Application of NEDD4L gene in preparation of medicine for treating vascular endothelial cell proliferation related diseases

Abstract

The invention relates to the technical field of biological medicines, and discloses application of an NEDD4L gene in preparation of a medicine for treating vascular endothelial cell proliferation related diseases. The invention provides application of an NEDD4L gene in preparation of drugs for treating vascular endothelial cell proliferation related diseases, the nucleotide sequence of the NEDD4L gene is as shown in SEQ ID NO: 1, and the amino acid sequence of protein coded by the NEDD4L gene is as shown in SEQ ID NO: 2. It is found for the first time that the overexpressed NEDD4L gene can inhibit proliferation of vascular endothelial cells, lower the invasion ability of the vascular endothelial cells, lower the migration rate of the vascular endothelial cells and induce autophagy of the endothelial cells, and on the basis, the NEDD4L gene is used for preparing the medicine for treating vascular endothelial cell hyperplasia related diseases and has a good application prospect. A new strategy is expected to be provided for treatment of vascular endothelial cell proliferative diseases.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and specifically discloses the application of NEDD4L gene in preparing medicine for treating diseases related to vascular endothelial cell proliferation. Background Art

[0002] Vascular endothelial cell proliferative diseases refer to a class of diseases involving abnormal proliferation of vascular endothelial cells. Vascular endothelial cells (VECs) have important functions such as maintaining vascular tone, angiogenesis, and hemostasis. They are also important guardians of vascular health and are essential for maintaining vascular health and function. Vascular endothelial cell dysfunction may manifest as abnormal endothelial cell proliferation, endothelium-dependent vasodilation disorders, enhanced oxidative stress, chronic inflammation, leukocyte adhesion, increased vascular permeability, endothelial cell aging, metabolic abnormalities, and endothelial cell-mesenchymal transformation. These dysfunctions are associated with a variety of vascular diseases, such as atherosclerosis, hypertension, diabetes, and infectious diseases.

[0003] Abnormal proliferation of vascular endothelial cells is a process that is regulated by multiple factors. The process is mainly affected by the local microenvironment composed of vascular endothelial cell heterogeneity, blood flow shear force, active substances secreted by endothelial cells, vascular endothelial growth factor, etc. In the progression of vascular endothelial cell proliferative diseases, a key link is the change in vascular endothelial cell function, which leads to the transcriptional activation of pro-proliferation genes, the silencing of pro-apoptotic genes, and ultimately leads to the activation of proliferation signaling pathways such as Akt and the initiation of cell migration. Therefore, abnormal proliferation and migration of vascular endothelial cells are closely related to vascular endothelial cell proliferative diseases such as atherosclerosis, hypertension, and diabetes. How to effectively inhibit abnormal proliferation and migration of vascular endothelial cells has become an important strategy for the treatment of these diseases.

[0004] At present, the conventional treatment methods for vascular endothelial cell proliferative diseases mainly include drug therapy, surgical treatment, interventional treatment, laser treatment, etc. Drug treatment mainly focuses on controlling the symptoms of the disease and slowing down the progression of the disease. For example, antihypertensive drugs are used to control hypertension, and hypoglycemic drugs are used to control diabetes. However, drug treatment is basically unable to cure the disease and may be accompanied by drug side effects. Although surgical treatment can directly remove the diseased tissue, it has the disadvantages of excessive bleeding, poor appearance, and easy recurrence. Interventional treatment and laser treatment have the risk of causing damage to normal tissues, so finding new molecules or proteins that inhibit the proliferation and migration of vascular endothelial cells has become a problem that needs to be solved urgently in the clinic. Summary of the invention

[0005] In view of the deficiencies in the conventional methods for treating vascular endothelial cell proliferative diseases in the prior art, the present invention aims to provide a gene drug that has the effects of inhibiting the proliferation, migration and invasion of vascular endothelial cells and inducing cell autophagy, and further provides the use of the NEDD4L gene in the preparation of drugs for treating vascular endothelial cell proliferative-related diseases, in order to open up new treatment strategies for vascular endothelial cell proliferative diseases.

[0006] In order to achieve the above-mentioned object of the invention, the present invention adopts the following technical solutions: In a first aspect, the present invention provides the use of NEDD4L gene in the preparation of a drug for treating diseases related to vascular endothelial cell proliferation.

