Human umbilical vein endothelial cells overexpressing integrin and their application

Abstract

The present invention discloses a human umbilical vein endothelial cell overexpressing integrin and its application. The integrin is αvβ3; the human umbilical vein endothelial cell is a human umbilical vein endothelial fusion cell. The present invention uses human umbilical vein endothelial cells, such as fusion HUVEC cells, whose integrin αvβ3 expression level is 1-6 times that of normal primary or immortalized HUVEC cells as a cell model for screening drugs, which can make the functional experimental results more obvious and is not easy to screen out drugs that act on the cells.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a human umbilical vein endothelial cell overexpressing integrin and an application thereof. Background Art

[0002] The growth process of a tumor can be divided into two stages: the prevascular stage and the vascular stage. In the prevascular stage, due to lack of blood supply, the tumor is dormant, usually 1-2 mm in diameter, and does not develop disease. In the vascular stage, due to adequate blood supply, the tumor volume increases rapidly, infiltration and metastasis occur, and angiogenesis is induced. Tumor angiogenesis refers to the process of microvessel growth induced by tumor cells and the establishment of blood circulation in the tumor. The process of tumor angiogenesis is regulated by factors that promote and inhibit angiogenesis. Angiogenesis inhibitors inhibit the formation of blood vessels in tumor tissues, cut off blood supply, and indirectly inhibit the growth of tumor cells. The use of angiogenesis inhibitors to treat tumors has the advantages of a broad anti-tumor spectrum, low toxicity and side effects, and not easy to develop drug resistance.

[0003] Human umbilical vein endothelial cells (HUVEC) are a commonly used cell screening model for screening drugs that inhibit angiogenesis, but primary cells must be used. Primary HUVECs will experience growth stagnation after about 10 generations, so experiments can only be performed with cells within 10 generations. Cells need to be purchased for each experiment, and the screening cost is high. In addition, the limited culture generations of primary cells may hinder their use in cell transfection research. Summary of the invention

[0004] The technical problem to be solved by the present invention is that the cell model used in the prior art for screening drugs inhibiting angiogenesis has the defects of high cost and high screening cost, and provides a human umbilical vein endothelial cell overexpressing integrin and its application. The present invention uses the human umbilical vein endothelial cell, such as EA.hy926, to prepare an anti-tumor drug screening model. When performing functional experiments, the cell is more sensitive to drugs and can be passaged for a long time.

[0005] In order to overcome the problem of the low number of HUVEC passages in the prior art, the inventors evaluated and screened the growth state and experimental conditions of the specified parameters of HUVEC that can be used for the construction of model cell lines, and obtained HUVEC that can be passaged for a long time. After culture, the stable expression technology was then used to prepare and construct integrin αvβ3 overexpressing cell lines with fused HUVEC cells as transfected target cells. Cells were infected with different MOIs and flow sorted, and pressure screening was performed to obtain cell lines with different expression levels. Cells with different integrin αvβ3 expression levels were added with positive drugs for scratch, tube formation, and migration tests, and the cell lines with the most obvious drug effects were selected for screening anti-tumor drugs.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] A first aspect of the present invention provides a human umbilical vein endothelial cell that overexpresses integrin, wherein the integrin is αvβ3; the human umbilical vein endothelial cell is a human umbilical vein endothelial fusion cell.

[0008] In some embodiments of the present invention, the human umbilical vein endothelial fusion cells are EA.hy926.

[0009] In some embodiments of the present invention, the expression level of the integrin in human umbilical vein endothelial cells is 1-6 times that of normal primary or immortalized HUVEC cells.

[0010] In some embodiments of the present invention, the expression level of the integrin in human umbilical vein endothelial cells is 2-4 times that of normal primary or immortalized HUVEC cells.

[0011] In some embodiments of the present invention, the human umbilical vein endothelial cells are human umbilical vein fusion cells, such as EA.hy926.

[0012] In some specific embodiments of the present invention, the integrin is αvβ3.

[0013] In some specific embodiments of the present invention, in integrin αvβ3, the coding sequence of av is shown as SEQ ID NO:3, and the amino acid sequence is shown as SEQ ID NO:1; the coding sequence of β3 is shown as SEQ ID NO:4, and the amino acid sequence is shown as SEQ ID NO:2.

[0014] In some specific embodiments of the present invention, the human umbilical vein endothelial cells are human umbilical vein endothelial cells with CCTCC number C2022394.

[0015] The second aspect of the present invention provides a cell model for screening anti-tumor drugs, wherein the cell model comprises the human umbilical vein endothelial cells as described in the first aspect, and the cell model screens the anti-tumor drugs by measuring the effect of the drug to be tested on the human umbilical vein endothelial cells.

[0016] In some embodiments of the present invention, the effect is selected from the group consisting of cell scratch healing effect, cell tube formation effect and cell migration effect.

[0017] The third aspect of the present invention provides a method for constructing the cell model as described in the second aspect, the method comprising:

[0018] Human umbilical vein endothelial cells overexpressing integrins are cultured in culture and parameters selected from one or more of the following are identified:

[0019] (1) Healing area in cell scratch test;

[0020] (2) Tube formation indicators in cell tube formation assay;

[0021] (3) cell migration rate in a cell migration assay; and

[0022] (4) cell adhesion rate in cell migration assay;

[0023] When at least one parameter of the cell is within a specified range, selecting the cell as a cell model;

[0024] The specified range of the healing area is at least 1-20 times that of the normal primary or immortalized HUVEC cells used as a control; the specified range of the tube formation index is that the number of nodes, the tube length, and the average value of the tube area are at least 1-20 times that of the normal primary or immortalized HUVEC cells used as a control; the specified range of the cell migration rate or cell adhesion rate is at least 1-20 times that of the normal primary or immortalized HUVEC cell control group used as a control.

