Application of FILIP1L as a target and its recombinant virus in the treatment of hypertension

Abstract

The present invention relates to the field of biomedical technology, and particularly relates to the application of FILIP1L as a target and its recombinant virus in the treatment of hypertension. The present invention relates to the application of FILIP1L as a target in the treatment of hypertension, and for the first time, it is proposed that smooth muscle FILIP1L is related to hypertension, and the specific regulatory mechanism of the FILIP1L gene in the occurrence and development of hypertension is deeply explored, providing new insights into revealing the pathogenesis of hypertension. The FILIP1L can be used as a new molecular target for the prevention or treatment of hypertension, guiding the screening and research and development of related drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and particularly relates to the application of FILIP1L as a target and its recombinant virus in the treatment of hypertension. Background Art

[0002] In recent years, the prevalence of hypertension has been continuously rising and has become the leading factor for death and disability worldwide. With the increase in blood pressure, the risk of cardiovascular diseases also increases accordingly, often accompanied by important target organ damage and promoting the occurrence of serious clinical cardiovascular events, such as stroke, coronary heart disease and other diseases. Obviously, hypertension has become a major public health problem across the country and even the world. However, the prevention and control of hypertension remains a worldwide problem, and about one-third of patients cannot achieve the ideal blood pressure lowering goal even with the best treatment. Especially because the cause and mechanism of hypertension are unclear, there have been few breakthrough new drugs on the market in the past thirty years. Therefore, in-depth exploration of the pathogenesis of hypertension, finding exact and effective intervention targets and developing new drugs for the treatment of hypertension are of great significance for the prevention and treatment of hypertension and reducing cardiovascular risks.

[0003] Studies have shown that hypertension is a disease mainly characterized by an increase in the hydrostatic pressure acting on the blood vessel wall, and endothelial cells and smooth muscle cells are the main components of the blood vessel wall. High hydrostatic pressure can affect the remodeling of the extracellular matrix by regulating the processes of smooth muscle cell proliferation, apoptosis, differentiation, etc., and can also promote endothelial cell proliferation and increase its permeability, resulting in endothelial dysfunction, and thus participating in vascular remodeling.

[0004] AAV (Adeno-associated virus, AAV) belongs to the Parvoviridae family, is an unenveloped, single-stranded DNA virus. It can infect cells and stably express genes in cells for a long time with less harm. By replacing the AAV coding region gene sequence with the target gene and related functional fragments, the recombinant AAV (rAAV) vector gene therapy is achieved by infecting target cells through intravenous injection and other methods and expressing the corresponding protein. Moreover, different capsid serotypes of rAAV have different tissue infection tropisms. Due to its high transduction efficiency of therapeutic genes, persistent expression ability and good safety, the rAAV vector has been developed as a tool for the treatment of various diseases. So far, 8 rAAV-based gene therapy products have been approved by the regulatory authorities for marketing, and more than 200 rAAV gene therapy products are undergoing clinical trials. The rAAV gene therapy drugs show great development potential and transformation prospects. Summary of the Invention

[0005] To this end, the first object of the present invention is to provide an application of FILIP1L as a target in the treatment of hypertension-related diseases; the present invention first proposes that FILIP1L is related to hypertension and deeply explores the mechanism of FILIP1L in the occurrence and development of hypertension, providing new insights into revealing the pathogenesis of hypertension;

[0006] The second object of the present invention is to provide a recombinant virus that effectively expresses FILIP1L and further discloses its use for preparing a drug for treating hypertension.

[0007] To solve the above technical problems, the present invention relates to an application of FILIP1L as a target in the preparation of a drug for treating hypertension.

[0008] The present invention also discloses an application of FILIP1L as a target for preparing a product having at least one of the following effects (1)-(5):

[0009] (1) A product for monitoring the treatment status of people with hypertension-related diseases;

[0010] (2) A product for monitoring the prognosis of people with hypertension-related diseases;

[0011] (3) A product for preventing and / or treating hypertension-related diseases;

[0012] (4) A product for screening targets related to hypertension-related diseases;

[0013] (5) A product for screening drugs for hypertension-related diseases.

[0014] Specifically, the product includes a reagent, a kit or a drug.