[0007] After extensive and in-depth research, the present invention has discovered for the first time that the NEDD4L gene can be used as a target for the treatment of vascular endothelial cell proliferative diseases. After overexpression, the NEDD4L gene can inhibit the proliferation, migration and invasion of vascular endothelial cells and induce cell autophagy, so it can be used for related diseases caused by abnormal proliferation of vascular endothelial cells. The use of the NEDD4L gene to prepare drugs for the treatment of vascular endothelial cell proliferative diseases can overcome the shortcomings of existing related drugs such as large side effects and more surgical bleeding, and provides a new treatment idea based on existing conventional treatments, opening up a new direction for the treatment of vascular endothelial cell proliferative diseases.

[0008] As a first limitation to the above application, the nucleotide sequence of the NEDD4L gene is shown in SEQ ID NO:1.

[0009] There are 7 different transcripts of NEDD4L gene in NCBI database, and for genes with multiple transcripts, the amino acid sequences encoded by different transcripts are different, resulting in differences in the structural domains of the proteins, which may cause the final proteins to play different physiological roles. The present invention creatively discovered that the sequence of nucleotides 601-3165 in the CDS region derived from E3 ubiquitin-protein ligase NEDD4-like isoform 2 (NM_001144964.1) has the effect of inhibiting the proliferation, migration and invasion of vascular endothelial cells and inducing cell autophagy within the experimental scope of the present invention.

[0010] As a second limitation to the above application, the amino acid sequence of the protein encoded by the NEDD4L gene is shown in SEQ ID NO:2.

[0011] As a third limitation to the above application, the diseases associated with vascular endothelial cell proliferation include atherosclerosis, hypertension, diabetes or hemangioma.

[0012] In a second aspect, the present invention provides a drug for treating diseases related to vascular endothelial cell proliferation, wherein the drug for treating diseases related to vascular endothelial cell proliferation comprises a NEDD4L gene overexpression vector.

[0013] In a third aspect, the present invention provides a method for preparing the above-mentioned drug for treating vascular endothelial cell proliferation-related diseases, the preparation method comprising the following steps: Step 1, cloning the NEDD4L gene into an adenovirus-associated virus to obtain an adenovirus-associated virus containing the NEDD4L gene; Step 2, transforming the adenovirus-associated virus containing the NEDD4L gene into Escherichia coli to obtain a recombinant plasmid; Step 3: transfect human endothelial cells with the recombinant plasmid, expand the culture, purify and concentrate to obtain an adenovirus concentrate containing the NEDD4L gene, which is the NEDD4L gene overexpression vector.

[0014] As a first limitation to the above preparation method, in step 1, the adenovirus-associated virus includes at least one of adenovirus, lentivirus or adeno-associated virus; Among them, adenovirus includes ADV4, etc., lentivirus includes LV5, etc., and adeno-associated virus includes AAV-GP-1 and AAV-GP-12N, etc. The present invention is described by taking ADV4 as an example.

[0015] As a second limitation to the above preparation method, in step 3, the human endothelial cells include human umbilical vein endothelial cells, human embryonic kidney cells or human smooth muscle cells; In step three, before the expanded culture, the recombinant plasmid is also subjected to high-purity endotoxin-free extraction.

[0016] As a third limitation of the above preparation method, the virus titer of the adenovirus concentrate containing the NEDD4L gene is measured in the human endothelial cells.

[0017] In a fourth aspect, the present invention provides the use of the above-mentioned drug for treating diseases related to vascular endothelial cell proliferation, wherein the drug is injected into the body for at least 72 hours; further, the injection includes microinjection.

[0018] The present invention provides an application of the NEDD4L gene in the preparation of a drug for treating vascular endothelial cell proliferation-related diseases, and for the first time found that overexpression of the NEDD4L gene can inhibit the proliferation of vascular endothelial cells, downregulate the invasive ability of vascular endothelial cells, reduce the migration rate of vascular endothelial cells, and induce autophagy of endothelial cells. Therefore, the drug for treating vascular endothelial cell proliferation-related diseases provided by the present invention exerts a therapeutic effect in the above four aspects of inhibiting the proliferation, invasive ability and migration of vascular endothelial cells and inducing autophagy of endothelial cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0020] Figure 1-1 to Figure 1-3 It is a partial sequencing detection map of the recombinant plasmid gene in Example 1 of the present invention; Figure 2 This is a cell morphology diagram of vascular endothelial cells infected with the NEDD4L gene overexpression vector in Example 2 of the present invention; Figure 3 The expression of mNRA in the vascular endothelial cells after being induced by the NEDD4L gene overexpression vector in Example 2 of the present invention; Figure 4 The expression of NEDD4L protein in the vascular endothelial cells after being induced by the NEDD4L gene overexpression vector in Example 2 of the present invention; Figure 5 The results of the cell proliferation test of the vascular endothelial cells in Example 2 of the present invention after being induced by the NEDD4L gene overexpression vector; Figure 6 The results of the apoptosis test of vascular endothelial cells after being induced by the NEDD4L gene overexpression vector in Example 2 of the present invention; Figure 7 The results of the cell cloning experiment after the vascular endothelial cells were induced by the NEDD4L gene overexpression vector in Example 2 of the present invention; Figure 8 The results of the cell Transwell assay after the vascular endothelial cells were induced by the NEDD4L gene overexpression vector in Example 2 of the present invention; Fig. 9 The results of the cell scratch test after the vascular endothelial cells were induced by the NEDD4L gene overexpression vector in Example 2 of the present invention; Fig.10 The results of immunoblotting of vascular endothelial cells induced by NEDD4L gene overexpression vector in Example 2 of the present invention are shown in FIG. Fig.11 The vascular endothelial cells in Example 2 of the present invention are induced by the NEDD4L gene overexpression vector and infected with the mRFP-GFP-LC3 virus, and the cell autophagy; wherein, Fig.11 a is a microscope image after infection with mRFP-GFP-LC3 virus. Fig.11b is a quantitative graph of the percentage of autophagy positive spots / cell number. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0022] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, cell culture, recombinant DNA technology, and related technologies in the art.