[0025] In some embodiments of the present invention, the designated range of the healing area is at least 2-18 times that of normal primary or immortalized HUVEC cells as a control; preferably 4-12 times, and more preferably 6-9 times.

[0026] In some embodiments of the present invention, the specified range of the tube formation index is that the average value of the number of nodes, tube length and / or tube area is at least 2-18 times that of normal primary or immortalized HUVEC cells used as a control; preferably 4-12 times, and more preferably 6-9 times.

[0027] In some embodiments of the present invention, the specified range of the cell migration rate or cell adhesion rate is at least 2-18 times that of a normal primary or immortalized HUVEC cell control group as a control; preferably 4-12 times, and more preferably 6-9 times.

[0028] The fourth aspect of the present invention provides a method for screening anti-tumor drugs, the method comprising contacting the drug to be tested with the human umbilical vein endothelial cells as described in the first aspect or the cell model as described in the second aspect;

[0029] When the test drug is determined to have an anti-tumor effect on the human umbilical vein endothelial cells by one or more of the following parameters within a corresponding range, the test drug is judged to have an anti-tumor effect:

[0030] (1) The specified range of the healing area is at least 1-20 times that of normal primary or immortalized HUVEC cells used as a control;

[0031] (2) the specified range of the tube formation index is that the average value of the number of nodes, tube length and / or tube area is at least 1-20 times that of normal primary or immortalized HUVEC cells used as a control; and

[0032] (3) The specified range of the cell migration rate or cell adhesion rate is at least 1-20 times that of a normal primary or immortalized HUVEC cell control group as a control.

[0033] The specified range of the healing area, the specified range of the tube formation index and the specified range of the cell migration rate are as described in the third aspect.

[0034] The fifth aspect of the present invention provides a use of the human umbilical vein endothelial cells as described in the first aspect in constructing a drug screening model.

[0035] The sixth aspect of the present invention provides a method for preparing human umbilical vein endothelial cells as described in the first aspect, comprising the steps of infecting human umbilical vein endothelial cells with MOI and performing flow cytometry sorting.

[0036] The seventh aspect of the present invention provides a use of the human umbilical vein endothelial cells as described in the first aspect in the preparation of an anti-tumor drug detection reagent.

[0037] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0038] The reagents and raw materials used in the present invention are commercially available.

[0039] The positive and progressive effects of the present invention are:

[0040] The present invention uses human umbilical vein endothelial cells, such as fusion HUVEC cells, whose integrin αvβ3 expression level is 1-20 times that of normal primary or immortalized HUVEC cells as a cell model for screening drugs, which can make the functional experimental results more obvious and is less likely to screen out drugs that act on the cells.

[0041] Biomaterial Deposit Information

[0042] The human umbilical vein endothelial cells overexpressing integrin of the present invention were deposited in the China Center for Type Culture Collection (CCTCC) on December 29, 2022. The deposit address is Wuhan University, Luojia Mountain, Wuchang, Wuhan City, Hubei Province, China, Postal Code 430072, the deposit number is CCTCC NO: C2022394, and the culture name is hybridoma cell line TB-EA.hy926-αvβ3. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Schematic diagram of the empty viral vector plasmid.

[0044] Figure 2 Schematic diagram of the viral vector (pLVX) carrying the avβ3 encoding gene.

[0045] Figure 3 This is the cell growth chart of HUVEC primary cells, fusion cells and immortalized cells.

[0046] Figure 4 The results of scratch assay on primary cells.

[0047] Figure 5 The results of the tube formation experiment of HUVEC primary cells.

[0048] Figure 6 The results of the tube formation experiment of HUVEC immortalized cells.

[0049] Figure 7 The results of the tube formation experiment of HUVEC fusion cells.

[0050] Figure 8 The results of the cell migration experiment of HUVEC primary cells.

[0051] Fig. 9 The results of the cell migration experiment of HUVEC immortalized cells.

[0052] Fig.10 The results of the cell migration experiment of HUVEC fusion cells.

[0053] Figure 11-Figure 12 The results are the background expression of αvβ3 in HUVEC primary cells, immortalized cells, fusion cells and cells overexpressing integrin.

[0054] Fig.13 The results show that different peptides have an inhibitory effect on the proliferation of HUVEC.

[0055] Fig.14 The inhibitory effects of different peptides on HUVEC cell adhesion.

[0056] Fig.15 The effect of different concentrations of P50 on HUVEC cell adhesion.

[0057] Fig.16 These are the results of the HUVEC cell tube formation inhibition experiment. DETAILED DESCRIPTION

[0058] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0059] The main experimental reagents used in the examples are shown in Table 1, and the main experimental instruments are shown in Table 2.

[0060] Table 1 Main experimental reagents

[0061]

[0062]

[0063] Table 2 Main experimental instruments

[0064]

[0065]

[0066] Example 1: Preliminary experiments on primary, immortalized and fused HUVEC cell lines

[0067] 1. Lentivirus packaging

[0068] 1.1 Experimental procedures

[0069] 1.1.1 Lentivirus packaging (empty vector)

[0070] The empty vector (containing green fluorescent marker protein, plasmid map as Figure 1 The steps are as follows:

[0071] 1.1.2 Cell preparation

[0072] After 293T cells are revived, they are subcultured for 2-3 generations to ensure that the cells are in good condition. The cells are then divided into 10 cm dishes so that the cell density reaches about 80% on the next day.

