Angiogenesis model and preparation method and application thereof

Abstract

The invention discloses an angiogenesis model as well as a preparation method and application thereof. The method comprises the following steps: extracting from oral squamous cell carcinoma tissues to obtain cancer-related fibroblasts in the oral squamous cell carcinoma tissues; the human umbilical cord vein endothelial cells capable of stably expressing the m-Chryry red fluorescent protein are obtained; co-culturing cancer-related fibroblasts in the oral squamous cell carcinoma tissue and the human umbilical vein endothelial cells stably expressing the m-Chryry red fluorescent protein in an ultralow adsorption plate to obtain a co-cultured cell cluster; and adding a co-culture medium into the co-culture cell cluster system to obtain the angiogenesis model. The close contact type co-culture of the two cells is realized through close contact type co-culture of the two cells, and compared with a traditional indirect co-culture model for treating the cells by using a conditioned culture medium, the close contact type co-culture model disclosed by the invention can better simulate direct interaction between the cells in an in-vivo tumor microenvironment.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue engineering, and in particular to an angiogenesis model and a preparation method and application thereof. Background Art

[0002] Tumor angiogenesis plays a vital role in tumor progression and is an important target in cancer treatment. When the tumor grows to 2-3 mm, in order to meet its own nutritional needs, tumor cells secrete pro-angiogenic factors to stimulate endothelial cell proliferation and achieve angiogenesis. The imbalance of secretion of pro-angiogenic factors and angiogenesis inhibitors in the tumor leads to abnormal angiogenesis. However, tumor angiogenesis is not only mediated by tumor cells.

[0003] Cancer associated fibroblasts (CAFs) are one of the most important components of the tumor microenvironment and are the main source of extracellular matrix components such as collagen, fibronectin, and matrix metalloproteinases. CAFs can secrete a variety of cytokines, chemokines, and exosomes, and are widely involved in cell proliferation, differentiation, migration, and angiogenesis.

[0004] Targeting the interaction between stromal cells in the tumor microenvironment is of great significance for tumor treatment. In previous studies, in vitro experiments based on the study of tumor angiogenesis-related phenotypes and mechanisms mainly relied on endothelial cell proliferation experiments, matrix gel lumen formation experiments, and chick embryo chorioallantoic membrane vascular experiments.

[0005] However, the above-mentioned in vitro experiments based on the two-dimensional culture system cannot simulate the sprouting and lumen formation of tumor blood vessels in vivo. Therefore, it is necessary to develop an angiogenesis model that can better simulate the sprouting and lumen formation of tumor blood vessels in vivo. Summary of the invention

[0006] The purpose of the present invention is to provide an angiogenesis model and its preparation method and application. The angiogenesis model is obtained by co-culturing cancer-associated fibroblasts with human umbilical vein endothelial cells (HUVEC), which can observe and integrate important matrix components in the tumor microenvironment in real time. It can not only better simulate the sprouting and branching tubular structure of tumor blood vessels in vivo, but also provide a new mechanism research model for exploring the interaction between cancer-associated fibroblasts and endothelial cells.

[0007] In order to achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect of the present invention, a method for preparing an angiogenesis model is provided, the method comprising:

[0009] Extracted from oral squamous cell carcinoma tissue, cancer-associated fibroblasts in oral squamous cell carcinoma tissue were obtained;

[0010] Obtain human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein;

[0011] Co-culturing the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue and the human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein in an ultra-low adsorption plate to obtain a co-cultured cell cluster;

[0012] A co-culture medium is added to the co-culture cell cluster system to form a stable branch and tubular structure to obtain an angiogenesis model.

[0013] Furthermore, the method for preparing cancer-associated fibroblasts in oral squamous cell carcinoma tissue comprises:

[0014] Fresh oral squamous cell carcinoma tissue was obtained, washed and cut into small pieces, digested and added into culture medium to obtain cancer-associated fibroblasts in oral squamous cell carcinoma tissue.

[0015] Furthermore, the preparation method of human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein includes: inoculating umbilical vein endothelial cells in a six-well plate, and when the cells grow to a density of 30%, infecting them with a lentiviral vector expressing m-Cherry red fluorescent protein (Gene Gene, Shanghai, China), and when the cells converge to a density of 80%, adding puromycin to screen successfully infected cells.

[0016] Furthermore, the addition ratio of the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue to the human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein is (4-6):1 according to the number of cells.

