Application of FILIP1L serving as target spot and recombinant virus of FILIP1L in treatment of hypertension

Abstract

The invention relates to the technical field of biological medicine, in particular to application of FILIP1L serving as a target spot and a recombinant virus of the FILIP1L in treatment of hypertension. The invention relates to application of FILIP1L as a target spot in treatment of hypertension, proposes that smooth muscle FILIP1L is related to hypertension for the first time, deeply discusses a specific regulation mechanism of the FILIP1L gene in occurrence and development of hypertension, and provides a new insight for revealing pathogenesis of hypertension. The FILIP1L can be used as a novel molecular target for preventing or treating hypertension and is used for guiding screening and research and development of related drugs.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of FILIP1L as a target and a recombinant virus thereof in treating hypertension. Background Art

[0002] In recent years, the prevalence of hypertension has continued to rise, and it has become the leading cause of death and disability worldwide. With the increase in blood pressure, the risk of cardiovascular disease also increases accordingly, often accompanied by damage to important target organs and promoting clinical serious cardiovascular events, such as stroke, coronary heart disease and other diseases. Obviously, hypertension has become a major public health problem in China and even around the world. However, the prevention and control of hypertension remains a global problem. About 1 / 3 of patients cannot achieve the ideal blood pressure reduction target even with the best treatment. In particular, due to the unclear etiology and mechanism of hypertension, few breakthrough new drugs have been launched in the past three decades. Therefore, in-depth exploration of the pathogenesis of hypertension, finding accurate and effective intervention targets, and developing new hypertension treatment drugs are of great significance for the prevention and treatment of hypertension and the reduction of cardiovascular risks.

[0003] Studies have shown that hypertension is a disease characterized by increased hydrostatic pressure acting on the blood vessel wall. 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 proliferation, apoptosis, and differentiation of smooth muscle cells. It can also promote endothelial cell proliferation, increase its permeability, and lead to endothelial dysfunction, thereby participating in vascular remodeling.

[0004] AAV (Adeno-associated virus, AAV) belongs to the Parvoviridae family. It is a non-enveloped, single-stranded DNA virus that can infect cells and express genes in cells for a long time and stably with less harm. Recombinant AAV (rAAV) vector gene therapy is achieved by replacing the gene sequence of the AAV coding region with the target gene and related functional fragments, infecting the target cells by intravenous injection and expressing the corresponding proteins. Moreover, rAAV with different capsid serotypes has different tissue infection tropism. rAAV vectors have been developed as a tool for the treatment of various diseases due to their efficient transduction rate of therapeutic genes, persistent expression ability and good safety. To date, 8 rAAV-based gene therapy products have been approved for marketing by regulatory authorities, and more than 200 rAAV gene therapy products are undergoing clinical trials. rAAV gene therapy drugs show great development potential and transformation prospects. Summary of the invention

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

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

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

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

[0010] (2) Products for prognosis monitoring of populations with hypertension-related diseases;

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

[0012] (4) Products that screen for targets associated with hypertension-related diseases;

[0013] (5) Screening products for drugs for hypertension-related diseases.

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

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

[0016] Preferably, the recombinant virus comprises 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 obtained by constructing a three-plasmid packaging system consisting 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 produced by cloning the Filip1l gene into the pAAV-MCS vector plasmid, including the inverted repeat sequence ITR, CMV promoter, ployA signal sequence and related transcriptional regulatory elements. The AAV9 serotype plasmid includes Rep and Cap genes. The auxiliary plasmid contains genes related to enhancing viral replication, assembly and cell release processes.

[0020] Specifically, the rAAV includes AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4 1. at least one of 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, comprising the steps of using the FILIP1L as a therapeutic gene and constructing a three-plasmid packaging system with a vector plasmid, a serotype plasmid and an auxiliary plasmid;

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

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

[0024] Preferably, the serotype plasmid comprises AAV.

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

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

[0027] The invention also discloses the use of the recombinant virus in preparing drugs 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 preparations, granules, suspensions, syrups, decoctions, injections or oral solutions;

[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, the pharmaceutical preparation comprising one or a mixture of several of the following: a FILIP1L precursor protein or its encoding nucleic acid, a FILIP1L mature protein or its encoding nucleic acid, a vector expressing FILIP1L, and a recombinant virus expressing FILIP1L;

[0033] Preferably, its main component is a recombinant adeno-associated virus that can express 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 proposes that smooth muscle FILIP1L is related to hypertension, and deeply explores the specific regulatory mechanism of the FILIP1L gene in the occurrence and development of hypertension, providing new insights into the pathogenesis of hypertension. The FILIP1L can provide a new molecular target for the prevention or treatment of hypertension, and guide the screening and development of related drugs.