[0015] The present invention also discloses a recombinant virus that can express an effective amount of FILIP1L;

[0016] Preferably, the recombinant virus includes a recombinant adeno-associated virus, a recombinant lentivirus or a recombinant adenovirus.

[0017] Specifically, the recombinant virus includes rAAV-mFilip1l-Flag and / or rAAV-mFilip1l.

[0018] Specifically, the recombinant virus is constructed by a three-plasmid packaging system of a vector plasmid pAAV-MCS, a serotype plasmid AAV9 and an auxiliary plasmid, and the therapeutic gene is mFilip1l.

[0019] Preferably, pAAV-mFilip1l-Flag is generated by cloning the Filip1l gene into the pAAV-MCS vector plasmid, and includes inverted repeat sequences ITR, CMV promoter, polyA signal sequence, and related transcriptional regulatory elements, etc. The AAV9 serotype plasmid includes Rep and Cap genes. The helper plasmid contains genes related to enhancing virus replication, assembly, and cell release processes.

[0020] Specifically, the rAAV includes at least one of AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM41, AAV.hu37, AAV.Anc80, AAV.Anc80L65, rAAV.7m8, AAV.PHP.B, AAV.PHP.eB, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

[0021] The present invention also discloses a method for constructing the recombinant virus, including the step of constructing a three-plasmid packaging system with the vector plasmid, serotype plasmid, and helper plasmid using the FILIP1L as the therapeutic gene;

[0022] Preferably, the FILIP1L gene includes the FILIP1L genes of species such as mice and humans;

[0023] Preferably, the vector plasmid includes pAAV-MCS;

[0024] Preferably, the serotype plasmid includes AAV.

[0025] Preferably, pAAV-mFilip1l-Flag is generated by cloning the Filip1l gene into the pAAV-MCS vector plasmid, and includes inverted repeat sequences ITR, CMV promoter, polyA signal sequence, and related transcriptional regulatory elements, etc. The AAV9 serotype plasmid includes Rep and Cap genes. The helper plasmid contains genes related to enhancing virus replication, assembly, and cell release processes.

[0026] Preferably, after the adeno-associated virus plasmid pAAV-mFilip1l-Flag, the serotype plasmid, and the helper plasmid are configured in a certain proportion, 293 cells are transfected to produce a high-titer adeno-associated virus rAAV9-mFilip1l-Flag containing the target gene.

[0027] The present invention also discloses the use of the recombinant virus for preparing a drug for hypertension-related diseases.

[0028] The present invention also discloses a pharmaceutical preparation for treating hypertension-related diseases, comprising the recombinant virus;

[0029] Preferably, the pharmaceutical preparation and dosage form include at least one of tablets, pills, capsules, powders, drops, lyophilized products, granules, suspensions, syrups, decoctions, injections, or oral liquids;

[0030] Preferably, the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier and / or excipient;

[0031] Preferably, the pharmaceutical preparation further comprises at least one of a pharmaceutically acceptable diluent, excipient, buffer, disintegrant, absorption enhancer, surfactant, or adsorption carrier.

[0032] The present invention also discloses a pharmaceutical preparation for treating hypertension-related diseases, which comprises a mixture of one or more of FILIP1L precursor protein or its coding nucleic acid, FILIP1L mature protein or its coding nucleic acid, a vector expressing FILIP1L, and a recombinant virus expressing FILIP1L;

[0033] Preferably, its main component is a recombinant adeno-associated virus capable of expressing an effective amount of FILIP1L; more preferably, the recombinant virus expressing FILIP1L includes a recombinant adeno-associated virus, a recombinant lentivirus, or a recombinant adenovirus.

[0034] The present invention relates to the application of FILIP1L as a target in the treatment of hypertension, and for the first time, it is proposed that smooth muscle FILIP1L is related to hypertension, and the specific regulatory mechanism of the FILIP1L gene in the occurrence and development of hypertension is deeply explored, providing new insights into revealing the pathogenesis of hypertension. The FILIP1L can provide a new molecular target for the prevention or treatment of hypertension, guiding the screening and research and development of related drugs.