[0017] In a second aspect of the present invention, a angiogenesis model obtained by using the method is provided.

[0018] In the third aspect of the present invention, the application of the angiogenesis model in screening drugs for inhibiting and promoting angiogenesis is provided.

[0019] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0020] 1. The present invention provides an angiogenesis model and its preparation method and application. The present invention constructs an angiogenesis evaluation model by co-culturing two cells in close contact, thereby achieving close contact co-culture of two cells. Compared with the traditional indirect co-culture model using conditioned medium to treat cells, the present invention can better simulate the direct interaction between cells in the tumor microenvironment in vivo. This provides a model that is easier to promote, simpler and easier to operate for studying the interaction mechanism between cancer-associated fibroblasts and endothelial cells. Compared with 3D printing technology and microfluidic technology, the construction method of this model does not require complex equipment, is simple to operate and easy to promote.

[0021] 2. The present invention constructs endothelial cells that stably express m-Cherry red fluorescent protein, and the present invention can use a fluorescence microscope to observe in real time the process of endothelial cells forming sprouts and branched tubular structures in a simulated in vivo vascular environment. This makes up for the species differences and the limitations of inability to observe in real time in animal in vivo experiments, as well as the shortcomings of endothelial cells in the inability to simulate the formation of stable sprouts and branched tubular structures of in vivo blood vessels in lumen formation experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 This is a cell immunofluorescence identification diagram of cancer-associated fibroblast surface markers in the example (scale bar = 50 μm);

[0024] Figure 2 Figure 1 shows HUVEC stably expressing m-Cherry red fluorescent protein in the examples (scale bar = 50 μm);

[0025] Figure 3 It is a pattern diagram of the present invention in the embodiment;

[0026] Figure 4 Comparison of endothelial cell morphology under various culture conditions. AB: Red fluorescent protein-labeled endothelial cells matrix gel tube formation experiment; CD: Red fluorescent protein-labeled endothelial cells seeded in matrix gel; EF: Red fluorescent protein-labeled endothelial cells and CAFs co-seeded in ultra-low adhesion plates to form co-cultured cell clusters; GH: Co-cultured cell clusters seeded in matrix gel (scale bar = 100 μm);

[0027] Figure 5HE staining of tumor parent tissue sections and HE staining of cell cluster sections in the embodiment;

[0028] Figure 6 Immunofluorescence staining of cell cluster sections in the example (scale bar = 100 μm). DETAILED DESCRIPTION

[0029] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0030] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0031] It should be understood that the terms "length", "width", "up", "down", "front", "back", "first", "second", "vertical", "horizontal", "top", "bottom", "inside", "outside", etc., indicating the orientation or position relationship, are based on the orientation or position relationship shown in the drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0032] In addition, in the description of the present application, “multiple” and “several” mean two or more, unless otherwise clearly and specifically defined.

[0033] The overall idea of ​​the technical solution of this application is as follows:

[0034] According to a typical embodiment of the present invention, a method for preparing an angiogenesis model is provided, the method comprising:

[0035] Step S1, extracting from oral squamous cell carcinoma tissue to obtain cancer-associated fibroblasts in the oral squamous cell carcinoma tissue;

[0036] In the step S1,

[0037] As a specific embodiment, the step of extracting cancer-associated fibroblasts from oral squamous cell carcinoma tissue is:

[0038] Fresh oral squamous cell carcinoma tissue was obtained and washed with PBS containing 20% ​​antibiotics. The tissue was cut into small pieces (<1 mm 3) and add DMEM / F12 medium containing digestive enzymes (trypsin, type IV collagenase, hyaluronidase). Digest for 1 hour on a shaker (37°C), and place the remaining large pieces of tissue after digestion at the bottom of a T25 culture dish to allow it to adhere to the wall. Add DMEM / HG medium containing 10% serum, but do not touch the tissue. Turn the T25 culture dish upside down and place it overnight until the tumor tissue blocks adhere to the wall, then turn it back to the normal culture state. After 7-10 days, cancer-associated fibroblasts can be seen crawling out of the tumor tissue blocks.

[0039] Step S2, obtaining human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein;

[0040] As a specific embodiment, the method for preparing human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein comprises:

[0041] Umbilical vein endothelial cells were seeded in six-well plates. When the cells grew to 30% density, they were infected with a lentiviral vector expressing m-Cherry red fluorescent protein (Gene Gene, China, GV549). When the cells converged to 80% density, puromycin was added to screen the successfully infected cells.