[0073] 1.1.3 Preparation before transfection

[0074] On the second day after cell passage, the 293T cell culture medium was replaced with pure DMEM culture medium. Prepare the required plasmids and calculate the usage of each plasmid. The mass ratio of the viral vector (pLVX) carrying the avβ3 encoding gene (the coding sequence of av is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:1; the coding sequence of β3 is shown in SEQ ID NO:4, and the amino acid sequence is shown in SEQ ID NO:2) to psPAX2 and pMD2.0G is 3:2:1, and the total amount of the three plasmids is 20 μg; the map of the viral vector (pLVX) carrying the avβ3 encoding gene is shown in Figure 2 shown.

[0075] 1.1.4 Cell transfection

[0076] Prepare EP tubes according to the ratio of DMEM: plasmid: transfection reagent = 100μL: 1μg: 2μL. First add pure DMEM medium, then add the three plasmids calculated in advance, and finally add the transfection reagent. After mixing, vortex it slightly to mix it evenly, and then let it stand at room temperature for 15-20 minutes. Finally, add it dropwise to the cells to be transfected. After transfection for 6 hours, replace it with new 11mL DMEM medium containing 10% serum but no antibiotics.

[0077] 1.1.5 Collecting viruses

[0078] 48 hours after transfection, collect the virus-containing cell culture fluid into a 15mL centrifuge tube. Then, add 10mL of fresh DMEM medium containing 10% serum but without double antibody to the dish, and continue to culture for 24 hours before the second virus collection. For the collected virus, centrifuge at 4000rpm for 15min in a 4℃ centrifuge, and then filter the virus supernatant with a 0.45μm filter for use in infecting cells. In addition, the virus is stored in a -80℃ refrigerator after aliquoting.

[0079] 1.1.6 Lentivirus titer detection

[0080] Day 0: Digest and count the 293T cells that are growing well and dilute to 1×10 5 cells / mL, add to 96-well plate, 100 μL / well (1×10 4 cells), 6 wells are needed for each virus. Place in a 37°C, 5% CO2 incubator and culture overnight.

[0081] Day 1: Make 10-fold gradient dilutions in EP tubes, with 6 consecutive dilutions. The dilution method is as follows: prepare 6 1.5mL EP tubes for each virus, add 90μL complete medium to each tube, add 10μL of virus stock solution to the first tube, mix well, then pipette 10μL and add to the second tube to mix well. And so on, number them as tube ①, tube ②, tube ③, tube ④, tube ⑤ and tube ⑥ respectively. Then incubate the diluted virus and cells overnight.

[0082] Day 2: Aspirate the culture medium containing the virus and add 100 μL of complete culture medium to each well to facilitate cell growth.

[0083] Day 3: Observe fluorescence under a microscope. At this time, fluorescence will begin to express.

[0084] Day 4: Observe the results under a fluorescence microscope, count the cells in the wells with a suitable fluorescence ratio (between 10-30%), and calculate the titer. The calculation formula is as follows:

[0085] Titer (TU / mL) = (number of cells × fluorescence percentage / virus volume) × dilution factor

[0086] For example: The fluorescence ratio of the corresponding well of tube ③ is 30%, and the total number of cells is 8×10 4 ,

[0087] Titer (TU / mL) = (8 × 10 4 ×30%× / 0.1)×10 3 =2.4×10 8

[0088] If you want to infect 2×10 5 cells, MOI = 30 (1 cell corresponds to 30 virus particles) (see note below), then the required virus volume is = 2×10 5 ×30 / 2.4×10 8 (mL) = 0.025mL = 25μL.

[0089] Note: MOI value: multiplicity of infection, which actually means how many viable viruses are infected with each cell. The optimal MOI value for various cells is different. Before the formal experiment, a preliminary experiment should be conducted to find the optimal MOI.

[0090] The MOI value measured in this experiment is 3×10 6 TU / mL.

[0091] 2. Primary HUVEC cell culture and cell detection

[0092] 2.1 Primary HUVEC cell culture

[0093] Experimental steps:

[0094] The three purchased HUVEC strains (referred to as original cells) were cultured in primary, immortalized, and EA.hy926 culture media. The culture media was changed every three days. When the cell confluence reached 80%, the cells were subcultured. This experiment used cells of the 3rd and 4th generations (referred to as primary cells). The cells were cultured until the 22nd generation or until they could no longer be expanded (the cells at this time were referred to as end-point cells).

[0095] Experimental results:

[0096] Growth diagram of HUVEC primary cells, HUVEC immortalized cells and HUVEC fusion cells (ie EA.hy926) Figure 3 The results showed that the primary HUVEC cells stopped growing and died at P13 and could no longer be passaged; the immortalized HUVEC cells grew slowly at P13, and their growth was significantly weaker than that of the fusion HUVEC at P22; while the fusion HUVEC cells grew well at P22, maintaining good cell phenotype and proliferation activity, so the fusion cells were selected as transfected cells.

[0097] 2.2 Western blot analysis of the background expression level of integrin αvβ3 in HUVEC cells

[0098] HUVEC cell grouping: primary cell I and terminal cell I.

[0099] 2.2.1 Experimental procedures

[0100] 2.2.1.1 Preparation of cell lysate

[0101] Take 2 mL of cell supernatant, centrifuge, and discard the supernatant. Wash the cells once with PBS, and discard the PBS. Add cell lysis buffer (the amount of cell lysis buffer is 10 7 Add 1mL of lysate), then add 1μL of PMSF, place in an ice box, and put into an ultrasonic crusher for 3 minutes. Centrifuge at 10000rpm / s for 10 minutes and discard the precipitate. Add an appropriate amount of 5× loading buffer to the supernatant to dilute it to 1×. After the lysate is mixed, place it in a 95℃ metal bath for 10 minutes. The lysate is stored at -20℃ for a long time.

