CN119303113A

CN119303113A - Application of NEDD4L gene in the preparation of drugs for the treatment of vascular endothelial cell proliferation-related diseases

Info

Publication number: CN119303113A
Application number: CN202411227160.XA
Authority: CN
Inventors: 秦岩; 刘雨萌; 赵明慧; 史建红; 李靖华; 王馨悦; 王婷婷; 吴飞翔; 张岚; 郑立双
Original assignee: AFFILIATED HOSPITAL OF HEBEI UNIVERSITY
Current assignee: AFFILIATED HOSPITAL OF HEBEI UNIVERSITY
Priority date: 2024-09-03
Filing date: 2024-09-03
Publication date: 2025-01-14

Abstract

The present invention relates to the field of biomedicine technology, and discloses the use of NEDD4L gene in the preparation of drugs for treating diseases related to vascular endothelial cell proliferation. The present invention provides the use of NEDD4L gene in the preparation of drugs for treating diseases related to vascular endothelial cell proliferation, wherein the nucleotide sequence of the NEDD4L gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the NEDD4L gene is shown in SEQ ID NO: 2. It is first discovered that overexpression of the NEDD4L gene can inhibit the proliferation of vascular endothelial cells, downregulate the invasive ability of vascular endothelial cells, reduce the mobility of vascular endothelial cells, and induce autophagy of endothelial cells. Based on this, the present invention uses the NEDD4L gene in the preparation of drugs for treating diseases related to vascular endothelial cell proliferation, in order to provide a new strategy for the treatment of vascular endothelial cell proliferative diseases.

Description

Application of NEDD4L gene in preparing medicament for treating vascular endothelial cell proliferation related diseases

Technical Field

The invention relates to the technical field of biological medicines, and particularly discloses application of NEDD4L genes in preparation of medicaments for treating vascular endothelial cell proliferation related diseases.

Background

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 of maintaining vascular tone, angiogenesis, hemostasis, etc., and are also important daemons for vascular health, which are critical to maintaining vascular health and function. Vascular endothelial cell dysfunction may be manifested as abnormal proliferation of endothelial cells, endothelial-dependent vasodilation disorders, oxidative stress enhancement, chronic inflammation, leukocyte adhesion, increased vascular permeability, endothelial cell aging, metabolic abnormalities, and endothelial cell interstitial transformation, etc., which are associated with a variety of vascular diseases such as atherosclerosis, hypertension, diabetes, and infectious diseases, etc.

Abnormal proliferation of vascular endothelial cells is a process which is controlled by multiple factors and is mainly influenced by local microenvironment consisting of vascular endothelial cell heterogeneity, blood flow shearing force, active substances secreted by endothelial cells, vascular endothelial growth factors and the like. In the development process of vascular endothelial cell proliferative diseases, a key link is that vascular endothelial cell functions are changed, so that pro-proliferation genes are activated in a transcribed manner, pro-apoptosis genes are silenced, and activation of proliferation signal paths such as akt and initiation of cell migration processes are finally caused, so that abnormal proliferation and migration of vascular endothelial cells are closely related to vascular endothelial cell proliferative diseases such as atherosclerosis, hypertension and diabetes, and how to effectively inhibit the abnormal proliferation and migration of vascular endothelial has become an important strategy for treating the diseases.

At present, the conventional methods for treating the vascular endothelial cell proliferative diseases mainly comprise drug treatment, operation treatment, interventional treatment, laser treatment and the like, and the drug treatment mainly focuses on controlling symptoms of the diseases and slowing down the disease progression. For example, antihypertensive drugs are used to control hypertension, hypoglycemic drugs are used to control diabetes, etc. However, the drug treatment cannot fundamentally cure the disease, and side effects of the drug can be accompanied. Although the operation treatment can directly cut off pathological tissues, the operation treatment has the defects of more bleeding, poor appearance effect, easy recurrence and the like. Interventional therapy and laser therapy present a risk of possible damage to normal tissue, so finding new molecules or proteins that inhibit vascular endothelial cell proliferation and migration is a current clinical challenge.

Disclosure of Invention

Aiming at the defects of the conventional method for treating the vascular endothelial cell proliferative diseases in the prior art, the invention aims to provide a gene medicament with the effects of inhibiting the proliferation, migration and invasion of vascular endothelial cells and inducing autophagy, and further provides the application of NEDD4L genes in preparing medicaments for treating the vascular endothelial cell proliferative related diseases so as to open up a new treatment strategy for the vascular endothelial cell proliferative diseases.

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

In a first aspect, the present invention provides the use of the NEDD4L gene in the manufacture of a medicament for the treatment of a vascular endothelial cell proliferation-related disease.

Through extensive and intensive research, the invention discovers that the NEDD4L gene can be used as a target spot for treating vascular endothelial cell proliferation diseases for the first time, and the NEDD4L gene can inhibit proliferation, migration and invasion of vascular endothelial cells and induce autophagy after being over-expressed, so that the NEDD4L gene can be used for treating related diseases caused by abnormal proliferation of vascular endothelial cells. The NEDD4L gene is used for preparing medicaments for treating vascular endothelial cell proliferation related diseases, which can overcome the defects of large side effect, excessive surgical bleeding and the like in the prior related medicaments, provides a new treatment idea on the basis of the prior conventional treatment and opens up a new direction for treating the vascular endothelial cell proliferation diseases.

