Volume (μ l)
		Linearized GV687 vector	1
Thbs2-shRNA	2
		T4 buffer	1
T4 ligase	0.5
		ddH ₂ O	4.5

5. Transformation of

And 4. A conversion step in the step I.

Sanger sequencing

And (3) sequencing by Sanger to identify positive clones, wherein the sequencing primer is CATGGTGTCTGCGTGAGTTCGTG. As a result: and (6) detecting.

7. Plasmid extraction

In the same step I, 7 plasmid extraction step.

Fourth, the silencing effect detection of the RNAi adeno-associated virus vector (GV 687-CMV-shRNA (Thbs 2))

Cotransfection of Thbs2 overexpression vectors and RNAi adeno-associated viral vectors

The Thbs2 overexpression vector (GV 143-Thbs2 plasmid) and the RNAi adeno-associated virus vector (GV 687-CMV-shRNA (Thbs 2)) were co-transfected into 293T cells. The Transfection procedure was the same as that in step 1 and cell Transfection procedure in step two, except that the experimental grouping (shown in Table 14) and the amount of plasmid/Reagent (shown in Table 15) were used, and the co-Transfection Reagent Invitrogen lipofectamine 2000 was replaced with X-tremeGENE HP DNA Transfection Reagent (available from Roche, cat. No. 06366236001) to carry out plasmid Transfection, and the procedure was followed, and 10 hours of FBS-containing DMEM complete medium was cultured for 48 hours 6 hours after Transfection.

Table 14 group

Note: CON: empty cell group, i.e. group of 293T cells not transfected with any plasmid

NC: negative control GV687-CMV-NC plasmid group

KD is low: low concentration GV687-CMV-shRNA (Thbs 2) plasmid

KD is high: high concentration GV687-CMV-shRNA (Thbs 2) plasmid

OE：GV143-Thbs2

TABLE 15 dosage

Marking	concentration/Titer (ng/. Mu.l)	Dosage (mu l)
			OE	415	2.8
NC	1125	1.0
			High KD	584	2.0
Low KD	584	1.2

Verification of GV687-CMV-shRNA (Thbs 2) plasmid silencing efficiency by Western Blot

In the same manner as in step two, step 2 of Western Blot analysis, the primary antibody includes FLAG (Sigma, F1804, dilution ratio 1 2000) and endogenous reference β -actin (Santa-Cruz, sc-32233, dilution ratio 1 5000), and the secondary antibody includes goat anti-mouse secondary antibody (Santa-Cruz, sc-8432, dilution ratio 1.

The results are shown in FIG. 7, which indicates that the GV687-CMV-shRNA (Thbs 2) plasmid can effectively inhibit the expression of THBS2 protein at high concentration.

Example 2 hepatic fibrosis intervention by injecting two vectors into the tail vein of mouse

The 8-week-old male C57BL/6J mice were purchased from Beijing Witonglihua laboratory animal technology, inc., and the mouse-related experiments were performed at the laboratory animal center of Beijing friendship Hospital, affiliated to the university of capital medical science. Mice were divided into two groups, AAV6-CMV-NC control group (n = 5), AAV6-CMV-shRNA (Thbs 2) experimental group (n = 6). AAV6-CMV-NC vector is obtained by coating GV687-CMV-NC plasmid with type 6 adeno-associated virus (AAV 6), AAV6-CMV-shRNA (Thbs 2) vector is obtained by coating GV687-CMV-shRNA (Thbs 2) plasmid with AAV6, and adeno-associated virus vector coating is completed by Shanghai Jikai gene chemistry technology GmbH. AAV6-CMV-NC vector is injected into tail vein of a control group, AAV6-CMV-shRNA (Thbs 2) vector is injected into tail vein of an experimental group, and mouse tail vein injection amounts in two groups are respectively as follows: 1.53E +11v.g,1.61E +11v.g. 12.5% by intraperitoneal injection after the tail vein injection every other day ₄ Solutions (1/7 by volume in mineral oil) were administered twice a week for 6 weeks at 0.01ml/g body weight per dose. Harvesting liver after neck-breaking sacrifice of mice, fixing part of left side leaves with paraformaldehyde, embedding paraffin, and fixing part of left side leavesFreezing at-80 deg.C for storage. HE staining, sirius red staining and Western Blot experiment were performed to detect Thbs2, collagen I and alpha SMA protein expression, respectively.