[0102] 2.2.1.2 Electrophoresis

[0103] (1) Install the gel plate: Remove the sample comb, install the gel plate into the electrophoresis tank, and place electrophoresis liquid inside and outside the gel plate. Fill the inner tank to the highest level, and the outer tank to a level higher than the bottom edge of the gel plate. Check if the inner tank is leaking. If so, reinstall the gel plate.

[0104] (2) Loading: Add the sample to the gel well and add a protein marker.

[0105] (3) Gel running: After all samples to be tested have been loaded, cover the electrophoresis tank and connect the wires to the power supply. Turn on the power supply and adjust it to a constant voltage of 120V to run the gel. When both the bromophenol blue and the smallest protein marker bands have run to the bottom of the gel, turn off the power supply.

[0106] 2.2.1.3 Transfer

[0107] (1) Soak the PVDF membrane, filter paper and sponge with methanol in advance. Two pieces of filter paper and sponge are required for each piece of glue.

[0108] (2) Install the transfer sandwich. Remove the gel plate after running, clean it, take the gel off the glass plate and place it in the wet transfer apparatus in the form of a sandwich. The order is: electrode (-) - sponge - filter paper - gel - PVDF membrane - filter paper - electrode (+). Note that the gel is close to the negative electrode.

[0109] (3) Transfer: Pour the transfer solution into the transfer tank, and then place the transfer tank in an ice bath. Cover the transfer tank with a lid, connect the power cord to a power source, set a constant voltage of 100 V, and keep it on for 1.5 hours.

[0110] (4) Check the transfer efficiency. After the transfer is completed, remove the sandwich from the tank and observe the transfer of the pre-stained marker. The pre-stained marker should be transferred from the gel to the membrane. Stain the membrane with Ponceau red staining solution for 1-3 minutes, rinse with water and observe the actual transfer effect.

[0111] 2.2.1.4 Antibody incubation

[0112] (1) Cutting the membrane: Cut off the edge area where the protein is not transferred, and cut the membrane according to the antibody incubation situation. Make sure the blade and pad are clean. Rinse the membrane with distilled water.

[0113] (2) Blocking: Block with 5% skim milk and shake at room temperature for 1 h.

[0114] (3) Primary antibody incubation

[0115] Dilute the primary antibody in 1% BSA antibody diluent. After mixing the primary antibody, pour out the blocking solution, add the primary antibody diluent directly, and incubate overnight at 4°C on a shaker at low speed. After the primary antibody incubation is completed, discard the primary antibody, rinse the membrane with water twice, then wash it with TBST three times, and shake it quickly on a shaker for 3-5 minutes / time.

[0116] Note: During the antibody preparation process, pay attention to the use of the pipette to ensure that enough antibody is sucked up and that excess antibody does not stick to the tip of the pipette.

[0117] (4) Secondary antibody incubation

[0118] The secondary antibody is diluted in 1% BSA antibody diluent. Generally, horseradish peroxidase (HRP) labeled secondary antibody (0.5mg / mL) is used, 1:5000, and incubated at room temperature on a shaker at low speed for 1h. After the incubation, the secondary antibody is discarded, the membrane is first rinsed with clean water twice, and then washed with TBST 5 times, shaking quickly on a shaker for 3-5min each time. After the end, the membrane is placed in clean water for exposure.

[0119] 2.2.1.5 Substrate and exposure

[0120] (1) Preparation of substrate

[0121] Take out substrate A & B from the 4°C refrigerator, mix them evenly in a 1:1 ratio and set aside.

[0122] (2) Substrate incubation

[0123] Add the substrate evenly to a membrane. Incubate for 30 seconds to 1 minute. Remove excess liquid, seal the membrane in a plastic clip with a fluorescent ruler, and place it in an X-ray photography cassette.

[0124] (3) Exposure

[0125] After turning off the light, take out the film and press it on the plastic clip in the dark box. Close the dark box and start exposure. The exposure time is generally 5-10s. If the signal is weak, you can choose to press it for a longer period of time. The film cannot be moved during the exposure process.

[0126] (4) Development and fixing

[0127] After exposure, take the film out of the press box. Soak the film in developer and fixer in turn. The general development time is 30 seconds. If the development is obviously insufficient (the stripes are blurred), add 1 minute and replace the developer in time. The general fixing time is 30 seconds. If the fixing is insufficient (the background is yellow), add 1 minute and replace the fixer in time. After fixing, the film goes into the film processor for drying.

[0128] 2.2.1.6 Result processing

[0129] Cover the dried film on the plastic clip, align the scale on the film precisely with the fluorescent scale on the plastic clip, and mark the position of the WB membrane and the position of the pre-stained marker strip on the film. Scan the marked film into the computer using a scanner and save the image.

[0130] 2.3 Analysis of the background expression level of integrin αvβ3 in HUVEC cells by flow cytometry

[0131] 2.3.1 Experimental steps:

[0132] (1) Prepare a single cell suspension and pass it through a 300-mesh cell sieve.

[0133] (2) Cell counting: 2×10 cells were collected from each sample tube. 5 cells in EP tubes.

[0134] (3) The primary antibody was added and incubated for 40 min. The negative control was PBS incubated with cells for 40 min.

[0135] (4) After washing twice with PBS, add fluorescently labeled secondary antibody and incubate for 20-30 min.