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

The NEDD4L gene in NCBI database has 7 different transcripts, and for genes with multiple transcripts, the amino acid sequences encoded by different transcripts are different, resulting in different protein domains, which may cause the final protein to exert different physiological effects. The invention creatively discovers that the sequence comprising nucleotide numbers 601-3165 of CDS region 601-3165 derived from E3 ubiquitin-protein LIGASE NEDD-like isocord 2 (NM_ 001144964.1) has the effects of inhibiting proliferation, migration and invasion of vascular endothelial cells and inducing autophagy within the experimental scope of the invention.

As a second limitation to the above application, the NEDD4L gene encodes a protein having the amino acid sequence shown in SEQ ID NO. 2.

As a third limitation on the above application, the vascular endothelial cell proliferation-related disease includes atherosclerosis, hypertension, diabetes, or hemangioma.

In a second aspect, the present invention provides a medicament for treating vascular endothelial cell proliferation-related diseases, wherein the medicament for treating vascular endothelial cell proliferation-related diseases comprises a NEDD4L gene overexpression vector.

In a third aspect, the present invention provides a method for preparing the above-mentioned medicament for treating vascular endothelial cell proliferation-related diseases, the method comprising the steps of:

Cloning NEDD4L genes to adenovirus-related viruses to obtain adenovirus-related viruses containing the NEDD4L genes;

Step two, converting the adenovirus related virus containing the NEDD4L gene into escherichia coli to obtain a recombinant plasmid;

and thirdly, transfecting the recombinant plasmid into human endothelial cells, performing amplification culture, purifying and concentrating to obtain adenovirus concentrated solution containing the NEDD4L gene, namely the NEDD4L gene overexpression vector.

As a first limitation on the above preparation method, in the first step, the adenovirus-associated virus includes at least one of adenovirus, lentivirus, or adeno-associated virus;

wherein, the adenovirus comprises ADV4, the slow virus comprises LV5, and the adeno-associated virus comprises AAV-GP-1, AAV-GP-12N, and the like. The invention will be described with respect to ADV 4.

As a second limitation of the above preparation method, in the third step, the human endothelial cells include human umbilical vein endothelial cells, human embryonic kidney cells, human smooth muscle cells, or the like;

And step three, before the amplification culture, the method further comprises the step of extracting the recombinant plasmid with high purity and without endotoxin.

As a third limitation on the above preparation method, the viral titer of the NEDD4L gene-containing adenovirus concentrate is measured in the human endothelial cells.

In a fourth aspect, the invention provides the use of a medicament for treating a vascular endothelial cell proliferation-related disorder as described above, the medicament being injected into the body for at least 72 hours, further wherein the injection comprises microinjection.

The invention provides application of NEDD4L gene in preparing medicaments for treating vascular endothelial cell proliferation related diseases, and discovers that over-expression of the NEDD4L gene can inhibit proliferation of vascular endothelial cells, reduce invasion capacity of the vascular endothelial cells, reduce mobility of the vascular endothelial cells and induce autophagy of the endothelial cells for the first time. Therefore, the medicine for treating vascular endothelial cell proliferation related diseases provided by the invention plays a therapeutic role in inhibiting proliferation, invasion capacity and migration of vascular endothelial cells and inducing autophagy of endothelial cells.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIGS. 1-1 to 1-3 are partial sequencing detection maps of recombinant plasmid genes in example 1 of the present invention;

FIG. 2 is a diagram showing the morphology of cells after infection of vascular endothelial cells with the NEDD4L gene overexpression vector according to example 2 of the present invention;

FIG. 3 shows the expression of mNRA of vascular endothelial cells induced by NEDD4L gene overexpression vector in example 2 according to the present invention;

FIG. 4 shows the expression of NEDD4L protein from vascular endothelial cells induced by NEDD4L gene overexpression vector according to example 2 of the present invention;

FIG. 5 shows the results of experimental detection of proliferation of vascular endothelial cells induced by NEDD4L gene overexpression vector in example 2 according to the present invention;

FIG. 6 shows the results of apoptosis test after the vascular endothelial cells of example 2 of the present invention are induced by NEDD4L gene overexpression vector;

FIG. 7 shows the results of the test of the cell cloning experiments of the vascular endothelial cells of example 2 of the present invention after induction by NEDD4L gene overexpression vector;

FIG. 8 shows the results of a cell Transwell experiment performed on vascular endothelial cells induced by NEDD4L gene overexpression vector according to example 2 of the present invention;

FIG. 9 shows the results of the cell scratch test of vascular endothelial cells induced by NEDD4L gene overexpression vector in example 2 according to the present invention;

FIG. 10 shows the result of immunoblotting of vascular endothelial cells induced by NEDD4L gene overexpression vector in example 2 according to the present invention;

FIG. 11 is a graph showing the autophagy of vascular endothelial cells infected with mRFP-GFP-LC3 virus induced by NEDD4L gene overexpression vector in example 2 according to the present invention, wherein FIG. 11a is a microscopic image of the infected mRFP-GFP-LC3 virus, and FIG. 11b is a graph showing the autophagy positive spot/cell number ratio.