[0035] The recombinant virus rAAV-mFilip1l-Flag and / or rAAV-mFilip1l of the present invention is obtained by constructing a three-plasmid packaging system including a vector plasmid pAAV-MCS, a serotype plasmid AAV9, and an auxiliary plasmid. The therapeutic gene is mFilip1l. Since rAAV9 used in the present invention has vascular smooth muscle targeting specificity, and the rAAV vector has high transduction efficiency, persistent expression ability, and good safety for the therapeutic gene, the recombinant AAV virus of the present invention has a good therapeutic effect on hypertension and contributes to the research and development of hypertension treatment drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] To make the content of the present invention easier to understand, the following further details the present invention according to specific embodiments of the present invention in conjunction with the drawings, where

[0037] Figure 1 is the identification result of BamHI+SalI digestion of the pAAV-mFilip1l-Flag plasmid; it can be seen that after digestion of the pAAV-mFilip1l-Flag plasmid with BamHI+SalI, fragments with lengths of approximately 5000bp (vector) and 3000bp (target gene) are obtained, proving that the pAAV-mFilip1l-Flag-21# clone is a positive clone;

[0038] Figure 2 is the plasmid map of the pAAV-mFilip1l-Flag vector plasmid;

[0039] Figure 3 is the result of overexpression of smooth muscle-specific FILIP1L reducing vascular remodeling;

[0040] Figure 4 is the result of overexpression of smooth muscle-specific FILIP1L significantly reducing the blood pressure of hypertensive mice;

[0041] Figure 5 is the result of overexpression of FILIP1L inhibiting the proliferation and migration of smooth muscle cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0042] The following further details the present invention in conjunction with specific embodiments, but the embodiments of the present invention are not limited thereto.

[0043] In the following examples of the present invention, the experimental animals and cells are as follows:

[0044] (1) Experimental animals and feeding

[0045] Mice at 8 weeks of age, weighing 20 - 30 g, with a genetic background of C57BL / 6 (WT, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.). All mice used in the study were specific pathogen-free (SPF) mice;

[0046] (2) Experimental cells

[0047] The human aortic smooth muscle cell line was purchased from the Cell Resource Center of the Institute of Basic Medicine, Chinese Academy of Medical Sciences & School of Basic Medicine, Peking Union Medical College.

[0048] In the following examples of the present invention, the main experimental equipment and main experimental reagents are as follows:

[0049] (1) Experimental equipment

[0050] Large refrigerated high-speed centrifuge ThermoSorvall Evolution TM RC (Thermo);

[0051] Ultra-micro high-precision spectrophotometer Nanodrop ND-2000 (Thermo);

[0052] Bio-Rad DNA electrophoresis system Sub-Cell GT Cell electrophoresis tank (Bio-Rad);

[0053] Bio-Rad gel imaging system Gel ImBamHr, Gel-Doc (Bio-Rad);

[0054] Eppendorf small refrigerated high-speed centrifuge 5417R (eppendorf);

[0055] Thermo floor-standing culture shaker Forma 481 (Thermo);

[0056] (2) Experimental reagents:

[0057] JM109 competent cells (Wuhan Vazyme Biotech Co., Ltd.);

[0058] Small plasmid extraction kit, product number: EM101 (TransGen Biotech);

[0059] Agarose gel recovery kit, product number: D2500 (Omega);

[0060] Trans2K Plus II DNA Marker, product number: BM121 (TransGen Biotech);

[0061] DNA primer synthesis (invitrogen);

[0062] DNA sequencing (Invitrogen);

[0063] Restriction endonucleases BamHI, SalI (NEB);

[0064] T4 DNA Ligase (NEB);

[0065] Qiagen large-scale plasmid extraction kit (Qiagen);

[0066] CPT high-efficiency transfection kit, product number: R001 (Wuhan Winosis Bio);

[0067] Millex-HV 0.45um PVDF filters, product number: SLHV-033-RB (Millipore);

[0068] ViraBind TM AAV Purification Kits (Cell BioLabs, VPK-141).

[0069] Example 1

[0070] In this example, the cloning and construction of the adeno-associated virus vector were carried out.

[0071] In this example, the plasmid pAAV-mFilip1l-Flag was constructed, where the name of the expressed gene is mouse Filip1l (NM_001040397.4), the name of the vector is pAAV-MCS, and the cloning sites are BamHI + SalI.

[0072] The plasmid vector pAAV-MCS was taken and digested with BamHI and SalI. The digestion reaction was carried out in a 37°C water bath for 3 h, and the large fragment of the plasmid vector pAAV-MCS digested with BamHI + SalI was recovered by 1% agarose gel electrophoresis.