[0035] The recombinant virus rAAV-mFilip1l-Flag and / or rAAV-mFilip1l described in the present invention is obtained by constructing 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. Since the rAAV9 used in the present invention has vascular smooth muscle targeting specificity and the rAAV vector has a high transduction rate, a lasting expression ability and good safety for the therapeutic gene, the recombinant AAV virus described in the present invention has a good therapeutic effect on hypertension and is helpful for the research and development of hypertension therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to make the content of the present invention easier to understand, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0037] Figure 1 This is the result of BamHI+SalI digestion of pAAV-mFilip1l-Flag plasmid. It can be seen that after BamHI+SalI digestion of pAAV-mFilip1l-Flag plasmid, fragments of about 5000 bp (vector) and 3000 bp (target gene) were obtained, proving that pAAV-mFilip1l-Flag-21# clone is a positive clone.

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

[0039] Figure 3 The smooth muscle-specific FILIP1L overexpression alleviates vascular remodeling outcomes;

[0040] Figure 4 The results showed that smooth muscle-specific FILIP1L overexpression significantly reduced blood pressure in hypertensive mice;

[0041] Figure 5 The results showed that overexpression of FILIP1L inhibited smooth muscle cell proliferation and migration. DETAILED DESCRIPTION

[0042] The present invention is further described in detail below in conjunction with specific implementation modes, but the implementation modes of the present invention are not limited thereto.

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

[0044] (1) Experimental animals and their breeding

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

[0046] (2) Experimental cells

[0047] Human aortic smooth muscle cell line was purchased from the Cell Resource Center of the Basic Medical College, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College.

[0048] In the following embodiments 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-volume 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 Vinosai Biotechnology);

[0058] Small-scale plasmid extraction kit, catalog number: EM101 (Quanshijin Biotechnology);

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

[0060] Trans2K Plus II DNA Marker, catalog number: BM121 (Quanshijin Biotechnology);

[0061] DNA primer synthesis (Invitrogen);

[0062] DNA sequencing (Invitrogen);

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

[0064] T4 DNALigase (NEB);

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

[0066] CPT high-efficiency transfection kit, catalog number: R001 (Wuhan Vinocell Biotechnology);

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

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

[0069] Example 1

[0070] This example is about cloning and constructing the adeno-associated virus vector.

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

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

[0073] The target gene plasmid pUC57-mFilip1l was taken and double-digested with BamHI and SalI. The digestion reaction was reacted in a 37°C water bath for 3 h, and the plasmid pUC57-mFilip1lBamHI+SalI digested large fragment was recovered by 1% agarose gel electrophoresis.

[0074] The large fragment recovered from the above plasmid pAAV-MCS was ligated with the fragment recovered from mFilip11, and the ligation reaction was carried out at 22°C for 3 hours.

[0075] Transformation of ligation product: Mix 10ul of ligation product with 100ul of JM109 competent bacteria, place on ice for 30min, heat shock at 42℃ for 45s, immediately place on ice for 2min, add 400ul of LB medium preheated to room temperature, culture at 37℃ constant temperature shaker for 1h, centrifuge at 4000rpm for 1min, discard 400ul of culture supernatant, mix the remaining 100ul with a pipette, and evenly spread on an LB plate containing 100μg / ml Ampicillin resistance, and culture inverted in a 37℃ constant temperature incubator overnight.

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

[0077]

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

[0079] The positive clone bacterial solution pAAV-mFilip1l-Flag-21# was sent for sequencing. The plasmid map of pAAV-mFilip1l-Flag is as follows: Figure 2 As shown, the comparison results show that the sequencing results are consistent with the designed sequence.

[0080] Example 2

[0081] This example is used to package rAAV9-mFilip1l-Flag adeno-associated virus.

[0082] According to 1.5×10 per dish 7 293AAV cells were inoculated into 18 15 cm cell culture dishes and cultured overnight in a 37°C, 5% CO2 incubator. After 24 hours of culture, each dish was replaced with 15 ml of high-glucose DMEM+10% FBS+P / S culture medium.

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

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

[0085] The above centrifuge tube was placed in -80℃ / 37℃ and repeatedly frozen and thawed for 3 times, centrifuged at 3000rpm for 10min, the supernatant was collected, and filtered with a 0.45um PVDF filter to remove cell debris. The virus supernatant was filtered by ViraBind TMThe virus was purified using AAV Purification Kits (Cell BioLabs, VPK-141), and finally eluted with 1.5 ml of Buffer for virus titer detection.