Detailed Description

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques of molecular biology, cell culture, recombinant DNA technology, and related art conventional techniques that are conventional in the art.

In order to better illustrate the embodiments of the present invention, the following is further illustrated by examples.

Example 1

The embodiment provides a preparation method of NEDD4L gene overexpression vector, which comprises the following specific steps:

1. obtaining the Gene fragment of interest

Oligo design, NEDD4L gene upstream and downstream primers plus EcoRI and BamHI and protective bases for subcloning of vector, oligo sequences are shown in Table 1 below.

TABLE 1

The primers were synthesized by the company, inc. of Ji Ma gene, suzhou, according to the sequences shown in Table 1.

2. Dissolving the above oligo to 50. Mu.M;

3. the PCR was performed using the synthesized primers and NEDD4L gene as a template, and the PCR system was as shown in Table 2 below.

TABLE 2

In the PCR, the cycle conditions are shown in Table 3 below.

TABLE 3 Table 3

After the PCR reaction is completed, the target gene fragment, namely the human NEDD4L gene (herein referred to as NEDD4L gene) is obtained by Agarose electrophoresis and gel cutting to recover the gene fragment.

2. NEDD4L Gene clone into the vector sheep adenovirus type 4 (ADV 4)

1. The NEDD4L gene fragment obtained above was digested with EcoRI and BamHI, and digested at 37℃for 2 hours, and the digestion system was as shown in Table 4 below.

TABLE 4 Table 4

Wherein, the concentration of NEDD4L gene in Table 4 was 50. Mu.M.

2. Vector ADV4 was digested with EcoRI and BamHI, at 37 ℃ for 2 hours, and the digestion system is shown in table 5 below.

TABLE 5

Wherein the concentration of the ADV4 vector in Table 5 is 50. Mu.M.

3. Electrophoresis, and recovery of the NEDD4L gene and the vector ADV4 in the enzyme-sectioned fragment using a DNA gel recovery kit.

4. Ligation was performed for 2 hours at 22℃using the NEDD4L gene fragment obtained by ligation with T4 DNA LIGASE to obtain a ligation product, and the ligation system is shown in Table 6 below.

TABLE 6

5. Preparing competent cells of the escherichia coli by referring to page 55 of the second edition of the molecular cloning experiment guide;

6. Adding 10 mu L of the connection product into a centrifuge tube filled with competent cells of escherichia coli, gently mixing the mixture, placing the mixture in ice for 30 minutes, placing the centrifuge tube on a test tube rack placed in a water bath kettle preheated to 42 ℃, and placing the centrifuge tube for 90 seconds without shaking the centrifuge tube. The centrifuge tube was quickly transferred to an ice bath and the cells were allowed to cool for 3 minutes. 800. Mu.L of LB medium (without antibiotics) was added to each of the tubes, and the tubes were then transferred to a shaker at 37℃and 250rpm for 45 minutes to resuscitate the bacteria.

7. 200. Mu.L of the cultured cells were uniformly spread on LB plates containing 50. Mu.g/ML AMPICILLIN. After the liquid on the plate was absorbed, the plate was placed in a 37℃incubator and incubated for 16 hours. Clone colonies were picked from the plates, plasmids were minidrawn and identified, and positive clones were picked.

8. 4 Individual, full colonies were picked from the cultured plates, placed in tubes containing 5ml (50. Mu.g/ML AMPICILLIN) LB medium, and placed in a shaker for 16 hours at 37℃and 250 rpm.

9. And extracting plasmids from the cultured bacterial liquid by using a plasmid small extraction kit (Tiangen biochemistry, DP 104-02) to obtain recombinant plasmids.

3. Sequencing verification and obtaining of recombinant plasmid

Taking 200 mu L of bacterial liquid corresponding to positive clone, carrying out sequencing, comparing a sequencing result with a target gene sequence, checking the sequencing result, and obtaining a corresponding positive clone strain as a recombinant strain;

The recombinant strain is amplified and cultured in LB culture medium, and plasmid is extracted to obtain sufficient recombinant plasmid.

The nucleotide sequence of the NEDD4L gene is shown as SEQ ID NO. 1, the amino acid sequence of the protein encoded by the NEDD4L gene (marked as NEDD4L protein) is shown as SEQ ID NO. 2, and the partial sequencing detection map of the recombinant plasmid gene is shown as figures 1-3.

As can be seen from NCBI database, NEDD4L gene has 7 different transcripts, the nucleotide sequence of NEDD4L gene of the invention comprises the sequence of CDS region 601-3165 nucleotide from E3 ubiquitin-protein LIGASE NEDD-like isoferm 2 (NM_ 001144964.1), and for the genes with multiple transcripts, the amino acid sequences encoded by different transcripts are different, so that the structural domain difference of protein can cause the final protein to exert different physiological effects.

