Method for domesticating HUVECs to simulate colorectal cancer vascular endothelial cells and domesticating culture medium

Abstract

The invention relates to the technical field of tumor in-vitro models, in particular to a method for domesticating HUVECs to simulate colorectal cancer vascular endothelial cells and a domestication culture medium. The colorectal cancer vascular endothelial cell specific secretion factor IGFBP5 is obtained through single cell sequencing analysis, the IGFBP5 and the HUVECs are added into an HUVECs complete culture medium, an in-vitro model with better tumor vascular characteristics can be formed, colorectal cancer blood vessels are better simulated, and a more simulated colorectal cancer in-vitro model is constructed.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro tumor models, and more specifically, it relates to a method for domesticating HUVECs to simulate colorectal cancer vascular endothelial cells and a domestication culture medium. Background Art

[0002] Angiogenesis plays an important role in the growth, invasion, and metastasis of colorectal cancer. There is a complex interaction between angiogenesis and colorectal cancer. Therefore, in vitro studies of colorectal cancer rely on the construction of a good functional vascularized in vitro model. Among these endothelial cell-based vascularized models, human umbilical vein endothelial cells (HUVECs) are most widely used, which have the characteristics of being close to autologous blood vessels, having the ability to form tubes, and being easily sourced. However, there are significant gene expression differences between tumor vascular endothelial cells and normal vascular endothelial cells. At the same time, compared with normal blood vessels, tumor blood vessels also show obvious structural and functional abnormalities. Tumor blood vessels are tortuous, with a thinner effective diameter, more irregularly connected branches, weakened inter-endothelial cell junctions, increased vascular gaps, resulting in increased vascular permeability. Therefore, the current colorectal cancer vascular models have a large gap from the ideal in vitro model both in terms of morphology and function. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the problem that the vascular model constructed with HUVECs in the existing in vitro colorectal cancer model lacks the characteristics of colorectal cancer blood vessels and cannot simulate colorectal cancer blood vessels well. By adding the colorectal cancer-specific secreted factor IGFBP5 to the complete medium of HUVECs, domesticate HUVECs to simulate colorectal cancer vascular endothelial cells, so as to construct a colorectal cancer in vitro vascular model with more tumor characteristics.

[0004] To achieve the above object, the present invention adopts the following technical solutions:

[0005] First, screen colorectal cancer-specific secreted factors through single-cell analysis technology. The relevant steps are as follows:

[0006] (1) Select the colorectal cancer single-cell transcriptome database;

[0007] (2) Obtain the transcriptome expression datasets of vascular endothelial cells from colorectal cancer patients and paired normal tissues;

[0008] (3) Screen out differentially expressed genes and rank them; among which the gene with the most obvious differential expression is IGFBP5.

[0009] Secondly, add IGFBP5 to the complete medium of HUVECs to domesticate HUVECs. The relevant steps are as follows:

[0010] (1)Prepare a complete medium for HUVECs containing endothelial cell basal medium (ECM), fetal bovine serum, penicillin / streptomycin solution, and vascular endothelial growth factor;

[0011] (2)Add 0 - 1000 ng / mL of IGFBP5 to the complete medium for HUVECs respectively to obtain the acclimation medium;

[0012] (3)Digest and plate HUVECs, and culture them using the acclimation medium supplemented with different concentrations of IGFBP5.

[0013] (4)Detect the proliferation of HUVECs under different culture conditions using CCK8.

[0014] Then analyze the angiogenesis experiment of HUVECs before and after acclimation. The relevant steps are as follows:

[0015] (1)Uniformly coat the Matrigel in a 48 - well plate;

[0016] (2)Digest the acclimated HUVECs;

[0017] (3)Resuspend with the acclimation medium supplemented with IGFBP5;

[0018] (4)Inject the resuspended HUVEC solution onto the surface of the Matrigel in the 48 - well plate;

[0019] (5)Digest the unacclimated HUVECs;

[0020] (6)Resuspend with the complete medium for HUVECs without IGFBP5;

[0021] (7)Inject the resuspended HUVEC solution onto the surface of the Matrigel in the 48 - well plate;

[0022] (8)Take pictures at 4 h and 24 h respectively;

[0023] (9)Perform quantitative analysis using ImageJ software.

[0024] The present invention has the following beneficial effects: Through single - cell sequencing, the colorectal cancer vascular endothelial cell - specific secreted factor IGFBP5 is screened out in this application and added to the complete medium for HUVECs, significantly improving the simulation ability of the in vitro vascular model for tumor vascular characteristics. The acclimated HUVECs show a morphology closer to that of real colorectal cancer blood vessels in the angiogenesis experiment (the number of nodes, branches, and grids increases, and the average blood vessel length shortens), solving the problem of the lack of tumor specificity in the existing model, providing a highly simulated in vitro model for the mechanism research, drug screening, and anti - angiogenesis treatment of colorectal cancer, and having important scientific research and clinical application values. Brief Description of the Drawings

[0025] Figure 1 It is a visualization diagram of the differential analysis of single-cell transcriptomes between colorectal cancer vascular endothelial cells and normal endothelial cells;

[0026] Figure 2 It is a diagram of the differential expression of different secreted factors in colorectal cancer vascular endothelial cells;

[0027] Figure 3 It is a comparison diagram of the vascular network morphology of HUVECs on Matrigel before and after domestication;

[0028] Figure 4 It is a diagram of the quantitative results of the vascular formation experiment. Detailed Embodiments

[0029] The present invention will be further described below in conjunction with embodiments, but the scope of protection required by the present invention is not limited to the scope described in the embodiments.