[0136] (5) After washing twice with PBS, add 300-500 μL PBS to resuspend the cells and perform testing on the microscope.

[0137] (6) Copy the experimental data to a USB flash drive and analyze the data using FlowJo software.

[0138] 2.3.2 Experimental Results

[0139] 2.4 HUVEC cell detection (first functional experiment)

[0140] The first HUVEC cell experiments (scratch, tube formation, migration) were divided into the following groups: primary cell I, primary cell I + positive drug.

[0141] The concentrations of Cilengitide used were 0.5 μg / ml and 3 μg / ml, named Cilengitide-1 and Cilengitide-2, respectively.

[0142] 2.4.1 Cell scratch assay

[0143] HUVEC cell grouping: primary cell I, primary cell I + positive drug (three cell lines).

[0144] 2.4.1.1 Experimental steps:

[0145] (1) Use a marker pen to draw horizontal lines evenly on the back of a 24-well plate. The lines are spaced about 0.3 cm apart and at least two lines are drawn in each well.

[0146] (2) Original HUVECs were 5×10 5 cells / well were seeded in 24-well plates and cultured overnight;

[0147] (3) Use a sterile 10 μL pipette tip to scratch against a ruler. The ruler should be perpendicular to the horizontal line on the back of the well plate. Keep the pipette tip as vertical as possible and avoid tilting it.

[0148] (4) Wash the detached cells twice with PBS, and add 0.5 mL / well of serum-free culture medium containing the corresponding positive drug concentration to the 24-well plate containing cells, in triplicate;

[0149] (5) Randomly sample three points from each well at 0, 12, and 24 h, along the cross, and take photos. Measure the distance and area of ​​each scratch. The scratch area was determined using Image J software. Cell migration rate (%) = (1-scratch area / original scratch area) × 100.

[0150] 2.4.1.2 Experimental results

[0151] 2.4.1.2.1 Primary cells

[0152] The results of the scratch test on primary cells are as follows Figure 4 As shown. Since the cell state of primary cells treated with drugs for 12h and 24h was not good, no effective results could be formed, so they were not shown.

[0153] 2.4.1.2.2 Fusion cells

[0154] In the scratch test of fused cells, the analysis of cell migration rate at different time points in the control group is shown in Table 3.

[0155] Table 3 Cell migration rate of the control group at different time points

[0156] 0h control 24.688 25.156 25.391 25.08 Cell migration rate 12h control 22.266 22.812 22.188 22.42 10.61％ 24h control 19.188 19.328 19.50 19.34 22.89％

[0157] The analysis of cell migration rate at different time points in the Cilengitide-1 group is shown in Table 4 , and the analysis of cell migration rate at different time points in the Cilengitide-2 group is shown in Table 5 .

[0158] Table 4 Cell migration rate at different time points in the Cilengitide-1 group

[0159] 0hCilengetide-1 27.188 27.109 27.422 27.24 Cell migration rate 12h Cilengitide-1 26.094 26.562 25.859 26.17 3.93％ 24h Cilengitide-1 24.688 24.062 24.609 24.45 10.24％

[0160] Table 5 Cell migration rate at different time points in the Cilengitide-2 group

[0161] 0h Cilengitide-2 28.516 28.281 28.594 28.46 Cell migration rate 12h Cilengitide-2 27.188 27.422 27.656 27.42 3.65％ 24h Cilengitide-2 26.406 26.953 26.264 26.54 6.75％

[0162] Conclusion: Compared with the control group, cilengitide has the ability to inhibit the migration of HUVEC fusion cells, and the higher the dose, the stronger the ability to inhibit cell migration.

[0163] 2.4.1.2.3 Immortalized cells

[0164] The analysis of cell migration rate at different time points in the control group is shown in Table 6, the analysis of cell migration rate at different time points in the Cilengitide-1 group is shown in Table 7, and the analysis of cell migration rate at different time points in the Cilengitide-2 group is shown in Table 8.

[0165] Table 6 Cell migration rate of the control group at different time points

[0166] 0h control 35.142 35.815 35.155 35.37 Cell migration rate 12h control 31.991 31.695 31.885 31.86 9.92％ 24h control 25.890 25.771 25.068 25.58 27.68％

[0167] Table 7 Cell migration rate at different time points in the Cilengitide-1 group

[0168] 0hCilengetide-1 32.791 33.470 32.963 33.07 Cell migration rate 12h Cilengitide-1 31.470 31.327 31.755 31.52 4.69％ 24h Cilengitide-1 29.878 29.689 30.035 29.87 9.68％

[0169] Table 8 Cell migration rate at different time points in the Cilengitide-2 group

[0170] 0h Cilengitide-2 31.284 31.577 31.175 31.35 Cell migration rate 12h Cilengitide-2 30.117 30.378 30.253 30.25 3.51％ 24h Cilengitide-2 29.984 29.527 29.547 29.69 5.30％

[0171] Conclusion: Compared with the control group, cilengitide has the ability to inhibit the migration of HUVEC immortalized cells, and the higher the dose, the stronger the ability to inhibit cell migration.

[0172] 2.4.2 Cell tube formation experiment

[0173] HUVEC cell grouping: primary cell I, primary cell I + positive drug (three cell lines).

[0174] 2.4.2.1 Experimental procedures

[0175] (1) Matrigel preparation: Place Matrigel in an ice box one day before the experiment and place it in a 4°C refrigerator overnight to allow the gel to melt slowly;

[0176] (2) Gel: The next day, after the Matrigel is frozen and thawed, prepare for aliquoting. Mix the Matrigel with a pre-cooled pipette tip, pre-cool the EP tube in advance, and aliquot 500 μL each. Keep the Matrigel in an ice box during the operation.