The nucleotide sequence of the NEDD4L gene selected in the invention is shown as SEQ ID NO. 1, and the SEQ ID NO. 1 specifically comprises the following components:

atggcgaccgggctcggggagccggtctatggactttccgaagacgagggagagtcccgtattctcagagtaaaagttgtttctggaattgatctcgccaaaaaggacatctttggagccagtgatccgtatgtgaaactttcattgtacgtagcggatgagaatagagaacttgctttggtccagacaaaaacaattaaaaagacactgaacccaaaatggaatgaagaattttatttcagggtaaacccatctaatcacagactcctatttgaagtatttgacgaaaatagactgacacgagacgacttcctgggccaggtggacgtgccccttagtcaccttccgacagaagatccaaccatggagcgaccctatacatttaaggactttctcctcagaccaagaagtcataagtctcgagttaagggatttttgcgattgaaaatggcctatatgccaaaaaatggaggtcaagatgaagaaaacagtgaccagagggatgacatggagcatggatgggaagttgttgactcaaatgactcggcttctcagcaccaagaggaacttcctcctcctcctctgcctcccgggtgggaagaaaaagtggacaatttaggccgaacttactatgtcaaccacaacaaccggaccactcagtggcacagaccaagcctgatggacgtgtcctcggagtcggacaataacatcagacagatcaaccaggaggcagcacaccggcgcttccgctcccgcaggcacatcagcgaagacttggagcccgagccctcggagggcggggatgtccccgagccttgggagaccatttcagaggaagtgaatatcgctggagactctctcggtctggctctgcccccaccaccggcctccccaggatctcggaccagccctcaggagctgtcagaggaactaagcagaaggcttcagatcactccagactccaatggggaacagttcagctctttgattcaaagagaaccctcctcaaggttgaggtcatgcagtgtcaccgacgcagttgcagaacagggccatctaccaccgcccagtgccccagctgggagagcgcgttcatcaactgtcacgggtggtgaggaaccaacgccatcagtggcctatgtacataccacgccgggtctgccttcaggctgggaagaaagaaaagatgctaaggggcgcacatactatgtcaatcataacaatcgaaccacaacttggactcgacctatcatgcagcttgcagaagatggtgcgtccggatcagccacaaacagtaacaaccatctaatcgagcctcagatccgccggcctcgtagcctcagctcgccaacagtaactttatctgccccgctggagggtgccaaggactcacccgtacgtcgggctgtgaaagacaccctttccaacccacagtccccacagccatcaccttacaactcccccaaaccacaacacaaagtcacacagagcttcttgccacccggctgggaaatgaggatagcgccaaacggccggcccttcttcattgatcataacacaaagactacaacctgggaagatccacgtttgaaatttccagtacatatgcggtcaaagacatctttaaaccccaatgaccttggcccccttcctcctggctgggaagaaagaattcacttggatggccgaacgttttatattgatcataatagcaaaattactcagtgggaagacccaagactgcagaacccagctattactggtccggctgtcccttactccagagaatttaagcagaaatatgactacttcaggaagaaattaaagaaacctgctgatatccccaataggtttgaaatgaaacttcacagaaataacatatttgaagagtcctatcggagaattatgtccgtgaaaagaccagatgtcctaaaagctagactgtggattgagtttgaatcagagaaaggtcttgactatgggggtgtggccagagaatggttcttcttactgtccaaagagatgttcaacccctactacggcctctttgagtactctgccacggacaactacacccttcagatcaaccctaattcaggcctctgtaatgaggatcatttgtcctacttcacttttattggaagagttgctggtctggccgtatttcatgggaagctcttagatggtttcttcattagaccattttacaagatgatgttgggaaagcagataaccctgaatgacatggaatctgtggatagtgaatattacaactctttgaaatggatcctggagaatgaccctactgagctggacctcatgttctgcatagacgaagaaaactttggacagacatatcaagtggatttgaagcccaatgggtcagaaataatggtcacaaatgaaaacaaaagggaatatatcgacttagtcatccagtggagatttgtgaacagggtccagaagcagatgaacgccttcttggagggattcacagaactacttcctattgatttgattaaaatttttgatgaaaatgagctggagttgctcatgtgcggcctcggtgatgtggatgtgaatgactggagacagcattctatttacaagaacggctactgcccaaaccaccccgtcattcagtggttctggaaggctgtgctactcatggacgccgaaaagcgtatccggttactgcagtttgtcacagggacatcgcgagtacctatgaatggatttgccgaactttatggttccaatggtcctcagctgtttacaatagagcaatggggcagtcctgagaaactgcccagagctcacacatgctttaatcgccttgacttacctccatatgaaacctttgaagatttacgagagaaacttctcatggccgtggaaaatgctcaaggatttgaaggggtggattaa.