[0086] Example 3

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

[0088] In this example, subcutaneous implantation of angiotensin II (AngII) osmotic pumps in mice was performed to induce a mouse hypertension model. The AngII dose required for each mouse is 1000 ng / kg / min. The required amount of AngII powder is calculated, added to an appropriate amount of normal saline, and slowly shaken to fully dissolve it. Inject the dissolved AngII drug into the injection hole of the ALZET2004 osmotic pump, and avoid bubbles during injection. Invert the osmotic pump in a centrifuge tube filled with sterile normal saline and incubate it in a 37°C water bath overnight. 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 has no obvious discomfort.

[0089] After 28 days of implantation, the intervention was terminated and all mice were sampled. During the model induction period, if a mouse died, an autopsy was performed immediately, and the cause of death was investigated by laparotomy, observed, and recorded.

[0090] Blood vessel sampling: The samples were fixed by perfusion with physiological saline and precooled 4% paraformaldehyde (PFA) solution. The mice were anesthetized with 1.25% avertin and the heart and the entire length of the aorta were isolated.

[0091] Blood vessel fixation: Place the blood vessels in 4% PFA fixative and fix them at room temperature for 12 hours. Then embed, dehydrate, and slice them for subsequent staining and other experiments.

[0092] Example 4

[0093] This example is based on the above-mentioned hypertension modeling, in which the renal artery and mesenteric artery of mice were isolated and pathologically stained to examine the results of smooth muscle-specific FILIP1L overexpression in alleviating vascular remodeling.

[0094] HE staining

[0095] Take out the paraffin sections, use xylene to remove the paraffin and make the tissue sections transparent. Dehydrate with anhydrous ethanol, 95% ethanol, 70% ethanol and distilled water respectively. Soak the tissue sections in hematoxylin solution for 5 minutes, and soak the tissue sections in eosin solution for 3 minutes, and then dehydrate and make them transparent with 95% ethanol, anhydrous ethanol and xylene respectively. Finally, soak in xylene for 2-3 minutes. Seal the tissue sections with sealing agent, and then observe and take pictures with a microscope. The experimental results are shown in the attached Figure 3 Middle B, shows that smooth muscle-specific overexpression of FILIP1L alleviates vascular remodeling.

[0096] Masson staining

[0097] The sections were baked at 68°C for 1 hour and dewaxed to water. After the mordant solution was left overnight at room temperature, it was stained with the prepared Weigert iron hematoxylin staining solution for 5 minutes. Differentiation was performed with acidic ethanol differentiation solution and washed with water. Masson bluing solution was used to return to blue and washed with distilled water for 1 minute. Staining was performed with Ponceau fuchsin staining solution for 10 minutes. Weak acid working solution was prepared in a ratio of distilled water: weak acid solution = 2:1, and washed with weak acid working solution for 1 minute. Washing was performed with phosphomolybdic acid solution for 5 minutes, and washing was performed with weak acid working solution for 1 minute. Staining was performed with aniline blue staining solution for 2 minutes, and washing was performed with weak acid working solution for 1 minute. Rapid dehydration was performed with 95% ethanol, dehydration was performed with anhydrous ethanol 3 times for 10 seconds each time, transparentization was performed with xylene 3 times for 2 minutes each time, and sealing was performed with neutral gum. Experimental results Figure 3 Middle C, shows 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 the thickness of the vascular media was reduced in the smooth muscle-specific FILIP1L overexpression group ( Figure 3 Middle B), decreased fibrosis ( Figure 3 Middle C), indicating attenuated vascular remodeling.

[0099] Example 5

[0100] This example is based on the above-mentioned hypertension modeling, in which the renal arteries and mesenteric arteries of mice were isolated to perform vascular tension function tests, and the results of smooth muscle-specific FILIP1L overexpression were investigated to alleviate vascular remodeling.

[0101] The system of 500ml of liquid preparation in this embodiment is as follows:

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

[0103] 10×kreb A solution: NaCl 34.615g, KCl 1.768g, KH2PO4 0.812g, MgSO4.7H2O1.469g, CaCl2 1.407g.

[0104] The liquid required for the experiment is (taking 1L as an example): 100ml liquid A + 100ml liquid B + 2.18g glucose, make up to 1L, and pass binary gas half an hour in advance.

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

[0106] Sampling: Mice were anesthetized or killed by cervical dislocation; blood vessels (superior mesenteric artery or its branches, aorta, renal artery, etc.) from corresponding parts of the mice were taken and placed on an ice box to maintain vascular activity.