[0073] The plasmid pUC57-mFilip1l containing the target gene was taken and digested with BamHI and SalI. The digestion reaction was carried out in a 37°C water bath for 3 h, and the large fragment of the plasmid pUC57-mFilip1l digested with BamHI + SalI was recovered by 1% agarose gel electrophoresis.

[0074] The large fragment recovered from the above plasmid pAAV-MCS was ligated with the mFilip1l recovered fragment, and the ligation reaction was carried out at 22°C for 3 hours.

[0075] Transformation of the ligation product: Take 10 μl of the ligation product and mix it with 100 μl of JM109 competent bacteria. Incubate on ice for 30 min, heat shock at 42 °C for 45 s, immediately place on ice for 2 min, add 400 μl of LB medium pre-warmed to room temperature, culture in a 37 °C constant temperature shaker for 1 h, centrifuge at 4000 rpm for 1 min, discard 400 μl of the culture supernatant, mix the remaining 100 μl with a pipette and evenly spread it on an LB plate containing 100 μg / ml Ampicillin resistance, and incubate upside down in a 37 °C constant temperature incubator overnight.

[0076] Pick 3 single colonies and inoculate them into 5 ml of LB culture medium containing 100 μg / ml Ampicillin resistance, culture in a 37 °C constant temperature shaker at 250 rpm overnight, extract the plasmid using a small-scale plasmid extraction kit, then perform enzyme digestion identification with BamHI + SalI, and finally send the positive clone bacterial solution for sequencing. The sequencing primer is CMV-F (5‘-CGCAAATGGGCGGTAGGCGTG-3’), and the sequencing result of the positive clone is shown as SEQ ID No.1.

[0077]

[0078] Appendix Figure 1 This is the result of BamHI + SalI digestion and identification of the pAAV - mFilip1l - Flag plasmid. It can be seen that after digestion of the pAAV - mFilip1l - Flag plasmid with BamHI + SalI, fragments with lengths of approximately 5000 bp (vector) and 3000 bp (target gene) are obtained, demonstrating that the pAAV - mFilip1l - Flag - 21# clone is a positive clone.

[0079] Send the positive clone bacterial liquid pAAV - mFilip1l - Flag - 21# for sequencing. The plasmid map of pAAV - mFilip1l - Flag is as Figure 2 shown. The comparison result shows that the sequencing result is consistent with the designed sequence.

[0080] Example 2

[0081] In this example, rAAV9 - mFilip1l - Flag adeno - associated virus is packaged.

[0082] Inoculate 1.5×10 7 293AAV cells per dish into 18 15 - cm cell culture dishes and culture overnight in an incubator at 37°C and 5% CO2. After 24 hours of culture, change the medium in each dish to 15 ml of high - glucose DMEM + 10% FBS + P / S medium.

[0083] Plasmid transfection (for each 15 - cm culture dish): Take two clean 2 - ml EP tubes, label them as tube A and tube B respectively. Add 1 ml of CPT Buffer A to tube A, add the plasmid and related reagents to tube B. Mix tubes A and B and let them stand for 30 minutes, then add the mixed solution drop by drop evenly into the 15 - cm cell culture dish and culture in an incubator at 37°C and 5% CO2 for 6 hours.

[0084] After 6 hours of culture, change the medium to 20 ml of high - glucose DMEM + 10% FBS + P / S medium and culture overnight in an incubator at 37°C and 5% CO2. After continuing to culture for 60 hours, use a 10 - ml pipette to repeatedly pipette the cells to completely detach all cells from the culture dish, and collect the cells and culture supernatant into a new 50 - ml centrifuge tube.

[0085] Place the above - mentioned centrifuge tube at - 80°C / 37°C and freeze - thaw it 3 times, centrifuge at 3000 rpm for 10 minutes, collect the supernatant, and filter it through a 0.45 - um PVDF filter to remove cell debris. The virus supernatant passes through ViraBind TMPurify the AAV Purification Kits (Cell BioLabs, VPK-141), and finally elute with 1.5 ml of Buffer, and perform virus titer detection.

[0086] Example 3

[0087] In this example, a hypertensive animal model was constructed, and the aorta was harvested and fixed.