SEQ ID NO. 2 specifically shows:

MATGLGEPVYGLSEDEGESRILRVKVVSGIDLAKKDIFGASDPYVKLSLYVADENRELALVQTKTIKKTLNPKWNEEFYFRVNPSNHRLLFEVFDENRLTRDDFLGQVDVPLSHLPTEDPTMERPYTFKDFLLRPRSHKSRVKGFLRLKMAYMPKNGGQDEENSDQRDDMEHGWEVVDSNDSASQHQEELPPPPLPPGWEEKVDNLGRTYYVNHNNRTTQWHRPSLMDVSSESDNNIRQINQEAAHRRFRSRRHISEDLEPEPSEGGDVPEPWETISEEVNIAGDSLGLALPPPPASPGSRTSPQELSEELSRRLQITPDSNGEQFSSLIQREPSSRLRSCSVTDAVAEQGHLPPPSAPAGRARSSTVTGGEEPTPSVAYVHTTPGLPSGWEERKDAKGRTYYVNHNNRTTTWTRPIMQLAEDGASGSATNSNNHLIEPQIRRPRSLSSPTVTLSAPLEGAKDSPVRRAVKDTLSNPQSPQPSPYNSPKPQHKVTQSFLPPGWEMRIAPNGRPFFIDHNTKTTTWEDPRLKFPVHMRSKTSLNPNDLGPLPPGWEERIHLDGRTFYIDHNSKITQWEDPRLQNPAITGPAVPYSREFKQKYDYFRKKLKKPADIPNRFEMKLHRNNIFEESYRRIMSVKRPDVLKARLWIEFESEKGLDYGGVAREWFFLLSKEMFNPYYGLFEYSATDNYTLQINPNSGLCNEDHLSYFTFIGRVAGLAVFHGKLLDGFFIRPFYKMMLGKQITLNDMESVDSEYYNSLKWILENDPTELDLMFCIDEENFGQTYQVDLKPNGSEIMVTNENKREYIDLVIQWRFVNRVQKQMNAFLEGFTELLPIDLIKIFDENELELLMCGLGDVDVNDWRQHSIYKNGYCPNHPVIQWFWKAVLLMDAEKRIRLLQFVTGTSRVPMNGFAELYGSNGPQLFTIEQWGSPEKLPRAHTCFNRLDLPPYETFEDLREKLLMAVENAQGFEGVD.

4. Obtaining NEDD4L gene over-expression vector

The recombinant plasmid is subjected to high-purity endotoxin-free extraction and then transfected into 293A cells, the cells are transfected for 6 hours and then replaced by ECM complete culture medium, the culture is carried out for 7-15 days, fresh culture medium is supplemented every 3 days, then the cells and supernatant are collected and placed into a centrifuge tube, freeze thawing is carried out for three times, centrifugation is carried out at 4000rpm for 10 minutes, and the supernatant is taken as adenovirus liquid primary stock solution. And (3) amplifying for three times continuously, collecting viruses, purifying and concentrating the viruses to obtain adenovirus concentrated solution containing NEDD4L genes with high titer, and obtaining the NEDD4L gene overexpression vector. Further, it was assayed and virus titers were calibrated in 293A cells. According to the determination, the virus titer in the NEDD4L gene overexpression vector obtained by the invention is 10 ⁸~10¹⁰ PFU/mL.

Example 2

This example examined the effect of NEDD4L protein on vascular endothelial cell proliferation, apoptosis, cloning, migration, and invasiveness. The specific contents are as follows:

1. The virus infects the cells. Experiment design NEDD4L overexpressing group (i.e., NEDD4L gene overexpressing vector prepared in example 1, virus titer was 1X 10 ¹⁰ PFU/mL), negative control group (without virus infection), normal control group (infected with ADV4 without NEDD4L gene). 3X 10 ⁶~5×10⁶ target cells/well (the target cells selected in this example are exemplified by human vena cava endothelial cells (HUVEC cells)) were inoculated on the day before the test in 6-well culture plates, and the added medium was ECM medium with a volume of 2mL. The fusion degree of cells during virus infection is 60% -80%. The cell supernatant was aspirated and 2mL of the desired medium was added, three wells per group. 3 sterile EP tubes were prepared, 3. Mu.L of 1X 10 ¹⁰ PFU/ml virus (pre-removed from-80 ℃, thawed rapidly, 4℃cold-run) was added to the broth and mixed. Cells were returned to the incubator for incubation at 37 ℃. Until 24 hours later, the medium was replaced with fresh medium. After 72 hours of infection, the cell status and fluorescent expression were observed.

The cell morphology of NEDD4L gene overexpression vector infected vascular endothelial cells (human umbilical vein endothelial cells) is shown in FIG. 2. As can be seen from FIG. 2, there was no significant difference in cell status after infection, and NEDD4L-GFP protein was expressed in more than 90% of endothelial cells.