[0107] On the machine: A tungsten wire was passed through the lumen of the corresponding blood vessel and fixed in the corresponding channel of the machine. After 30 minutes of equilibration, high potassium (4M), phenylephrine (PE), acetylcholine (Ach), and sodium nitroprusside (SNP) were given successively from low concentration to high concentration (10 -9 ~10 -5 ), and after different concentrations stabilized, the corresponding values ​​were recorded and data analysis was performed.

[0108] In this embodiment, the vascular tension function test results are shown in the attached Figure 3 Middle A, the results showed that the vasoconstriction function of mice in the smooth muscle-specific FILIP1L overexpression group (AAV9-FILIP1L) was reduced, and the endothelium-independent relaxation function was increased.

[0109] Example 6

[0110] This embodiment performs blood pressure monitoring based on implant telemetry.

[0111] Before the operation, the surgical instruments and related supplies were autoclaved and disinfected, and the mice were fasted for half a day in advance (they could be fasted the night before the operation, but they were given drinking water).

[0112] Implantation: Fix the mouse on the dissection board and place it under the stereoscope. Use micro forceps to separate the thymus and muscle tissue to fully expose the carotid artery. Insert the implant into the corresponding position, suture the mouse and place it on the heating pad to make it wake up faster. Data collection can be started 5 days after surgery. The experimental results are attached. Figure 4 .

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

[0114] Example 7

[0115] In this example, immunoblotting was performed.

[0116] In this embodiment, the corresponding concentrated gel (see Table 1 below) and separation gel (see Table 2 below) are prepared according to the gel preparation scheme:

[0117] Table 15% concentrated gel (12ml)

[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 Separation gel system

[0120] Reagents 8% separation gel (30ml) 10% separation gel (30ml) 12% separation gel (30ml) <![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 7-8ml of separation gel and 3-4ml of concentrated gel. After adding separation gel, be careful not to leak. After standing for 20 minutes, add a layer of concentrated gel and insert a 1.5cm comb.

[0122] Electrophoresis: Prepare electrophoresis buffer: 28.8 g of glycine, 6 g of Tris-base, 2 g of SDS, add 2 L of deionized water to fully dissolve.

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

[0124] Electrotransfer: Prepare electrotransfer solution: 28.8g of glycine, 6g of Tris-base, 400ml of methanol, add deionized water to 2L, and fully dissolve. Place the gel and NC membrane using the sandwich method, i.e., black clips-sponge-filter paper-gel-NC membrane-filter paper-sponge-white clips. Place the clips with gel and NC membrane in the electrotransfer tank and add enough electrotransfer solution. Place the electrotransfer tank in an ice box and add enough ice to maintain a low temperature. Generally, proteins are electrotransferred at 200mA for 2 hours.

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

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

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

[0128] Place the NC membrane in skimmed milk powder blocking solution and incubate on a shaker at room temperature for 1 hour. After blocking, wash the membrane with TBST 3 times, 5 minutes each time. Apply primary antibody and incubate overnight in a shaker in a 4°C chromatography cabinet. Apply secondary antibody the next day for 1 hour at room temperature. After TBST elution is completed, place the membrane in an exposure machine and add luminescent liquid for exposure imaging. The experimental results involved in this embodiment are shown in the attached Figure 5 A and D. It was demonstrated that overexpression of FILIP1L can inhibit smooth muscle cell proliferation and phenotypic transformation, while knockdown of FILIP1L can promote smooth muscle cell proliferation and phenotypic transformation.

[0129] Example 8

[0130] In this example, plasmid and siRNA transfection was performed.

[0131] Cell plating: Cells were plated after counting according to the cell growth rate to ensure that the cell density reached about 80%-90% during transfection the next day.

[0132] Cell transfection: Use lip3000 as the transfection reagent. Replace the cell culture medium (without double antibody and serum) before transfection. Dilute the plasmid or siRNA with opti-MEM, and dilute lip3000 with opti-MEM. Let the two stand for 5 minutes, then mix them in the same tube. Avoid rough blowing when mixing (because it may cause the liposome to fail). Let it stand for 15 minutes, add the prepared transfection solution to the cell culture plate, and shake it gently. Change the medium in time 6 hours after transfection (replace the serum-free medium with complete medium).

[0133] Detection: 24 hours after successful transfection, remove the cells from the incubator for subsequent related detection.