[0177] (3) Matrigel plating: Pre-cool the 24-well plate. Add 300 μL / well Matrigel to the 24-well plate (avoid bubbles when adding), and place in a cell culture incubator for 45-60 minutes to solidify.

[0178] (4) Cell plating: When the cells are 70%-80% full, discard the culture medium and wash twice with PBS, add 2mL of trypsin containing EDTA to digest the cells. After complete digestion, add serum-containing culture medium to terminate digestion, count and resuspend the cells with culture medium and adjust the cell density to 2×105 cells / mL. After the gel solidifies, take out the 24-well plate, add 500μL of cell suspension to each well, mark and place in the incubator for regular culture, and make 3 replicates;

[0179] (5) During the cell culture period, observe the cell tube formation at 4 h, 6 h, 12 h, and 24 h. The specific observation time depends on the cell tube formation status. If tube formation is observed, take photos and save them.

[0180] (6) Result analysis: Randomly select 5 visual fields to observe and count the number of endothelial cell tubes, and give the visual field diagram and tube count diagram. Tube formation rate = number of tubes in the experimental group / number of tubes in the control group*100%.

[0181] 2.4.2.2 Experimental results

[0182] The results of the tube formation experiments of HUVEC primary cells, immortalized cells and fusion cells are shown in Figure 5 , Figure 6 and Figure 7 The results showed that cilengitide inhibited the tube formation of HUVECs compared with the control group.

[0183] 2.4.3 Cell migration

[0184] HUVEC cell grouping: primary cell I, primary cell I + positive drug (three cell lines).

[0185] 2.4.3.1 Experimental procedures

[0186] (1) Cell treatment: Digest the cells in the logarithmic growth phase, centrifuge and remove the supernatant after terminating the digestion, wash with PBS 1-2 times, resuspend in serum-free medium, and adjust the cell density to 5×10 5 / mL;

[0187] (2) Inoculation of cells: Take 100 μL of cell suspension and add it to the Transwell chamber. Each well of a 24-well plate should contain approximately 2.0*10 5 Cells, make 3 duplicate wells; 600 μL of culture medium supplemented with 10% FBS and positive drug solution is added to the lower chamber of the 24-well plate (bubbles often appear between the lower culture medium and the small chamber. If bubbles appear, the chemotaxis of the lower culture medium will be weakened. Therefore, special attention should be paid when seeding the plate. Once bubbles appear, the small chamber should be lifted, the bubbles should be removed, and then the small chamber should be placed in the culture plate);

[0188] (3) Cell culture: conventional culture, tentatively analyzing cell migration after 24 h (the specific time depends on different cell passages);

[0189] (4) Fixation: Remove the Transwell chamber, discard the culture medium, rinse with PBS, and fix the cells with 4% paraformaldehyde for 30 min;

[0190] (5) Staining: Stain with 0.1% crystal violet for 20 min, discard the crystal violet solution, wash twice with PBS, gently wipe off the upper layer of non-migrated cells with a cotton swab, and wash three times with PBS. Observe and count the number of migrated cells under a microscope and count them.

[0191] 2.4.3.2 Experimental results

[0192] The results of cell migration experiments of HUVEC primary cells, immortalized cells and fusion cells are shown in Figure 8 , Fig. 9 and Fig.10 The results showed that compared with the control group, cilengitide inhibited cell migration; compared with the control group, TB01 promoted the migration of HUVEC immortalized cells, while cilengitide inhibited cell migration; compared with the control group, TB01 promoted the migration of HUVEC fusion cells, while cilengitide inhibited cell migration.

[0193] 3. Transfection of empty plasmid cell culture and detection

[0194] 3.1 Lentivirus infection of cells

[0195] (1) Primary, immortalized, and fused HUVEC cells (5×10 5 cells) were inoculated in 6-well plates and cultured until the confluence reached 70%-80%;

[0196] (2) Infect HUVEC cells with negative control lentivirus at an MOI of 1:20 (depending on the specific experiment), and perform pressure screening by treating HUVEC cells transfected with empty plasmid with different concentration gradients of puromycin (0, 1, 2, 4, 8, and 16 μg / mL) to determine the optimal puromycin dose (depending on the specific experimental results). Remove the infected negative cells based on the optimal puromycin dose (the infected positive cells are referred to as primary cells II);

[0197] (3) After 48 h, the expression of zsGreen1 was observed under a fluorescence microscope, and cells with a fluorescent cell ratio of more than 85% were selected for this study.

[0198] 3.2 Transfection of empty plasmid cell culture

[0199] HUVEC cells transfected with empty plasmid were cultured in the corresponding culture medium. The culture medium was changed every three days. When the cell confluence reached 80%, the cells were subcultured. This experiment used cells of the 3rd and 4th generations. Cells that successfully expressed the empty vector were cultured until the 22nd generation or until they could no longer be amplified (end point cells II). The end point cells were again fluorescently photographed to detect the positive rate of empty vector expression.

[0200] 3.3 Western blot analysis of the background expression level of integrin αvβ3 in primary cell II and terminal cell II

[0201] HUVEC cell grouping: primary cell II and terminal cell II.

[0202] Western blot analysis of the expression level of integrin αvβ3 in primary cell II and terminal cell II. The specific operation steps are the same as 2.2.1.

[0203] 3.4 Flow cytometry analysis of the background expression level of integrin αvβ3 in primary cell II and terminal cell II

[0204] HUVEC cell grouping: primary cell II and terminal cell II.

[0205] The experimental process is the same as 2.3.1.