2. Influence of NEDD4L Gene overexpression vector on vascular endothelial cell mRNA

Total RNA of the cells after infection in the first part of this example was extracted, and the infection efficiency was evaluated by detecting the expression of the target gene by Real-time PCR. After digestion of endothelial cells with pancreatin, the cell pellet was collected by centrifugation and resuspended sufficiently with 20 μl PBS. Adding 150 μl Solution R1, vortex mixing for 30s to lyse cells, placing the EP tube in a 4 deg.C centrifuge, centrifuging 13000 rpm s for 30s, sucking the supernatant into a new EP tube, adding 500 μl Solution R2, mixing upside down, and pouring into an adsorption column. Centrifuge 13000 rpm at 4℃for 30s, pouring out the waste liquid, adding 500. Mu. L RNA WASHING Buffer to the adsorption column, and centrifuging 13000 at rpm at 4℃for 15s. Repeating the steps once, putting the adsorption column back into a centrifuge again, centrifuging for 1min at the temperature of 4 ℃ with a centrifuge 13000 rpm, thoroughly drying residual ethanol, putting the adsorption column core into a 1.5 mLNuclease Free collecting pipe, adding 20 mu L Nuclease Free H ₂ O into the adsorption column core, standing for 1min at room temperature, centrifuging for 1min at the speed of 13,000 rpm with a centrifuge at the temperature of 4 ℃, obtaining eluent, measuring the concentration and purity of RNA, and then carrying out RNA reverse transcription reaction and fluorescent quantitative PCR for amplification according to the following system:

RNA thermal denaturation

RNA thermal denaturation was carried out according to the reaction system described in Table 7 below.

TABLE 7

The reaction condition is 42 ℃ for 2min.

Reverse transcription of RNA

Reverse transcription of RNA was performed according to the reaction system shown in Table 8 below.

TABLE 8

The reverse transcription reaction conditions of RNA were 37℃for 15min and 85℃for 5sec.

3. Fluorescent quantitative PCR reaction for detecting expression of NEDD4L gene

Primer sequences for NEDD4L and beta-actin are shown in Table 9 below.

TABLE 9

The fluorescent quantitative PCR reaction system of this part is shown in Table 10 below.

Table 10

For fluorescence quantification, the reaction conditions were pre-deformation of 95℃30 sec, and cycling reactions of 95℃10 sec,60℃30 sec,20 cycles. The relative expression of NEDD4L in endothelial cells was calculated according to 2 ^{- ∆∆ Ct}.

After the vascular endothelial cells are induced by NEDD4L gene overexpression vectors, the expression situation of mNRA is shown in figure 3. In FIG. 3, normal, control, negative Control, OE-NEDD4L, NEDD4L over-expression group. As can be seen from FIG. 3, the mRNA expression level of NEDD4L in the NEDD4L overexpressed group was up-regulated about 22-fold as compared to the normal group and the control group.

3. Influence of NEDD4L Gene overexpression vector on expression of NEDD4L protein in vascular endothelial cells

The total protein of the endothelial cells after infection in a part of this example was extracted and Western blot was performed to detect the expression level of NEDD4L gene. After digestion of endothelial cells with pancreatin, the cell pellet was collected by centrifugation, 100. Mu.L of cold lysis buffer (PMSF: RIPA 1:100) was added to the pellet, the cell suspension was vortexed for 30s, five minutes once, centrifuged at 13,000 rpm for 3min at 4℃centrifuge, the supernatant was taken in a fresh centrifuge tube, protein concentration was determined by BCA method, 5×loading buffer 25. Mu.L was added, and metal bath was kept at 85℃for 5min, -20 ℃. Taking an equivalent protein sample, performing SDS-polyacrylamide gel electrophoresis for 90min at constant pressure, taking out gel for transferring membrane, soaking with methanol for 2min before PVDF membrane is used for 120min at constant flow, taking out PVDF membrane after transferring membrane, sealing at room temperature for 60min in 5% skimmed milk powder, taking out PVDF membrane for six times of washing, performing primary antibody and secondary antibody binding reaction (wherein the primary antibody is NEDD4L rabbit anti-human monoclonal antibody, and can be specifically bound with the target protein, and the secondary antibody is HRP labeled goat anti-rabbit antibody, and can be specifically bound with the primary antibody, finally displaying a strip under the action of ECL luminous solution (containing peroxide reagent, luminol reinforcing agent and the like)), and finally scanning a luminous film, and analyzing the molecular weight and net optical density value of a target strip by using a gel image processing system.

The expression of NEDD4L protein of vascular endothelial cells after induction by NEDD4L gene over-expression vector is shown in FIG. 4. In FIG. 4, normal, control, negative Control, OE-NEDD4L, NEDD4L over-expression group.

In the invention, the virus infection time is set to be 72 hours, the cell state and the infection efficiency of virus infection are observed through fluorescence after the infection is finished, the cell state is stable and the infection efficiency reaches more than 90%, and the subsequent experiment is carried out. Since the adenovirus over-expression vector cannot be integrated into the genome after transfection to establish a stable cell line, the efficiency of cell infection is critical to the influence of the subsequent results, and on the premise of ensuring the infection efficiency, as can be seen from fig. 4, the expression level of NEDD4L protein in endothelial cells is obviously increased by about 6.15 times in the NEDD4L over-expression group compared with the normal group and the control group.

4. CCK8 method for detecting influence of NEDD4L gene over-expression vector on endothelial cell proliferation

Referring to the first part of this example, NEDD4L over-expression, negative control and normal control groups were set. Cells at log phase after adenovirus infection were plated in 96-well plates at 3000 cell/well, 6 replicates per group, 10 μl CCK8 reagent was added per well 3h before termination of culture at 0d,1d, 2d, 3d, OD values were detected 3h later at wavelength 450 nm using a microplate reader, the proliferation inhibition of cells was analyzed statistically and plotted.