[0088] In this example, a subcutaneous implant of a mouse angiotensin II (Angiotensin II, AngII) osmotic pump was performed to induce a mouse hypertension model. The required dose of AngII for each mouse was 1000 ng / kg / min. Calculate the required amount of AngII powder, add it to an appropriate amount of physiological saline, and slowly oscillate to dissolve it fully. Inject the dissolved AngII drug into the injection hole of the ALZET 2004 osmotic pump, avoiding the generation of air bubbles during injection. Invert the osmotic pump in a centrifuge tube filled with sterile physiological saline and incubate it overnight in a 37°C water bath. Implant the ALZET 2004 osmotic pump into the subcutaneous tissue of the mouse. Pay attention to disinfection and observation to prevent mouse infection. Observe for 30 minutes, and leave after the mouse shows no obvious discomfort.

[0089] Twenty-eight days after the pump implantation, the intervention ended, and all mice were sacrificed. During the model induction period, if a mouse died, immediate autopsy was required to explore the cause of death by laparotomy, and observe and record.

[0090] Vascular harvesting: Perfuse with physiological saline and pre-cooled 4% paraformaldehyde (PFA) solution for fixation and harvesting. After anesthetizing the mouse with 1.25% avertin, isolate the heart and the entire aorta.

[0091] Vascular fixation: Place the blood vessels in 4% PFA fixative and fix at room temperature for 12 hours. Embed, dehydrate, and section for subsequent staining and other experiments.

[0092] Example 4

[0093] In this example, based on the separation of the renal artery and mesenteric artery of the mouse after the aforementioned hypertension modeling, pathological staining was performed to investigate the result that overexpression of smooth muscle-specific FILIP1L alleviates vascular remodeling.

[0094] HE staining

[0095] Take out the paraffin sections, remove the paraffin with xylene and make the tissue sections transparent. Dehydrate with absolute ethanol, 95% ethanol, 70% ethanol and distilled water respectively. Immerse the tissue sections in hematoxylin solution for 5 minutes and in eosin solution for 3 minutes, then dehydrate and clarify with 95% ethanol, absolute ethanol and xylene respectively. Finally, immerse in xylene for 2 - 3 minutes. Seal the tissue sections with a mounting medium, and then observe and photograph under a microscope. The experimental results are shown in Figure 3 Figure B in the appendix, showing that smooth muscle-specific overexpression of FILIP1L reduces vascular remodeling.

[0096] Masson staining

[0097] The sections were baked at 68 °C for 1 h routinely and dewaxed to water. After mordant solution was incubated overnight at room temperature, it was stained with the prepared Weigert iron hematoxylin staining solution for 5 min. Differentiate with acidic ethanol differentiating solution and wash with water. Blue back with Masson blueing solution and wash with distilled water for 1 min. Stain with Ponceau fuchsin staining solution for 10 min. Prepare a weak acid working solution according to the ratio of distilled water: weak acid solution = 2:1, and wash with the weak acid working solution for 1 min. Wash with phosphomolybdic acid solution for 5 min and with the weak acid working solution for 1 min. Stain with aniline blue staining solution for 2 min and wash with the weak acid working solution for 1 min. Dehydrate quickly with 95% ethanol, dehydrate with absolute ethanol 3 times, 10 s each time, clarify with xylene 3 times, 2 min each time, and mount with neutral gum. The experimental results Figure 3 Figure C in the appendix, showing that smooth muscle-specific overexpression of FILIP1L reduces the degree of vascular fibrosis.

[0098] In summary, the pathological staining results in this example suggest that in the group with smooth muscle-specific FILIP1L overexpression, the thickness of the vascular media decreases ( Figure 3 Figure B in the appendix), and the degree of fibrosis decreases ( Figure 3 Figure C in the appendix), indicating weakened vascular remodeling.

[0099] Example 5

[0100] In this example, based on the separation of mouse renal arteries and mesenteric arteries after the above-mentioned hypertension model establishment, vascular tone function detection was carried out to investigate the result of smooth muscle-specific FILIP1L overexpression in reducing vascular remodeling.

[0101] The formulation system for 500 ml in this example is as follows:

[0102] 10×kreb B solution: 10.5 g of NaHCO3;

[0103] 10×kreb A solution: 34.615 g of NaCl, 1.768 g of KCl, 0.812 g of KH2PO4, 1.469 g of MgSO4·7H2O, 1.407 g of CaCl2.