[0023] In order to better illustrate the embodiments of the present invention, further examples are given below.

[0024] Example 1 This example provides a preparation of a NEDD4L gene overexpression vector, and the specific method is as follows: 1. Obtaining the target gene fragment 1. Oligo design, NEDD4L gene upstream and downstream primers plus EcoRI and BamHI and protective bases, used for vector subcloning, the oligo sequence is shown in Table 1 below.

[0025] Table 1

[0026] According to the sequences in Table 1, Suzhou Genetech Co., Ltd. was commissioned to synthesize the above primers.

[0027] 2. Dissolve the above oligo into 50µM; 3. Use the synthesized primers and NEDD4L gene as a template for PCR reaction. The PCR system is shown in Table 2 below.

[0028] Table 2

[0029] In the above PCR, the cycling conditions are shown in Table 3 below.

[0030] Table 3

[0031] 4. After the PCR reaction is completed, the gene fragment is recovered by agarose electrophoresis and gel cutting to obtain the target gene fragment, namely the human NEDD4L gene (referred to as NEDD4L gene in the present invention).

[0032] 2. Cloning of NEDD4L gene into vector ovine adenovirus type 4 (ADV4) 1. The NEDD4L gene fragment obtained above was digested with EcoRI and BamHI at 37°C for 2 hours. The digestion system is shown in Table 4 below.

[0033] Table 4

[0034] Among them, the concentration of NEDD4L gene in Table 4 is 50 µM.

[0035] 2. The vector ADV4 was digested with EcoRI and BamHI at 37°C for 2 hours. The digestion system is shown in Table 5 below.

[0036] Table 5

[0037] Wherein, the concentration of the ADV4 vector in Table 5 is 50µM.

[0038] 3. Perform electrophoresis and use a DNA gel recovery kit to recover the NEDD4L gene and vector ADV4 fragments.

[0039] 4. The NEDD4L gene fragment obtained by enzyme digestion and the linearized vector ADV4 were connected with T4 DNA ligase at 22°C for 2 hours to obtain the connection product. The connection system is shown in Table 6 below.

[0040] Table 6

[0041] 5. Refer to page 55 of Molecular Cloning Experiment Guide (Second Edition) to prepare competent E. coli cells; 6. Add 10 μL of the above ligation product to the centrifuge tube containing the competent E. coli cells, gently mix the contents, and place in ice for 30 minutes; place the centrifuge tube in a test tube rack in a water bath preheated to 42°C for 90 seconds without shaking the centrifuge tube. Quickly transfer the centrifuge tube to an ice bath and cool the cells for 3 minutes. Add 800 μL of LB medium (without antibiotics) to each centrifuge tube, and then transfer the centrifuge tube to a shaker at 37°C and 250 rpm for 45 minutes to allow the bacteria to recover.

[0042] 7. Take 200 μL of the cultured cells and evenly spread them on the LB plate containing 50 μg / ml Ampicillin. After the liquid on the plate is absorbed, place the plate upside down in a 37°C incubator and culture for 16 hours. Pick the clone colonies from the plate, extract the plasmid and identify it, and select the positive clones.

[0043] 8. Pick 4 separate, full colonies from the cultured plate, place them in a test tube containing 5 ml of LB medium (containing 50 µg / ml Ampicillin), and culture them in a shaker at 37°C, 250 rpm for 16 hours.

[0044] 9. Use a plasmid extraction kit (Tiangen Biochemical, DP104-02) to extract the plasmid from the cultured bacterial solution to obtain the recombinant plasmid.