The test results of the cell proliferation experiment of the vascular endothelial cells after being induced by NEDD4L gene overexpression vector are shown in figure 5. In FIG. 5, normal, control, negative Control, OE-NEDD4L, NEDD4L over-expression group.

As can be seen from FIG. 5, the NEDD4L over-expression group has significantly inhibited endothelial cell growth by 80% compared with the normal group and the control group.

5. Effect of NEDD4L Gene overexpression vector on endothelial apoptosis

Referring to the first part of this example, NEDD4L over-expression, negative control and normal control groups were set. Cells were plated in 1X 10 ⁵ cells/well 6-well plates after adenovirus infection, and after 24h the cells were collected in 5ml centrifuge tubes with 3 duplicate wells per group. Provided that the number of cells is ∈ 5×10 ⁶/treatment. Centrifugation, pre-cooling at 4 ℃, washing 1 time with PBS, centrifuging, washing the cell pellet once with 1 Xbinding buffer, centrifuging, collecting the cells, and adding 200. Mu.L of 1 Xbinding buffer to resuspend the cells. Add 10. Mu.L Annexin V-APC staining, keep out of light for 10-15min at room temperature. FACS flow cytometer detection, data analysis, mapping.

The test results of apoptosis experiments of vascular endothelial cells after being induced by NEDD4L gene overexpression vector are shown in figure 6. In fig. 6, the apoptosis rates of the negative control group, the normal cell group and the NEDD4L overexpressing group were 5.517% ± 1.231%, 5.723% ± 0.586% and 31.760% ± 1.603% in this order. It is known that the apoptosis rate of endothelial cells in NEDD4L over-expression group is significantly higher than that in negative control group and normal cell group, and the apoptosis rate of NEDD4L over-expression group is about 6 times that of negative control group and normal cell group.

6. Clone formation method for detecting influence of NEDD4L gene over expression vector on endothelial cell proliferation capacity

Referring to the first part of this example, NEDD4L over-expression, negative control and normal control groups were set. And (3) after virus infection of cells, paving 6 pore plates on cells in the logarithmic phase, adding 400-1000 cells into each pore, and arranging 3 compound pores in each pore. And continuously culturing the cells until the number of 10 d-14 d or the number of most single clone cells is more than 50. Clones were photographed by fluorescence microscopy before termination of the experiment and washed once with PBS. 1mL of 4% paraformaldehyde is added to each well, and cells are fixed for 30-60 min and washed once with PBS. Each well was stained with 500. Mu.L of GIEMSA dye solution for 20min. ddH ₂ O washes cells, dries in air, photographs with a digital camera, cloning techniques, and plots.

The test results of the cell cloning experiment of the vascular endothelial cells after being induced by NEDD4L gene overexpression vector are shown in figure 7. In FIG. 7, the number of cell clones in the negative control, normal and NEDD4L overexpressing groups was 66.33.+ -. 11.37, 72.33.+ -. 7.506 and 21.33.+ -. 3.22 in order. As can be seen, the number of endothelial clones in the NEDD4L over-expressed group was significantly lower than that in the negative control group and the normal cell group.

7. Transwell migration experiment for detecting influence of NEDD4L gene overexpression vector on endothelial cell invasion capacity

Referring to the first part of this example, NEDD4L over-expression, negative control and normal control groups were set. The cells were collected after 24h of culture in serum-free medium, the concentration was adjusted to 1X 10 ⁶, 200. Mu.L of 10 ⁵ cells mixed with ECM complete medium were added to the upper chamber of each Boyden cell (pore size: 8 μm), 500. Mu.L of serum-containing complete medium was added to the lower chamber of each Boyden cell for 8h of culture, the lower chamber medium was removed, sodium chloride-alcohol was fixed for 10min, hematoxylin was stained for 10min, the upper chamber medium was removed, the upper chamber cells were scraped off with a cotton swab, the image was taken by counting with an inverted microscope, and 8 visual field counts were selected with 200X light and the average value was taken.

The result of the cell Transwell experiment is shown in FIG. 8 after the vascular endothelial cells are induced by NEDD4L gene overexpression vector. Transwell migration experiments in which Normal, control, negative Control, OE-NEDD4L, NEDD4L over-expression groups. As can be seen from the results of FIG. 8, the NEDD4L over-expression group showed a significant decrease in the number of the transmembrane migration cells, which decreased the number of the invasion cells by about 3 times or more as compared with the negative control group and the normal group.

8. Cell scratch test for detecting influence of NEDD4L gene over-expression vector on endothelial cell migration capacity

Referring to the first part of this example, NEDD4L over-expression, negative control and normal control groups were set. Human umbilical vein endothelial cells infected by NEDD4L gene over-expressed adenovirus are inoculated in a 6-well plate, 3 multiple holes/group are used for overnight culture, when the cell confluence reaches 90%, a straight line is drawn at the center position by using a 100ul pipette gun head, the fallen cells are washed out by PBS, and after 24 hours, photographing and statistics are carried out under an inverted microscope.