[0206] 3.5 Detection of HUVEC cells transfected with empty plasmid (second functional assay)

[0207] The second HUVEC cell experiment (scratch, tube formation, migration) was divided into the following groups: primary cell I, primary cell I + positive drug; endpoint cell I (P22), endpoint cell I (P22) + positive drug; endpoint cell II (P22), endpoint cell II (P22) + positive drug.

[0208] The concentrations of Cilengitide used were 0.5 μg / ml and 3 μg / ml, named Cilengitide-1 and Cilengitide-2, respectively.

[0209] The experimental process was the same as the first experiment.

[0210] Experimental Results

[0211] 1. The results of the cell scratch test of primary cell II are shown in Tables 9 to 11, and the results of the cell scratch test of endpoint cell II are shown in Tables 12 to 14.

[0212] Table 9 Migration rate of primary cells in the control group at different time points

[0213] 0h control 31.889 31.228 31.320 31.48 Cell migration rate 12h control 28.472 28.901 28.239 28.54 9.34％ 24h control 27.026 26.832 26.638 26.83 14.77％

[0214] Table 10 Migration rate of primary cells at different time points in the Cilengitide-1 group

[0215] 0hCilengetide-1 37.711 37.954 37.759 37.81 Cell migration rate 12h Cilengitide-1 36.558 36.172 36.815 36.52 3.41％ 24h Cilengitide-1 33.318 33.159 33.489 33.32 11.88％

[0216] Table 11 Migration rate of primary cells in the Cilengitide-2 group at different time points

[0217] 0h Cilengitide-2 37.396 36.095 36.269 36.59 Cell migration rate 12h Cilengitide-2 36.194 35.900 35.633 35.91 1.86％ 24h Cilengitide-2 33.032 33.717 33.902 33.55 8.31％

[0218] Table 12 End point cell migration rate of the control group at different time points

[0219] 0h control 35.040 35.199 36.606 35.62 Cell migration rate 12h control 34.314 34.058 34.553 34.31 3.68％ 24h control 32.129 32.771 33.544 32.81 7.89％

[0220] Table 13 End point cell migration rate of Cilengitide-1 group at different time points

[0221] 0hCilengetide-1 35.244 35.387 36.021 35.55 Cell migration rate 12h Cilengitide-1 34.532 34.551 34.118 34.40 3.23％ 24h Cilengitide-1 33.593 34.025 33.624 33.75 5.06％

[0222] Table 14 End point cell migration rate of Cilengitide-2 group at different time points

[0223] 0hCilengetide-2 36.253 36.379 36.401 36.34 Cell migration rate 12h Cilengitide-2 35.993 35.886 36.022 35.97 1.02％ 24h Cilengitide-2 34.229 34.685 34.266 34.39 5.37％

[0224] 4. Screening and detection of overexpression cell lines

[0225] 4.1 Lentivirus infection of cells

[0226] (1) Primary, immortalized, and fused HUVEC cells (5×10 5 cells) were inoculated in 6-well plates and cultured until the confluence reached 70%-80%;

[0227] (2) Infect cells with lentivirus overexpressing integrin αvβ3 at an MOI (1:20) (depending on the specific experiment), and perform pressure screening by treating confluent HUVEC cells overexpressing integrin αvβ3 with different concentration gradients of puromycin (2, 4, 8, and 16 μg / mL) (referred to as primary cell III);

[0228] (3) After 48 h, the expression of zsGreen1 was observed under a fluorescence microscope, and cells with a fluorescent cell ratio of more than 85% were selected for this study.

[0229] 4.2 Cell culture overexpressing integrin αvβ3

[0230] Confluent HUVEC cells overexpressing integrin αvβ3 were cultured in the corresponding culture medium. The culture medium was changed every three days. When the cell confluence reached 80%, the cells were passaged. The cells were again photographed with fluorescence to detect the positive rate of empty vector expression.

[0231] 4.3 Western blot analysis of the background expression level of integrin αvβ3 in primary cell line III, the specific operation steps are the same as 2.2.1.

[0232] 4.4 Flow cytometry analysis of the background expression level of integrin αvβ3 in primary cell line III

[0233] The experimental process is the same as 2.3.1.

[0234] The streaming results are as follows Fig.11 and Fig.12 The results of Western blot and flow cytometry showed that compared with the positive control (SKOV3) cells, the background expression of integrin in HUVEC primary cells was low, while the immortalization and fusion of HUVEC were higher than those of the positive cell group, indicating relatively high expression.

[0235] 4.5 HUVEC cell detection (the third functional experiment)

[0236] HUVEC cell groups: primary cell II, primary cell II + positive drug, primary cell III, primary cell III + positive drug (for the experimental process, please refer to the first functional experiment).

[0237] Experimental results:

[0238] The results of the test on the tube-forming ability of fusion HUVEC cells showed that: under the same dose of positive drugs, when the expression level of integrin αvβ3 in fusion HUVEC cells was 1-20 times that of normal primary or immortalized HUVEC cells, the positive drugs had the most significant effect on fusion HUVEC cells, and the number of tubes formed was significantly reduced.

[0239] The results of the scratch repair test on fused HUVEC cells showed that under the same dose of positive drugs, when the expression level of integrin αvβ3 in fused HUVEC cells was 1-20 times that of normal primary or immortalized HUVEC cells, the positive drugs had the most significant effect on fused HUVEC cells, and the healing area was significantly reduced.