[0045] 3. Sequencing verification and acquisition of recombinant plasmid Take 200 μL of the bacterial solution corresponding to the positive clone and send it for sequencing. Compare the sequencing result with the target gene sequence. After verification, the corresponding positive clone strain is the recombinant strain; The recombinant strain was expanded and cultured in LB medium, and the plasmid was extracted to obtain a sufficient amount of recombinant plasmid.

[0046] The nucleotide sequence of the NEDD4L gene is shown in SEQ ID NO: 1; the amino acid sequence of the protein encoded by the NEDD4L gene (denoted as NEDD4L protein) is shown in SEQ ID NO: 2; the partial sequencing detection map of the recombinant plasmid gene is shown in Figure 1-1~1-3 shown.

[0047] It can be known from the NCBI database that the NEDD4L gene has 7 different transcripts. The nucleotide sequence of the NEDD4L gene of the present invention includes the sequence of nucleotides 601-3165 in the CDS region derived from E3 ubiquitin-protein ligase NEDD4-like isoform 2 (NM_001144964.1). For genes with multiple transcripts, the amino acid sequences encoded by different transcripts are different, resulting in differences in the structural domains of the proteins, which may cause the final proteins to exert different physiological effects.

[0048] The nucleotide sequence of the NEDD4L gene selected in the present invention is shown in SEQ ID NO: 1, and SEQ ID NO: 1 is specifically:

[0049] SEQ ID NO:2 is specifically: MATGLGEPVYGLSEDEGESRILRVKVVSGIDLAKKDIFGASDPYVKLSLYVADENRELALVQTKTIKKTLNPKWNEEFYFRVNPSNHRLLFEVFDENRLTRDDFLGQVDVPLSHLPTEDPTMERPYTFKDFLLRPRSHKSRVKGFLRLKMAYMPKNGGQDEENSDQRDDMEHGWEVVDSNDSASQHQEELPPPPLPPGWEEKVDNLGRTYYVNHNNRTTQWHRPSLMDVSSESDNNIRQINQEAAHRRFRSRRHISEDLEPEPSEGGDVPEPWETISEEVNIAGDSLGLALPPPPASPGSRTSPQELSEELSRRLQITPDSNGEQFSSLIQREPSSRLRSCSVTDAVAEQGHLPPPSAPAGRARSSTVTGGEEPTPSVAYVHTTPGLPSGWEERKDAKGRTYYVNHNNRTTTWTRPIMQLAEDGASGSATNSNNHLIEPQIRRPRSLSSPTVTLSAPLEGAKDSPVRRAVKDTLSNPQSPQPSPYNSPKPQHKVTQSFLPPGWEMRIAPNGRPFFIDHNTKTTTWEDPRLKFPVHMRSKTSLNPNDLGPLPPGWEERIHLDGRTFYIDHNSKITQWEDPRLQNPAITGPAVPYSREFKQKYDYFRKKLKKPADIPNRFEMKLHRNNIFEESYRRIMSVKRPDVLKARLWIEFESEKGLDYGGVAREWFFLLSKEMFNPYYGLFEYSATDNYTLQINPNSGLCNEDHLSYFTFIGRVAGLAVFHGKLLDGFFIRPFYKMMLGKQITLNDMESVDSEYYNSLKWILENDPTELDLMFCIDEENFGQTYQVDLKPNGSEIMVTNENKREYIDLVIQWRFVNRVQKQMNAFLEGFTELLPIDLIKIFDENELELLMCGLGDVDVNDWRQHSIYKNGYCPNHPVIQWFWKAVLLMDAEKRIRLLQFVTGTSRVPMNGFAELYGSNGPQLFTIEQWGSPEKLPRAHTCFNRLDLPPYETFEDLREKLLMAVENAQGFEGVD。

[0050] IV. Acquisition of NEDD4L gene overexpression vector The above recombinant plasmid was extracted with high purity and endotoxin-free, and then transfected into 293A cells. After 36 hours of transfection, the medium was replaced with ECM complete medium and cultured for 7-15 days. Fresh medium was added every 3 days or so, and then the cells and supernatant were collected and placed in a centrifuge tube. The cells were frozen and thawed three times, and centrifuged at 4000rpm for 10 minutes. The supernatant was taken as the primary stock solution of adenovirus liquid. After three consecutive generations of amplification, the virus was collected, purified and concentrated to obtain a high-titer adenovirus concentrate containing the NEDD4L gene, that is, the NEDD4L gene overexpression vector. Further, the virus titer was measured and calibrated in 293A cells. It was determined that the virus titer in the NEDD4L gene overexpression vector obtained by the present invention was 10 8 ~10 10 PFU / mL.