HE staining: paraffin sections are dewaxed conventionally to water, hematoxylin is stained for 10min,1% hydrochloric acid ethanol is differentiated for several seconds, eosin is stained for 5min, the sections are dehydrated and transparent, and the sections are observed and photographed by a common optical microscope, and the result is shown in A in figure 8, and AAV6-CMV-shRNA (Thbs 2) vector mouse tail intravenous injection can obviously inhibit the infiltration of the hepatic histiitis cells caused by 6 weeks of CCl4 intraperitoneal injection.

Sirius red staining: paraffin section is dewaxed to water conventionally, sirius red staining liquid is stained for 1h and then dehydrated and transparent sealing sheet is removed conventionally, and observation and photographing are carried out under a polarized light microscope, the result is shown as A in figure 8, AAV6-CMV-shRNA (Thbs 2) vector mouse tail vein injection mice can both inhibit CCl remarkably ₄ Hepatic fibrosis was progressed by intraperitoneal injection for 6 weeks, and hepatic fibrosis inhibition efficiency was 73.2% (fig. 8B).

Western Blot experiment: weighing 100mg of small frozen liver tissue of left lateral lobe, adding 1ml of RIPA (Shanghai Bin Yuntian biotechnology Co., ltd., P0013B) and 10 μ l of protease inhibitor (Shanghai Biotechnology science Co., ltd., 20124ES 03) tissue homogenizer to pulverize the liver tissue until the liver tissue is invisible to naked eyes, standing on ice for 30min, and shaking for 5s every 10 min. 13500rpm, centrifugating at 4 ℃ for 15min, discarding the precipitate, and collecting the supernatant as protein solution. Protein concentration was determined by the classical BCA method. The protein loading was 60 μ g, and the primary antibodies were respectively THBS2 (Abcam, ab112543, dilution ratio 1. The secondary antibodies are goat-anti-mouse and goat-anti-rabbit secondary antibodies (China fir gold bridge, dilution ratio 1. The images were developed using the Super ECL Detection Reagent ECL chemiluminescence Super-sensitive color development kit (assist in san Jose Biotech Co., ltd.). The results are shown in FIG. 9, AAV6-CMV-shRNA (Thbs 2) vector mouse injected with rat tail vein can significantly inhibit CCl ₄ The expression of the Collagen I and the alpha SMA protein in the liver tissue of 6 weeks after intraperitoneal injection, and the inhibition efficiency of the expression of the THBS2 protein is 76.9 percent.

In conclusion, the AAV6-CMV-shRNA (Thbs 2) vector can target hepatic stellate cells and inhibit the expression of THBS2 protein by virtue of rat tail intravenous injection, and has good anti-hepatic fibrosis and anti-inflammatory effects.

It should be understood that the above-described embodiments of the present invention are examples for clearly illustrating the invention, and are not to be construed as limiting the embodiments of the present invention, and it will be obvious to those skilled in the art that various changes and modifications can be made on the basis of the above description, and it is not intended to exhaust all embodiments, and obvious changes and modifications can be made on the basis of the technical solutions of the present invention.

Claims

Application of an inhibitor of Thbs2 gene or THBS2 protein in preparing a medicament for resisting hepatic fibrosis.
2. The use of claim 1, wherein the inhibitor is an siRNA, dsRNA, shRNA, miRNA that targets the THBS2 protein or a transcript thereof and is capable of inhibiting the expression of the THBS2 protein or transcription of the Thbs2 gene.
3. The use according to claim 2, wherein the inhibitor is any one of:

a) The target sequence of the siRNA is shown as SEQ ID NO. 1;

b) shRNA, wherein the sequence of the shRNA is shown as SEQ ID NO. 2;

c) A vector capable of expressing the siRNA of a) or the shRNA of b);

d) A virus comprising the vector of c).
4. An siRNA molecule, characterized in that the target sequence of the siRNA molecule is shown in SEQ ID NO. 1.
5. An siRNA molecule according to claim 4, characterized in that the targeting leader of said siRNA molecule is as shown in SEQ ID No. 3.
6. An shRNA molecule is characterized in that the sequence of the shRNA molecule is shown in SEQ ID NO. 2.
7. An expression vector capable of expressing said siRNA molecule or said shRNA molecule.
8. A virus comprising the expression vector of claim 7.
9. The virus of claim 8, wherein the virus is an adeno-associated virus type 6.
10. An anti-liver fibrosis drug comprising the siRNA molecule of claim 4 or 5, the shRNA molecule of claim 6, the expression vector of claim 7 and/or the virus of claim 8 or 9, and a pharmaceutically acceptable carrier.