[0104] The liquids required for the experiment (taking 1L as an example) are: 100 ml of Solution A + 100 ml of Solution B + 2.18 g of glucose. Make up the volume to 1L and pass binary gas half an hour in advance.

[0105] Instruments required for the experiment: silicone plate, micro scissors, stereomicroscope, ice box, acupuncture needle, tungsten wire, etc.

[0106] Sample collection: Anesthetize the mouse or kill it by cervical dislocation; take the blood vessels at the corresponding part of the mouse (superior mesenteric artery or its branches, aorta, renal artery, etc.) and place them on the ice box to maintain the vascular activity.

[0107] Put on the machine: Pass a tungsten wire through the lumen of the corresponding blood vessel and fix it in the corresponding channel of the machine. Balance for 30 min. After stabilization, successively administer high K (4M), phenylephrine (PE), acetylcholine (Ach), and sodium nitroprusside (SNP), from low concentration to high concentration (10 -9 ~10 -5 ), and record the corresponding values and perform data analysis after stabilization at different concentrations.

[0108] In this embodiment, the results of the vascular tension function detection are shown in Appendix Figure 3 A. The results show that in the group of mice with overexpression of smooth muscle-specific FILIP1L (AAV9-FILIP1L), the vascular contraction function is decreased and the endothelium-independent relaxation function is increased.

[0109] Example 6

[0110] This embodiment is based on the implantable telemetry method for blood pressure monitoring.

[0111] Before the operation, sterilize the surgical instruments and related supplies by high-pressure steam sterilization, and fast the mice half a day in advance (the mice can be fasted the night before the operation, but water should be provided).

[0112] Implantation of the implant: Fix the mouse on the dissection board, place it under the stereomicroscope, separate the thymus and muscle tissues with micro forceps to fully expose the carotid artery. Insert the implant into the corresponding position, suture it, and then put the mouse on the heating pad to make the mouse wake up faster. Data can be collected 5 days after the operation. The experimental results are shown in Appendix Figure 4 .

[0113] Figure 4Results of significantly reduced blood pressure in smooth muscle-specific FILIP1L overexpressing hypertensive mice; It can be seen that AAV9-FILIP1L and AAV9-green fluorescent protein (GFP) were respectively injected into the experimental group and the control group of mice via the tail vein. After 2 months, an osmotic pump was subcutaneously implanted with AngII (1000 ng / kg / min) to construct a hypertensive animal model, and blood pressure was continuously monitored by the tail cuff method and the implanted telemetry method. The results showed that compared with the mice in the control group (AAV9-GFP), the blood pressure of the mice in the smooth muscle-specific FILIP1L overexpressing group (AAV9-FILIP1L) was significantly reduced. It can be seen that smooth muscle-specific overexpression of FILIP1L can effectively reduce blood pressure.

[0114] Example 7

[0115] In this example, immunoblotting technique was performed.

[0116] In this example, the corresponding stacking gel (see Table 1 below) and separating gel (see Table 2 below) were prepared according to the gel preparation protocol:

[0117] Table 1 5% stacking gel (12 ml)

[0118] <![CDATA[ddH2O]]> 8.2ml 30% acrylamide solution 2.0ml 1M Tris-Cl, pH 6.8 1.5ml 10% SDS 0.12ml 10% AP 0.12ml TEMED 0.012ml

[0119] Table 2 Separating gel system

[0120] reagent 8% separating gel (30 ml) 10% separating gel (30 ml) 12% separating gel (30 ml) <![CDATA[ddH2O]]> 13.8ml 11.8ml 9.9ml 30% acrylamide 8.0ml 10ml 12ml 1.5M Tris-Cl 7.6ml 7.6ml 7.5ml 10% SD 0.3ml 0.3ml 0.3ml 10% AP 0.3ml 0.3ml 0.3ml TEMED 0.018ml 0.012ml 0.012ml

[0121] Take the volume of the separating gel to be 7 - 8 ml and the volume of the stacking gel to be 3 - 4 ml. After adding the separating gel, be sure to pay attention not to leak. After standing for 20 minutes, add the upper layer of stacking gel and insert a 1.5 cm comb.