[0051] Example 2 This example investigates the effects of NEDD4L protein on the proliferation, apoptosis, cloning, migration, and invasion of vascular endothelial cells. The specific contents are as follows: 1. Virus infection of cells. Experimental design: NEDD4L overexpression group (i.e., the NEDD4L gene overexpression vector prepared in Example 1, with a virus titer of 1×10 10 PFU / mL), negative control group (not infected with virus), and normal control group (infected with ADV4 without NEDD4L gene). 6 ~5×10 6 Target cells / well [the target cells selected in this example are human intravenous endothelial cells (HUVEC cells) as an example] are placed in a 6-well culture plate, and the added culture medium is ECM culture medium with a volume of 2 mL. When the virus is infected, the cell confluence is 60%~80%. Remove the cell supernatant, add 2 mL of the required culture medium, and add three replicates per group. Prepare 3 sterile EP tubes, and take 3 μL of 1×10 10 Add PFU / ml of virus (previously taken out from -80℃, quickly thawed, and operated at 4℃) to the culture medium and mix well. Return the cells to the incubator and incubate at 37℃. Replace with fresh culture medium after 24 hours. Observe the cell status and fluorescence expression 72 hours after infection.

[0052] The cell morphology of vascular endothelial cells (human umbilical vein endothelial cells) infected with the NEDD4L gene overexpression vector is shown in the figure Figure 2 As shown. Figure 2 It can be seen that there is no significant difference in the cell state after infection, and NEDD4L-GFP protein is expressed in more than 90% of endothelial cells.

[0053] 2. Effects of NEDD4L gene overexpression vector on vascular endothelial cell mRNA Extract the total RNA of the infected cells in the first part of this example, and evaluate the infection efficiency by detecting the expression of the target gene by Real-time PCR. After the endothelial cells are digested with trypsin, collect the cell pellet by centrifugation, and fully resuspend the cell pellet with 20 μL PBS. Add 150 μL Solution R1 and vortex mix for 30 seconds to lyse the cells, put the EP tube into a 4℃ centrifuge, centrifuge at 13000 rpm for 30 seconds, and aspirate the supernatant into a new EP tube; add 500 μL Solution R2, mix evenly by turning upside down, and pour into the adsorption column. Centrifuge at 13000 rpm at 4℃ for 30 seconds, and discard the waste liquid; add 500 μL RNA Washing Buffer to the adsorption column, and centrifuge at 13000 rpm at 4℃ for 15 seconds. Repeat this step once; put the adsorption column back into the centrifuge, centrifuge at 13,000 rpm at 4°C for 1 min, and completely dry the residual ethanol; put the adsorption column core into a 1.5 mL Nuclease Free collection tube, add 20 μL Nuclease Free H2O to the adsorption column core, and leave it at room temperature for 1 min; centrifuge at 13,000 rpm at 4°C for 1 min. The eluate is the extracted RNA. After measuring the concentration and purity of the RNA, perform RNA reverse transcription reaction and fluorescent quantitative PCR for amplification according to the following system: 1. RNA thermal denaturation RNA thermal denaturation was performed according to the reaction system described in Table 7 below.

[0054] Table 7

[0055] The reaction conditions were 42°C for 2 min.

[0056] 2. RNA Reverse Transcription The reverse transcription reaction of RNA was carried out according to the reaction system described in Table 8 below.

[0057] Table 8

[0058] The reverse transcription reaction conditions of RNA were 37°C for 15 min and 85°C for 5 sec.

[0059] 3. Detection of NEDD4L gene expression by fluorescence quantitative PCR The primer sequences of NEDD4L and β-actin are shown in Table 9 below.

[0060] Table 9

[0061] The fluorescence quantitative PCR reaction system of this part is shown in Table 10 below.

[0062] Table 10

[0063] For fluorescence quantification, the reaction conditions were pre-deformation: 95°C for 30 sec; cycle reaction: 95°C for 10 sec, 60°C for 30 sec, 20 cycles. - ∆∆ Ct The relative expression of NEDD4L in endothelial cells was calculated.

[0064] After endothelial cells were induced by NEDD4L gene overexpression vector, the expression of mNRA was as follows Figure 3 shown. Figure 3 In the figure, Normal: normal cell group, Control: negative control group, OE-NEDD4L: NEDD4L overexpression group. Figure 3 It can be seen that compared with the normal group and the control group, the mRNA expression level of NEDD4L in the NEDD4L overexpression group was approximately upregulated by 22 times.