[0134] Example 9

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

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

[0137] Injection: Use a cotton swab dipped in 75% alcohol to disinfect the mouse tail. Use a knife to scrape off excess hair on the tail to fill the blood vessels in the tail. Fix the mouse with a visual tail vein injection fixture. Pinch the mouse tail with the index finger and thumb of the right hand. The mouse tail bends downward behind the thumb. Use a disposable 1ml insulin syringe to absorb the virus solution. The needle and the mouse tail vein are at an angle of about 30°. With the needle tip facing up, gently pierce the skin and immediately make the needle parallel to the blood vessel. When the needle has penetrated more than half of the blood vessel, gently shake the needle. After confirming that the needle is in the blood vessel, slowly push the drug solution to complete the tail vein injection.

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

[0139] Example 10

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

[0141] Plating: Resuspend the cells and plate them at a density that allows them to grow to full size the next day.

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

[0143] Take photos: Choose appropriate time points to take photos, such as 0h, 6h, and 12h under a microscope.

[0144] Data processing and analysis: Image J was used to analyze the width or area of ​​the scratch at each time point.

[0145] In this example, the results of the cell scratch test are shown in the attached Figure 5 As shown in Figures B and E, specific overexpression of FILIP1L can inhibit smooth muscle cell proliferation and migration, while specific knockdown of FILIP1L can promote smooth muscle cell proliferation and migration.

[0146] Embodiment 11

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

[0148] Inoculate into transwell chambers according to the groups, add 10% complete culture medium to the bottom well plate, and culture in an incubator for 24 hours. Discard the original culture medium, wash once with PBS, add 4% paraformaldehyde solution to each well, and fix at room temperature for 20 minutes. Discard the fixative, wash once with tap water, add 0.1% (1mg / mL) crystal violet solution to each well, wash once with tap water after staining for 2 minutes, wipe off the non-migrated cells in the chamber with a cotton swab, and dry. Use tweezers to peel off the membrane at the bottom of the chamber, transfer it to a glass slide with the bottom facing up, and seal it with neutral resin glue. Observe and count under a 200X microscope, and record the results for analysis.

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

[0150] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled 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 list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. An application of FILIP1L as a target in the preparation of a drug for treating hypertension.

2. Use of FILIP1L as a target for preparing a product having at least one of the following effects (1)-(5): (1) Products for monitoring the treatment status of people with hypertension-related diseases; (2) Products for prognosis monitoring of populations with hypertension-related diseases; (3) Products for preventing and / or treating hypertension-related diseases; (4) Products that screen for targets associated with hypertension-related diseases; (5) Screening products for drugs for hypertension-related diseases.

3. A recombinant virus, characterized in that: The recombinant virus can express an effective amount of FILIP1L; Preferably, the recombinant virus comprises a recombinant adeno-associated virus, a recombinant lentivirus or a recombinant adenovirus.

4. The recombinant virus according to claim 3, characterized in that The recombinant virus includes rAAV-mFilip1l-Flag and / or rAAV-mFilip1l.

5. The recombinant virus according to claim 4, characterized in that The rAAV includes AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAVrh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4 1. at least one of 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.

6. A method for constructing the recombinant virus according to any one of claims 3 to 5, characterized in that: The method comprises the steps of using the FILIP1L as a therapeutic gene and constructing a three-plasmid packaging system using a vector plasmid, a serotype plasmid and an auxiliary plasmid; Preferably, the FILIP1L gene includes FILIP1L genes of species such as mice and humans; Preferably, the vector plasmid includes pAAV-MCS; Preferably, the serotype plasmid comprises AAV.

7. Use of the recombinant virus according to any one of claims 3 to 5 for preparing drugs for hypertension-related diseases.

8. A pharmaceutical preparation for treating hypertension-related diseases, characterized in that: The pharmaceutical preparation comprises one or a mixture of several of the following: FILIP1L precursor protein or its encoding nucleic acid, FILIP1L mature protein or its encoding nucleic acid, a vector expressing FILIP1L, and a recombinant virus expressing FILIP1L.

9. The pharmaceutical preparation for treating hypertension-related diseases according to claim 8, characterized in that: The recombinant virus expressing FILIP1L includes the recombinant virus according to any one of claims 3 to 5.

10. The pharmaceutical preparation for treating hypertension-related diseases according to claim 8 or 9, characterized in that: The pharmaceutical preparation and dosage form include at least one of tablets, pills, capsules, powders, drops, lyophilized preparations, granules, suspensions, syrups, decoctions, injections or oral solutions; and / or, The pharmaceutical preparation further comprises a pharmaceutically acceptable carrier and / or excipient; and / or, The pharmaceutical preparation further comprises at least one of a pharmaceutically acceptable diluent, excipient, buffer, disintegrant, absorption promoter, surfactant or adsorption carrier.

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