[0042] Step S3, co-culturing the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue and the human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein in an ultra-low adsorption plate to obtain a co-cultured cell cluster;

[0043] In the step S3,

[0044] The addition ratio of the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue to the human umbilical vein endothelial cells stably expressing the m-Cherry red fluorescent protein is (4-6):1 according to the number of cells.

[0045] Co-culturing according to the above ratio can better simulate the ratio of fibroblasts to endothelial cells in tumor tissues. If the addition ratio is not within the above range, the tube-forming ability of endothelial cells decreases, and even the angiogenesis model cannot be successfully obtained.

[0046] The culture medium used was DMEM / HG culture medium (Sciencecell, China, catalog number sh30022.01) and ECM culture medium (Sciencecell, USA, catalog number 1001) in a volume ratio of (4-6):1.

[0047] Step S3, co-culturing the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue and the human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein in an ultra-low adsorption plate to obtain a co-cultured cell cluster;

[0048] According to another typical embodiment of the present invention, there is provided an angiogenesis model obtained by using the method described above.

[0049] The innovation of the present invention lies in: by constructing endothelial cells that stably express m-Cherry red fluorescent protein, and then by close contact co-culturing of the endothelial cells that stably express m-Cherry red fluorescent protein and cancer-associated fibroblasts in oral squamous cell carcinoma tissue, an angiogenesis evaluation model is constructed, and the close contact co-culture of the two cells is achieved. Compared with the traditional indirect co-culture model using conditioned medium to treat cells, the present invention can better simulate the direct interaction between cells in the tumor microenvironment in vivo.

[0050] According to another typical embodiment of the present invention, there is provided application of the angiogenesis model in screening drugs for inhibiting and promoting angiogenesis.

[0051] The angiogenesis model of the present application and its preparation method and application will be described in detail below with reference to the embodiments and drawings.

[0052] Example 1: Human oral squamous cell carcinoma angiogenesis evaluation model and its construction method

[0053] 1. Extraction of human oral squamous cell carcinoma cancer-associated fibroblasts: Wash oral squamous cell carcinoma tissue with PBS containing 20% ​​antibiotics, cut it into small pieces with scissors, add DMEM / F12 medium containing digestive enzymes (trypsin, type IV collagenase, hyaluronidase), place on a shaker (37°C) for digestion for 1 hour, place the remaining large pieces of tissue after digestion on the bottom of a T25 culture dish to make it adhere to the wall, add DMEM / HG medium containing 10% serum, but do not touch the tissue, turn the T25 culture dish over and place it, wait overnight until the tumor tissue pieces adhere to the wall, turn it back to the normal culture state, and wait 7-10 days until CAFs can be seen crawling out of the tumor tissue pieces;

[0054] 2. Identification of surface markers of human oral squamous cell carcinoma-associated fibroblasts: Figure 1 As shown, CAFs were seeded in 24-well plates one day in advance. The next day, the cells were washed 2-3 times with PBS, fixed with 4% paraformaldehyde, permeabilized with 0.5% TritonX-100 at room temperature for 20 minutes, blocked with 10% secondary antibody homologous serum, added with primary antibody dilution, and incubated overnight at four degrees. The next day, a secondary antibody homologous to the primary antibody was added, incubated at room temperature for 1 hour, the secondary antibody was washed off, DAPI staining solution was added, and the slides were sealed with anti-fluorescence quenching sealing agent. Observe and collect images under a fluorescence microscope;

[0055] 3. Transfect cells:

[0056] The HUVEC stably expressing mCherry red fluorescent protein was established by lentiviral transfection method, and the stably transfected cells were screened by puromycin. Figure 2 As shown;

[0057] 4. Construction of CAFs and HUVEC co-culture model:

[0058] like Figure 4 As shown in AB, endothelial cells labeled with red fluorescent protein were seeded on preheated and solidified matrix gel, and observed and photographed under a microscope for 6-8 hours; Figure 4 As shown in CD, 3 μl of red fluorescent protein-labeled endothelial cell single-cell suspension was taken, added to 300 μl of matrix gel and mixed thoroughly on ice, inoculated into a preheated 24-well plate, and immediately placed in a 37°C incubator and added with ECM culture medium after solidification. After 24 hours, photos were taken to record the results; Figure 3 and Figure 4 As shown in CD, CAFs and HUVEC cells were digested and added to the ultra-low adsorption plate at a ratio of 5:1. After 24 hours, HUVECs were observed to form branches and tubular structures;