[0065] 3. Effect of NEDD4L gene overexpression vector on NEDD4L protein expression in vascular endothelial cells The total protein of the infected endothelial cells in part of this example was extracted, and the expression level of the NEDD4L gene was detected by Western blot. After the endothelial cells were digested with trypsin, the cell pellet was collected by centrifugation; 100 μL of cold lysis buffer (PMSF:RIPA 1:100) was added to the pellet; the cell suspension was vortexed for 30 seconds, once every five minutes, for a total of six times; centrifuged at 13,000 rpm at 4°C for 3 minutes, and the supernatant was taken into a new centrifuge tube; the protein concentration was determined by the BCA method; 25 μL of 5× loading buffer was added; the metal bath was 85°C for 5 minutes, and stored at -20°C. Take equal amounts of protein samples for SDS-polyacrylamide gel electrophoresis at a constant voltage of 120V for 90min; after electrophoresis, take out the gel for membrane transfer. Before using the PVDF membrane, soak it in methanol for 2min at a constant current of 320mA for 120min; after membrane transfer, take out the PVDF membrane and seal it in 5% skim milk powder at room temperature for 60min; take out the PVDF membrane and wash it six times in five minutes; perform primary and secondary antibody binding reactions (the primary antibody is NEDD4L rabbit anti-human monoclonal antibody, which can specifically bind to the target protein, and the secondary antibody is HRP-labeled goat anti-rabbit antibody, which can specifically bind to the primary antibody, and finally display the band under the action of ECL luminescent solution (containing peroxide reagent and luminol enhancer, etc.)); finally, scan the luminescent film and use the gel image processing system to analyze the molecular weight and net optical density value of the target band.

[0066] After endothelial cells were induced by NEDD4L gene overexpression vector, the expression of NEDD4L protein was as follows Figure 4 shown. Figure 4 In the figure, Normal: normal cell group, Control: negative control group, OE-NEDD4L: NEDD4L overexpression group.

[0067] In the present invention, the virus infection time is set to 72h. After the infection, the cell state and infection efficiency of the virus infection are observed by fluorescence. It is determined that the cell state is stable and the infection efficiency is more than 90%, and then the subsequent experiments are carried out. Since the adenovirus overexpression vector cannot be integrated into the genome to establish a stable cell line after transfection, the efficiency of cell infection is crucial to the subsequent results. Under the premise of ensuring the infection efficiency, the present invention is Figure 4 It can be seen that compared with the normal group and the control group, the expression level of NEDD4L protein in endothelial cells in the NEDD4L overexpression group was significantly increased, about 6.15 times.

[0068] IV. CCK8 assay to detect the effect of NEDD4L gene overexpression vector on endothelial cell proliferation Referring to the first part of this example, a NEDD4L overexpression group, a negative control group, and a normal control group were set. The logarithmic phase cells infected with adenovirus were plated in 96-well plates at 3000 cells / well, with 6 replicates per group. On the 0th day, 1st day, 2nd day, 3rd day, and 3 hours before the termination of culture, 10 μL of CCK8 reagent was added to each well. After 3 hours, the OD value was detected at a wavelength of 450 nm using an enzyme reader. The data were statistically analyzed for the cell proliferation inhibition rate and a curve graph was drawn.

[0069] After endothelial cells were induced by NEDD4L gene overexpression vector, the results of cell proliferation assay were as follows Figure 5 shown. Figure 5 In the figure, Normal: normal cell group, Control: negative control group, OE-NEDD4L: NEDD4L overexpression group.

[0070] Depend on Figure 5 It can be seen that compared with the normal group and the control group, the growth of endothelial cells in the NEDD4L overexpression group was significantly inhibited, and the cell inhibition rate reached 80%.

[0071] 5. Effect of NEDD4L gene overexpression vector on endothelial cell apoptosis Referring to the first part of this example, a NEDD4L overexpression group, a negative control group, and a normal control group were set up. 1×10 cells in the logarithmic phase infected with adenovirus were 5 5×10 cells / well were plated on a 6-well plate. After 24 hours, the cells were collected in a 5 ml centrifuge tube. Three replicate wells were set up for each group. The condition was that the number of cells was ≥5×10 6 / treatment. Centrifuge, precool at 4℃, wash once with PBS and centrifuge, wash the cell pellet once with 1×binding buffer, centrifuge, collect the cells and add 200 μL 1×binding buffer to resuspend the cells. Add 10 μL Annexin V-APC for staining and keep at room temperature away from light for 10-15 minutes. FACS flow cytometer detection, data analysis, and drawing.