[0030] Embodiment 1

[0031] In this embodiment, the colorectal cancer-specific secreted factor IGFBP5 is screened by single-cell analysis technology, and the specific steps are as follows:

[0032] (1) Select the colorectal cancer single-cell transcriptome database GSE144735, import the original count data into R, and use Seurat (version 4.2.0) 40 for analysis. Dead cells are removed according to the detected number of genes (<2,500) and the percentage of mitochondrial genes (>20%).

[0033] (2) Logarithmically normalize and scale the data, and use burgertools version 2.0.0 (https: / / github.com / nbroguiere / burgertools) and the following gene set for feature scoring: GSE144735 (CD3D, CD3E, CD79A, JCHAIN, NCAM1, FGFBP2, CD68, C1QA, TPSB2, TPSAB1, VWF, PECAM1, COL1A1, COL3A1, EPCAM, BGN, CALD1, DCN, MGP).

[0034] (3) Apply the tSNEPlot function to visualize the cells in two-dimensional space (see Figure 1 ).

[0035] (4)On this basis, genes with significant differences in the expression of colorectal cancer vascular endothelial cells and paired normal tissue vascular endothelium were further identified, and they were sorted according to the magnitude of differential expression LogFC. Among them, the gene with the highest expression and the most obvious differential expression in colorectal cancer vascular endothelial cells is IGFBP5 (see Figure 2 ).

[0036] Example 2

[0037] In this example, IGFBP5 was added to the complete medium of HUVECs to domesticate HUVECs. The specific steps are as follows:

[0038] (1)Prepare the complete medium for HUVECs

[0039] The preparation of the complete medium for HUVECs includes: endothelial cell basal culture ECM, 5% fetal bovine serum, 1% penicillin / streptomycin solution, and 1% endothelial cell growth factor.

[0040] (2)Add IGFBP5 secretion factor

[0041] Different concentrations of IGFBP5 were added to the prepared complete medium for HUVECs, and the concentrations were 0 ng / mL, 10 ng / mL, 20 ng / mL, 50 ng / mL, 80 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1000 ng / mL respectively to obtain 9 groups of domesticated media with different concentrations.

[0042] (3)Culture HUVECs with the domesticated medium

[0043] When the cell density reached about 70%-80% under the microscope, the domesticated media with different concentrations (a total of 9 groups) were aspirated, trypsin digestion solution was added, and it was placed in an incubator at 37°C with CO2 for 3 minutes. After taking it out, the corresponding concentration of domesticated medium was added, and it was gently pipetted. After the HUVECs detached, they were transferred to a 15 ml centrifuge tube and centrifuged at 300 g for 3 minutes.

[0044] The supernatant was removed, and the cells were resuspended with the corresponding concentration of domesticated medium and plated in a 96-well culture plate. The medium was changed every 2-3 days.

[0045] (4)Detect the proliferation ability of HUVECs under different culture conditions by CCK8

[0046] After taking out after the first medium change, add 10 μL of CCK-8 reagent to each well and incubate in the dark for 2 h. Detect the OD value on an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the cell proliferation ability: Cell proliferation ability (%) = [OD (plus IGFBP5) - OD (blank)] / [OD (without adding IGFBP5) - OD (blank)] × 100%. The detection and calculation results are shown in Table 1. The results show that when the added concentration of IGFBP5 is 50 ng / mL, the proliferation ability of HUVECs is the best. Therefore, the concentration of IGFBP5 added at 50 ng / mL is selected to domesticate HUVECs.

[0047] Table 1 Proliferation of HUVECs at different IGFBP5 concentrations

[0048] IGFBP5 concentration (ng / mL) 0 10 20 50 80 100 200 500 1000 Proliferation ability of HUVECs 100% 108% 111% 125% 120% 115% 105% 95% 90%

[0049] Example 3

[0050] In this example, the angioplasty experiments before and after domesticating HUVECs were analyzed. The specific steps are as follows:

[0051] (1) Plating of Matrigel

[0052] Place the 48-well plate in an incubator at 37 °C and 5% CO2 30 minutes in advance. Use a pre-cooled pipette tip to aspirate 200 μL of Matrigel and quickly and evenly spread it over the small holes in the 48-well plate to form a Matrigel plane with a consistent height.