[0122] Electrophoresis: Prepare electrophoresis buffer: Dissolve 28.8 g of glycine, 6 g of Tris-base, and 2 g of SDS in 2 L of deionized water.

[0123] Loading: Add the same mass of protein to each well, generally 25 - 50 μg. After loading, turn on the power supply, set the starting voltage to 80 V, and when the sample moves from the stacking gel to the separating gel, adjust the voltage to 120 V. When the target protein moves to the appropriate position, cut off the power supply.

[0124] Electroblotting: Prepare the electroblotting solution: 28.8 g of glycine, 6 g of Tris-base, 400 ml of methanol, make up to 2 L with deionized water and dissolve thoroughly. Place the gel and NC membrane using the sandwich method, i.e., black clip - sponge - filter paper - gel - NC membrane - filter paper - sponge - white clip. Put the clip with the gel and NC membrane in the electroblotting tank and add sufficient electroblotting solution. Place the electroblotting tank in an ice box and add enough ice to maintain a low temperature state. For general proteins, electroblot at 200 mA for 2 hours.

[0125] Blocking: Prepare the skim milk blocking solution: 2.5 g of skim milk, add it to a 50 ml clean centrifuge tube, then add 50 ml of 1×TBST solution (see Table 3 below), vortex and mix well.

[0126] Table 3 Preparation of 1×TBST Buffer (1 L)

[0127] NaCl 10g 1M Tris-HCl (pH 8.0) 20ml <![CDATA[ddH2O]]> 980ml Tween 20 50 μl

[0128] Put the NC membrane into the skim milk blocking solution and incubate on a shaker at room temperature for 1 hour. After blocking, wash the membrane 3 times with TBST, 5 minutes each time. Incubate with the primary antibody overnight in a shaker in a chromatography cabinet at 4°C. Incubate with the secondary antibody at room temperature for 1 hour the next day. After elution with TBST is completed, put the membrane into the exposure machine, add the luminescent solution and perform exposure imaging. In this example, the experimental results involved are shown in Appendices Figure 5 A and D. It is proved that overexpression of FILIP1L can inhibit the proliferation and phenotypic transformation of smooth muscle cells, and knockdown of FILIP1L can promote the proliferation and phenotypic transformation of smooth muscle cells.

[0129] Example 8

[0130] In this example, plasmid and siRNA transfection are carried out.

[0131] Cell seeding: After counting according to the cell growth rate, perform seeding to ensure that the cell density reaches about 80% - 90% when transfected the next day.

[0132] Cell transfection: Use lip3000 as the transfection reagent. Before transfection, change the cell culture medium (without antibiotics and serum). Dilute the plasmid or siRNA with opti-MEM, and dilute lip3000 with opti-MEM as well. After mixing the two and standing for 5 minutes, mix them in the same tube. Avoid rough pipetting when mixing (because it may cause the liposome to fail). Stand for 15 minutes, then add the prepared transfection solution to the cell culture plate and gently shake evenly. Replace the medium in a timely manner 6 hours after transfection (replace the serum-free medium with the complete medium).

[0133] Detection: After successful transfection for 24 hours, take the cells out of the incubator and perform subsequent relevant detections.

[0134] Example 9

[0135] In this example, an adeno-associated virus was constructed for mouse tail vein injection.

[0136] Calculation: The virus injection amount for each mouse was 1×10 11 vg, and the total required virus amount was calculated on the day of the experiment. After the virus was melted in an ice box at 4°C, it was dissolved in sterile normal saline for tail vein injection.

[0137] Injection: Dip a cotton swab in 75% alcohol to disinfect the mouse's tail, scrape off the excess hair on the tail with a small knife to make the tail blood vessels full, fix the mouse with a visible tail vein injection fixator for mice, hold the mouse's tail with the right index finger and thumb, bend the mouse's tail downward behind the thumb, draw the virus solution with a disposable 1 ml insulin syringe, keep the needle at an angle of about 30° to the mouse tail vein, with the needle tip bevel facing up, gently pierce the skin and then immediately keep the needle parallel to the blood vessel. After the needle penetrates more than half of the blood vessel, gently shake the needle. After confirming that the needle is in the blood vessel, slowly push the liquid medicine to complete the tail vein injection.

[0138] In this example, the experimental results are shown in the appendix Figure 4 . It can be seen that smooth muscle-specific overexpression of FILIP1L can effectively reduce blood pressure.