[0072] After endothelial cells were induced by NEDD4L gene overexpression vector, the results of apoptosis test were as follows Figure 6 shown. Figure 6 The apoptosis rates of the negative control group, normal cell group and NEDD4L overexpression group were 5.517%±1.231%, 5.723%±0.586% and 31.760%±1.603%, respectively. It can be seen that the apoptosis rate of endothelial cells in the NEDD4L overexpression group was significantly higher than that in the negative control group and the normal cell group, and the apoptosis rate in the NEDD4L overexpression group was about 6 times that of the negative control group and the normal cell group.

[0073] 6. Clone formation assay to detect the effect of NEDD4L gene overexpression vector on endothelial cell proliferation Referring to the first part of this embodiment, a NEDD4L overexpression group, a negative control group, and a normal control group were set up. After the virus infected the cells, the logarithmic phase cells were plated on a 6-well plate, 400-1000 cells were added to each well, and 3 replicates were set up in each well. The cells continued to be cultured until 10d-14d or the number of cells in most single clones was greater than 50. Before the experiment was terminated, the clones were photographed under a fluorescence microscope and washed once with PBS. 1mL of 4% paraformaldehyde was added to each well, and the cells were fixed for 30min-60min and washed once with PBS. 500 μL of GIEMSA dye was added to each well and the cells were stained for 20min. The cells were washed with ddH2O, dried, photographed with a digital camera, and cloning technology was used to draw charts.

[0074] After endothelial cells were induced by NEDD4L gene overexpression vector, the results of cell clone assay were as follows: Figure 7 shown. Figure 7 In the negative control group, the number of cell clones in the normal cell group and the NEDD4L overexpression group were 66.33±11.37, 72.33±7.506 and 21.33±3.22, respectively. It can be seen that the number of endothelial cell clones in the NEDD4L overexpression group was significantly lower than that in the negative control group and the normal cell group.

[0075] VII. Transwell migration assay to detect the effect of NEDD4L gene overexpression vector on endothelial cell invasion ability Referring to the first part of this example, a NEDD4L overexpression group, a negative control group, and a normal control group were set. After culturing the cells in serum-free medium for 24 h, the cells were collected and the concentration was adjusted to 1×10 6 Add 200 μL of ECM complete medium to each Boyden chamber (pore size 8 μm) and mix well. 5 cells, add 500 μL of complete medium containing serum to the lower chamber of the Boyden chamber and culture for 8 h; remove the medium in the lower chamber, fix with sodium chloride-alcohol for 10 min, stain with hematoxylin for 10 min, discard the medium in the upper chamber, wipe off the cells on the upper chamber with a cotton swab, count and take pictures under an inverted microscope, select 8 fields of view for counting under a 200× light microscope, and take the average value.

[0076] After endothelial cells were induced by NEDD4L gene overexpression vector, the results of cell Transwell assay were as follows: Figure 8 As shown in the Transwell migration experiment, Normal: normal cell group, Control: negative control group, OE-NEDD4L: NEDD4L overexpression group. Figure 8The results showed that the number of endothelial cell transmembrane migration cells in the NEDD4L overexpression group was significantly reduced, and the number of invasive cells in the transmembrane migration cells was reduced by more than 3 times compared with the negative control group and the normal cell group.

[0077] 8. Cell scratch assay to detect the effect of NEDD4L gene overexpression vector on endothelial cell migration ability Referring to the first part of this example, a NEDD4L overexpression group, a negative control group, and a normal control group were set. Human umbilical vein endothelial cells infected with adenovirus overexpressing the NEDD4L gene were inoculated in a 6-well plate, 3 replicates / group, and cultured overnight; when the cell confluence reached 90%, a straight line was drawn in the center using a 100ul pipette tip; PBS was used to rinse off the detached cells, and after 24 hours, photographed and counted under an inverted microscope.

[0078] After vascular endothelial cells were induced by NEDD4L gene overexpression vector, the results of cell scratch test were as follows Fig. 9 shown. Fig. 9 In the figure, Normal: normal cell group, Control: negative control group, OE-NEDD4L: NEDD4L overexpression group. Fig. 9 The results showed that the migration area of ​​the NEDD4L overexpression group was significantly lower than that of the negative control group and the normal cell group, indicating that the NEDD4L overexpression group can effectively inhibit the migration ability of endothelial cells.

[0079] IX. Immunoblotting and mRFP-GFP-LC3 virus infection assay to detect the effect of NEDD4L gene overexpression vector on endothelial cell autophagy Referring to the first part of this example, a NEDD4L overexpression group, a negative control group, and a normal control group were set up. Human umbilical vein endothelial cells infected with the NEDD4L gene overexpression vector were inoculated in a 6-well plate, 3 replicates / group, and cell pellets were collected 24 hours later to extract proteins for autophagy-related protein immunoblotting detection; when the cell confluence reached 90%, mRFP-GFP-LC3 virus was used, and 24 hours later, photos and statistics were taken under an inverted microscope.