The detection results of the cell scratch experiments of the vascular endothelial cells after being induced by the NEDD4L gene overexpression vector are shown in figure 9. In FIG. 9, normal, control, negative Control, OE-NEDD4L, NEDD4L over-expression group. As can be seen from the results of fig. 9, the migration area of the NEDD4L over-expressed group was significantly reduced compared with that of the negative control group and the normal cell group, indicating that the NEDD4L over-expressed group can effectively inhibit the migration ability of endothelial cells.

9. Immunoblotting and mRFP-GFP-LC3 virus infection experiment to detect influence of NEDD4L gene over-expression vector on endothelial cell autophagy ability

Referring to the first part of this example, NEDD4L over-expression, negative control and normal control groups were set. Human umbilical vein endothelial cells infected by NEDD4L gene overexpression vectors are inoculated into a 6-well plate, 3 multiple holes/groups are carried out, cell precipitation is collected after 24 hours to extract protein, autophagy related western immunoblotting detection is carried out, mRFP-GFP-LC3 virus is used when the cell confluency reaches 90%, and photographing and statistics are carried out under an inverted microscope after 24 hours.

The results of the autophagy-related western blotting detection of vascular endothelial cells after induction by NEDD4L gene overexpression vector are shown in FIG. 10. The vascular endothelial cells were infected with mRFP-GFP-LC3 virus after being induced by NEDD4L gene overexpression vector, and the autophagy condition thereof is shown in FIG. 11, wherein FIG. 11a is a microscopic image after infection with mRFP-GFP-LC3 virus, and FIG. 11b is a graph of autophagy positive point/cell number ratio quantification. In FIG. 10 or FIG. 11, normal cells, control, negative Control, OE-NEDD4L, NEDD4L over-expression.

From FIG. 10, NEDD4L overexpression group was able to effectively induce the expression of autophagy-related proteins ATG5 and Beclin 1. As can be seen from FIG. 11, the NEDD4L overexpression group showed a significant increase in autophagosomes represented by red spots and autophagosomes represented by yellow spots compared to the negative control group and the normal cell group after the mRFP-GFP-LC3 virus infection experiment, and obvious autophagy and autophagy flow occurred.

In the experimental range of the invention, the NEDD4L gene expression can be induced to induce the occurrence of endothelial autophagy, and the cell autophagy is the basis of degradation and recycling of cell components and is the main form of maintaining the steady state of cells, and the occurrence of the cell autophagy is influenced by multiple factors such as physiological environment and conditions. The NEDD4L gene may play different roles in different physiological environments.

Example 3

Since NEDD4L gene over-expression vector can inhibit proliferation, migration and invasion of vascular endothelial cells and induce autophagy, it can be developed into a medicine for treating vascular endothelial cell proliferative diseases.

The prepared medicine for treating vascular endothelial cell proliferation diseases may include over-expression vector of NEDD4L gene or over-expression product of NEDD4L gene and pharmaceutically acceptable supplementary material. The preparation form of the prepared medicament for treating vascular endothelial cell proliferative diseases is not particularly limited, and can be solid, liquid, gel, semifluid, aerosol and other various substance forms.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, or alternatives falling within the spirit and principles of the invention.

Claims

The NEDD4L gene is applied in preparing medicine for treating vascular endothelial cell proliferation related diseases.
2. The use according to claim 1, wherein the nucleotide sequence of the NEDD4L gene is shown in SEQ ID No. 1.
3. The use according to claim 2, wherein the NEDD4L gene encodes a protein having the amino acid sequence shown in SEQ ID NO. 2.
4. The use according to any one of claims 1 to 3, wherein the vascular endothelial cell proliferation-related disease comprises atherosclerosis, hypertension, diabetes or hemangioma.
5. A medicament for treating vascular endothelial cell proliferation related diseases, which is characterized in that the medicament comprises a NEDD4L gene overexpression vector.
6. The method for preparing a medicament for treating vascular endothelial cell proliferation-related diseases according to claim 5, wherein the method comprises the steps of:

Cloning NEDD4L genes to adenovirus-related viruses to obtain adenovirus-related viruses containing the NEDD4L genes;

Step two, converting the adenovirus related virus containing the NEDD4L gene into escherichia coli to obtain a recombinant plasmid;

and thirdly, transfecting the recombinant plasmid into human endothelial cells, performing amplification culture, purifying and concentrating to obtain the NEDD4L gene overexpression vector.
7. The method of claim 6, wherein in the first step, the adenovirus-associated virus comprises at least one of adenovirus, lentivirus, and adeno-associated virus.
8. The method of claim 6, wherein in the third step, the human endothelial cells include human umbilical vein endothelial cells, human embryonic kidney cells or human smooth muscle cells, and/or

And step three, before the amplification culture, the method further comprises the step of extracting the recombinant plasmid with high purity and without endotoxin.
9. The method of claim 6, wherein in step three, the viral titer in the NEDD4L gene overexpression vector is measured in the human endothelial cells.
10. The use of a medicament for the treatment of a vascular endothelial cell proliferation-related disease according to claim 5, wherein the medicament is injected into the body for at least 72 hours.