[0139] Example 10

[0140] In this example, a cell scratch assay was performed.

[0141] Plating: Resuspend the cells and plate them, controlling the density to be confluent the next day.

[0142] Scratching: Use a scratch instrument to make a scratch, try to complete it in one go, and then rinse with PBS to remove the floating cells. To reduce false positive results caused by cell proliferation, use a medium with 0 - 2% FBS to reduce the impact of cell proliferation on the experimental results.

[0143] Photographing: Select appropriate time points for photographing, such as 0 h, 6 h, 12 h, and photograph under a microscope.

[0144] Data processing and analysis: Use image j to analyze the width or area of the scratch at each time point.

[0145] In this example, the results of the cell scratch assay are shown in the appendix Figure 5 in B and E. It can be seen that specific overexpression of FILIP1L can inhibit the proliferation and migration of smooth muscle cells, and specific knockdown of FILIP1L can promote the proliferation and migration of smooth muscle cells.

[0146] Example 11

[0147] In this example, a Transwell migration experiment was conducted.

[0148] Cells were inoculated into the Transwell chambers according to the grouping, and complete medium with a concentration of 10% was added to the lower well plates. Then, they were placed in an incubator and cultured for 24 hours. The original medium was discarded, and the cells were washed once with PBS. A 4% paraformaldehyde solution was added to each well and fixed at room temperature for 20 minutes. The fixing solution was discarded, and the cells were washed once with tap water. A 0.1% (1 mg / mL) crystal violet solution was added to each well, and after staining for 2 minutes, the cells were washed once with tap water. The non-migrated cells in the chambers were wiped off with a cotton swab and left to dry. The membrane at the bottom of the chamber was removed with forceps, placed face up, transferred to a glass slide, and sealed with neutral resin glue. They were observed and counted under a 200X microscope, and the results were recorded and analyzed.

[0149] In this example, the results of the Transwell migration experiment are shown in Appendix Figure 5 C and F, demonstrating that specific overexpression of FILIP1L can inhibit the migration of smooth muscle cells, and specific knockdown of FILIP1L can promote the migration of smooth muscle cells.

[0150] Obviously, the above examples are merely illustrations for clear explanation and not limitations on the implementation methods. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A smooth muscle FILIP1L The invention discloses use of gene-specific overexpression recombinant adeno-associated virus for preparing medicine for treating hypertension.

2. The use according to claim 1, characterized in that The recombinant adeno-associated virus includes AAV9.

3. The use according to claim 1, characterized in that The smooth muscle FILIP1L The method for constructing a recombinant adeno-associated virus for specific overexpression of a gene comprises the following steps: FILIP1L For therapeutic genes, the vector plasmid, serotype plasmid and helper plasmid are used to construct a three-plasmid packaging system.

4. The use according to claim 3, characterized in that Said FILIP1L Genes include mouse or human FILIP1L Gene.

5. The use according to claim 3, characterized in that: The vector plasmid is pAAV-MCS.

6. The use according to claim 3, characterized in that: The serotype plasmid is AAV9.

7. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug includes at least one of tablets, pills, capsules, powders, drops, lyophilized preparations, granules, suspensions, syrups, decoctions, injections or oral solutions; The medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

8. The use according to any one of claims 1 to 6, characterized in that: The drug further comprises at least one of a pharmaceutically acceptable diluent, excipient, buffer, disintegrant, absorption promoter, surfactant or adsorption carrier.

9. A smooth muscle FILIP1L Use of a gene-specific overexpression recombinant adeno-associated virus for preparing a product for preventing and / or treating hypertension.

10. The use according to claim 9, characterized in that The recombinant adeno-associated virus includes AAV9.

11. The use according to claim 9, characterized in that The smooth muscle FILIP1L The method for constructing a recombinant adeno-associated virus for specific overexpression of a gene comprises the following steps: FILIP1L For therapeutic genes, the vector plasmid, serotype plasmid and helper plasmid are used to construct a three-plasmid packaging system.

12. The use according to claim 11, characterized in that Said FILIP1L Genes include mouse or human FILIP1L Gene.

13. The use according to claim 11, characterized in that The vector plasmid is pAAV-MCS.

14. The use according to claim 11, characterized in that The serotype plasmid is AAV9.

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