[0053] (2) Digesting HUVECs before and after domestication and plating

[0054] Under the microscope, when the cell density reaches about 70%-80%, aspirate the domestication medium at different concentrations (a total of 2 groups, the domestication medium with an IGFBP5 concentration of 0 ng / ml corresponds to before domestication, and the domestication medium with an IGFBP5 concentration of 50 ng / ml corresponds to after domestication). Add trypsin digestion solution and place it in an incubator at 37 °C and CO2 for 3 mins. After taking it out, add the domestication medium at the corresponding concentration and gently pipette until the HUVECs detach. Transfer them to a 15 ml centrifuge tube and centrifuge at 300 g for 3 mins. Discard the supernatant, resuspend the HUVECs with the domestication medium at the corresponding concentration, and the density is about 5×10 4 / mL, drop it into the small holes that have been pre-plated with Matrigel, and then place it in an incubator at 37 °C and 5% CO2.

[0055] (3) Analysis of angiogenesis experiment

[0056] The angioplasty experiment was photographed and recorded at 4 h and 24 h (see Figure 3), and quantitative analysis was performed using the Angiogenesis Analyzer plugin of Image J software. The specific indicators were the number of nodes, branches, meshes, and average vessel length in the vascular network. Through quantitative analysis, it was found that the vascular model formed by domesticated HUVECs had more nodes (more than 20% increase, approaching 100%), branches (more than 20% increase, approaching 100%), meshes (more than 100% increase), and shorter vessel length (more than 15% reduction, approaching 40%), being closer to the real tumor vascular model (see Figure 4 ).

[0057] Combined with Examples 1 - 3, it can be seen that in this application, colorectal cancer vascular endothelial cell-specific secreted factor IGFBP5 was screened out through single-cell sequencing and added to the complete medium of HUVECs, significantly improving the simulation ability of the in vitro vascular model for tumor vascular characteristics. The domesticated HUVECs showed a morphology closer to that of real colorectal cancer blood vessels in the vascular formation experiment (the number of nodes, branches, and meshes increased, and the average vessel length decreased), solving the problem of the lack of tumor specificity in existing models, providing a highly simulated in vitro model for the mechanism research, drug screening, and anti-angiogenic therapy of colorectal cancer, and having important scientific research and clinical application values.

[0058] The above are only the preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions falling within the idea of the present invention belong to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, several improvements and refinements made without departing from the principle of the present invention should also be regarded as within the protection scope of the present invention.

Claims

1. A method for domesticating HUVECs to simulate colorectal cancer vascular endothelial cells, characterized in that Comprising the following steps: S1: Screening for the colorectal cancer vascular endothelial cell-specific secreted factor IGFBP5 by single-cell sequencing technology combined with differential expression analysis; S2: Adding IGFBP5 to the complete medium for HUVECs to domesticate HUVECs to form an in vitro model with the characteristics of colorectal cancer blood vessels, wherein the added concentration of IGFBP5 is 10 - 200 ng / mL.

2. The method according to claim 1, characterized in that, The specific method for screening IGFBP5 in step S1 includes the following steps: S11: Obtaining single-cell transcriptome data of vascular endothelial cells from colorectal cancer patients and paired normal tissues; S12: Screening for genes highly expressed in colorectal cancer vascular endothelial cells by differential expression analysis; S13: Determining IGFBP5 as a specific secreted factor based on the degree of differential expression, where IGFBP5 is the highest in the differential expression LogFC ranking.

3. The method according to claim 2, characterized in that The single-cell transcriptome data is derived from the database GSE144735, and the Seurat tool is used for data normalization, dimensionality reduction, and differential gene analysis.

4. The method according to claim 1, wherein The added concentration of IGFBP5 is 20 - 100 ng / mL.

5. The method according to claim 4, characterized in that The added concentration of IGFBP5 is 50 ng / mL.

6. The method according to claim 1, characterized in that The complete medium for HUVECs includes: endothelial cell basal medium, 5% fetal bovine serum, 1% penicillin / streptomycin solution, and 1% vascular endothelial growth factor.

7. The method according to claim 1, wherein The domesticated HUVECs are verified for their tumor blood vessel characteristics through an angiogenesis experiment, and the angiogenesis experiment includes: S21: Inoculating the domesticated HUVECs on the surface of Matrigel; S22: Analyzing its similarity to colorectal cancer blood vessels by quantifying the number of nodes, branches, meshes, and average blood vessel length of the vascular network.

8. A domestication medium for constructing an in vitro vascular model of colorectal cancer, characterized in that, Including the complete medium for HUVECs and IGFBP5; the added concentration of IGFBP5 is 10 - 200 ng / mL; the complete medium for HUVECs contains endothelial cell basal medium, 5% fetal bovine serum, 1% penicillin / streptomycin solution, and 1% vascular endothelial growth factor.

9. The domesticated culture medium according to claim 8, characterized in that, The added concentration of IGFBP5 is 50 ng / mL.

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