[0240] The results of the Transwell assay for fusion HUVEC cell migration or the cell adhesion experiment showed that: under the same positive drug dose, when the expression level of integrin αvβ3 in fusion HUVEC cells was 2-20 times that of normal primary or immortalized HUVEC cells, the positive drug had the most significant effect on fusion HUVEC cells and could significantly inhibit the chamber migration or cell adhesion of fusion HUVEC cells.

[0241] Based on the above results, fused HUVEC cells with integrin αvβ3 expression levels 2-20 times that of primary or immortalized HUVEC cells were selected as a cell model for drug screening.

[0242] In this embodiment, fused HUVEC cells with an expression level of integrin αvβ3 in the range of 2-20 times that of primary or immortalized HUVEC cells, i.e., cells overexpressing αvβ3 on EA.hy926, were deposited in the China Center for Type Culture Collection (CCTCC) on December 29, 2022, with the deposit number CCTCC NO: C2022394.

[0243] Example 2 HUVEC cells for drug screening

[0244] This example is used to screen drugs with proliferation inhibition and adhesion effects using HUVEC cells.

[0245] The test drugs used were TB1030-TB1057 (abbreviated as P30-57), a total of 28 peptides, the specific sequences of which are shown in Table 23, and were synthesized by Zhuhai Tengbai Pharmaceutical Co., Ltd.

[0246] The positive drugs used were TB01, TB1013, cilengitide, and bevacizumab.

[0247] 1. HUVEC proliferation inhibition experiment

[0248] HUVEC cells were seeded into 96-well plates at a density of 6000 cells / well, and then treated with 200 μg / ml of each peptide for 24 hours. Each peptide was tested in triplicate (n=3). Cell viability values ​​were obtained using CCK8 assay, VEGF solution was used as blank control (CTL), **P<0.05, ***P<0.001, one-way ANOVA.

[0249] The results are as follows Fig.13 The results showed that at a concentration of 200 μg / ml, P36, P39, P40, P41, P44-P50 had a significant inhibitory effect on HUVEC.

[0250] 2. HUVEC cell adhesion assay

[0251] The effects of P41-P43 and P50-P57 on HUVEC cell adhesion were tested. Plates were coated with vitronectin in advance, cells were treated with peptides (100 μg / ml) for 2 hours, and non-adherent cells were washed off. The relative absorbance of adherent cells was measured by CCK8 method and recorded by microscopic photography. ****p<0.0001 is significant compared with the control group (CTL).

[0252] like Fig.14 As shown, the results showed that P50 and P42 could significantly inhibit the adhesion of HUVEC to vitronectin coating, especially P50 had a very good adhesion effect.

[0253] 3. Effects of different concentrations of P50 on HUVEC cell adhesion

[0254] Referring to the above experiment, HUVEC cells were treated with different concentrations (10, 30 and 100 μg / ml) of P50 and 100 μg / ml of TB01 for 2 hours, non-adherent cells were washed away, and microscopic photos were taken to record the results. Fig.15 As shown in A. The relative absorbance of adherent HUVEC cells was determined by CCK8 method as shown in Fig.15 B. The adhesion curves of HUVEC cells under the action of different concentrations of P50 are shown in Fig.15 C. *p<0.05 for the comparison between P50 (30μg / ml) and the control group (CTL), ****p<0.0001 for the comparison between P50 (100μg / ml) and the control group (CTL). The results showed that P50 could significantly inhibit the adhesion of HUVEC, and its IC50 was 68.57μg / ml.

[0255] 4. HUVEC cell tube formation inhibition experiment

[0256] The cells were seeded onto Matrigel and treated with different drugs (peptide concentration was 200 μg / ml, bevacizumab was 25 μg / ml), incubated for 6 hours and observed using a phase contrast microscope (10×). Fig.16 Where A is the branch length, B is the number of branches, and the results are expressed as the percentage of tube length and branches in the control group.

[0257] The results showed that P34, P40-P44, P46, P47, P48, P49, P50, P54 and P57 had an inhibitory effect on the tube formation of HUVECs.

[0258] Table 23 Peptide sequences used

[0259]

[0260]

Claims

1. Use of human umbilical vein endothelial cells overexpressing integrin or a cell model comprising the human umbilical vein endothelial cells in screening anti-tumor drugs; The human umbilical vein endothelial cells are human umbilical vein endothelial cells with a preservation number of CCTCC No: C2022394; The screening criteria for human umbilical vein endothelial cells as a cell model are: (1) Healing area: The specified range of the healing area is at least 2-18 times that of normal primary or immortalized HUVEC cells used as a control; (2) Tube formation index: The specified range of the tube formation index is that the average value of the number of nodes, tube length and / or tube area is at least 2-18 times that of normal primary or immortalized HUVEC cells used as a control; (3) Cell migration rate: The specified range of the cell migration rate is at least 2-18 times that of a normal primary or immortalized HUVEC cell control group as a control.

2. The use according to claim 1, characterized in that The specified range of the healing area is at least 4-12 times that of normal primary or immortalized HUVEC cells as a control; And / or, the specified range of the tube formation index is that the average value of the number of nodes, the tube length and / or the tube area is at least 4-12 times that of normal primary or immortalized HUVEC cells used as a control; And / or, the specified range of cell migration rate is at least 4-12 times that of a normal primary or immortalized HUVEC cell control group as a control.

3. The use according to claim 2, characterized in that The specified range of the healing area is at least 6-9 times that of normal primary or immortalized HUVEC cells as a control; And / or, the specified range of the tube formation index is that the average value of the number of nodes, the tube length and / or the tube area is at least 6-9 times that of normal primary or immortalized HUVEC cells used as a control; And / or, the specified range of cell migration rate is at least 6-9 times that of a normal primary or immortalized HUVEC cell control group as a control.

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