[0080] After endothelial cells were induced by NEDD4L gene overexpression vector, the results of autophagy-related protein immunoblotting were as follows: Fig.10 As shown. After endothelial cells were induced by NEDD4L gene overexpression vector and infected with mRFP-GFP-LC3 virus, the cell autophagy was shown in Fig.11 As shown; among them, Fig.11 a is a microscope image after infection with mRFP-GFP-LC3 virus. Fig.11 b is a quantitative graph showing the percentage of autophagy positive spots / cells. Fig.10or Fig.11 In the figure, Normal: normal cell group, Control: negative control group, OE-NEDD4L: NEDD4L overexpression group.

[0081] Depend on Fig.10 In the NEDD4L overexpression group, the expression of autophagy-related proteins ATG5 and Beclin1 was effectively induced. Fig.11 It can be seen that after the mRFP-GFP-LC3 virus infection experiment, the autophagic lysosomes represented by red spots and the autophagosomes represented by yellow spots in the NEDD4L overexpression group were significantly increased compared with the negative control group and the normal cell group, and obvious autophagy and autophagic flow appeared.

[0082] In the experimental scope of the present invention, it was found that inducing the expression of NEDD4L gene can induce the occurrence of endothelial cell autophagy. Since cell autophagy is the basis for the degradation and recycling of cell components and the main form of cell homeostasis, the occurrence of cell autophagy is affected by multiple factors such as physiological environment and conditions. NEDD4L gene may play different roles in different physiological environments.

[0083] Example 3 Given that the NEDD4L gene overexpression vector can inhibit the proliferation, migration and invasion of vascular endothelial cells and induce cell autophagy, it can be developed into a drug for the treatment of vascular endothelial cell proliferative diseases.

[0084] The prepared drug for treating vascular endothelial cell proliferative diseases may include a certain dose of a NEDD4L gene overexpression vector or an expression product containing NEDD4L gene overexpression, and may also include pharmaceutically acceptable excipients. The dosage form of the prepared drug for treating vascular endothelial cell proliferative diseases is not particularly limited, and may be in various material forms such as solid, liquid, gel, semi-fluid, aerosol, etc.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of NEDD4L gene in the preparation of drugs for the treatment of vascular endothelial cell proliferation-related diseases.

2. The use according to claim 1, characterized in that The nucleotide sequence of the NEDD4L gene is shown in SEQ ID NO:

1.

3. The use according to claim 2, characterized in that The amino acid sequence of the protein encoded by the NEDD4L gene is shown in SEQ ID NO:

2.

4. The use according to any one of claims 1 to 3, characterized in that: The diseases related to vascular endothelial cell proliferation include atherosclerosis, hypertension, diabetes or hemangioma.

5. A drug for treating vascular endothelial cell proliferation-related diseases, characterized in that: The medicine contains a NEDD4L gene overexpression vector.

6. The method for preparing a drug for treating vascular endothelial cell proliferation-related diseases according to claim 5, characterized in that: The preparation method comprises the following steps: Step 1, cloning the NEDD4L gene into an adenovirus-associated virus to obtain an adenovirus-associated virus containing the NEDD4L gene; Step 2, transforming the adenovirus-associated virus containing the NEDD4L gene into Escherichia coli to obtain a recombinant plasmid; Step 3: transfecting the recombinant plasmid into human endothelial cells, expanding the culture, purifying and concentrating to obtain the NEDD4L gene overexpression vector.

7. The method for preparing a drug for treating vascular endothelial cell proliferation-related diseases according to claim 6, characterized in that: In step 1, the adeno-associated virus includes at least one of adenovirus, lentivirus or adeno-associated virus.

8. The method for preparing a drug for treating vascular endothelial cell proliferation-related diseases according to claim 6, characterized in that: In step 3, the human endothelial cells include human umbilical vein endothelial cells, human embryonic kidney cells or human smooth muscle cells; and / or In step three, before the expanded culture, the recombinant plasmid is also subjected to high-purity endotoxin-free extraction.

9. The method for preparing a drug for treating vascular endothelial cell proliferation-related diseases according to claim 6, characterized in that: In step three, the virus titer in the NEDD4L gene overexpression vector is measured in the human endothelial cells.

10. Use of the drug for treating vascular endothelial cell proliferation-related diseases according to claim 5, characterized in that: The drug is injected into the body for at least 72 hours.

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