[0059] 5. Identification of CAFs and HUVEC co-culture model:

[0060] like Figure 5 As shown, CAFs-m-Cherry-HUVEC co-cultured cells were collected at the time point of interest, and the matrix gel-cell mixture was embedded in OCT gel as completely as possible to prepare frozen sections. PBS was added to the frozen sections for 3 times, and the residual liquid was shaken off. An appropriate amount of eosin stain was taken to cover the frozen sections, shaken for 1 minute, and the frozen sections were rinsed with running water. An appropriate amount of hematoxylin stain was taken and shaken slowly and evenly for 10 seconds, and then rinsed with running water. The HE staining results were observed under a microscope, the sections were sealed with neutral gum, and the results were recorded by taking pictures under a microscope;

[0061] like Figure 6 As shown, PBS was added to the frozen sections prepared above, rinsed 3 times, fixed with 4% paraformaldehyde, added with 1:200 diluted COL1A1 primary antibody, incubated at 4 degrees overnight, and the next day, added with green fluorescent secondary antibody from the same source as the primary antibody, incubated at room temperature for 1 hour, added with DAPI staining solution for 1 minute, and sealed with anti-fluorescence quenching sealing solution. The staining was observed under a fluorescence microscope and photographed to record the results. Endothelial cells formed branched tubular structures.

[0062] Example 2: Human oral squamous cell carcinoma angiogenesis evaluation model and its construction method

[0063] In this embodiment, the ratio of the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue to the human umbilical vein endothelial cells stably expressing the m-Cherry red fluorescent protein is 4:1 according to the number of cells, and the other steps are the same as in Embodiment 1.

[0064] Example 3: Human oral squamous cell carcinoma angiogenesis evaluation model and its construction method

[0065] In this embodiment, the addition ratio of the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue to the human umbilical vein endothelial cells stably expressing the m-Cherry red fluorescent protein is 6:1 according to the number of cells, and the other steps are the same as in Embodiment 1.

[0066] Comparative Example 1

[0067] In this comparative example, the addition ratio of the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue to the human umbilical vein endothelial cells stably expressing the m-Cherry red fluorescent protein is 1:1 based on the number of cells, and the other steps are the same as in Example 1.

[0068] Comparative Example 2

[0069] In this comparative example, the addition ratio of the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue to the human umbilical vein endothelial cells stably expressing the m-Cherry red fluorescent protein is 8:2 based on the number of cells, and the other steps are the same as in Example 1.

[0070] Experimental Example 1: Performance Measurement

[0071] The performance of the angiogenesis models obtained in Example 1 to Example 3 is shown in Table 1.

[0072] Table 1

[0073] Group Success rate of forming stable branched tubular structures Example 1 100％ Example 2 100％ Example 3 100％ Comparative Example 1 Unable to build successfully Comparative Example 2 Unable to build successfully

[0074] From Table 1, we can see that:

[0075] In comparative examples 1-2, the ratio of endothelial cells added was too high, which could not simulate the ratio of fibroblasts to endothelial cells in tumors and the endothelial cells could not form branched tubular structures. The angiogenesis models obtained in examples 1-3 had good endothelial cell tube-forming ability, with a success rate of up to 100%.

[0076] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0077] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0078] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing an angiogenesis model, characterized in that: The method comprises: Extracted from oral squamous cell carcinoma tissue, cancer-associated fibroblasts in oral squamous cell carcinoma tissue were obtained; Obtain human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein; Co-culturing the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue and the human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein in an ultra-low adsorption plate to obtain a co-cultured cell cluster; A co-culture medium is added to the co-culture cell cluster system to form a stable branch and tubular structure to obtain an angiogenesis model.

2. The method for preparing an angiogenesis model according to claim 1, characterized in that: The method for preparing cancer-associated fibroblasts in oral squamous cell carcinoma tissue comprises: Fresh oral squamous cell carcinoma tissue was obtained, washed and cut into small pieces, digested and added into culture medium to obtain cancer-associated fibroblasts in oral squamous cell carcinoma tissue.

3. The method for preparing an angiogenesis model according to claim 1, characterized in that: The method for preparing human umbilical vein endothelial cells stably expressing m-Cherry red fluorescent protein comprises: Umbilical vein endothelial cells were seeded in six-well plates. When the cells grew to a density of 30-40%, they were infected with a lentiviral vector expressing m-Cherry red fluorescent protein. When the cells converged to a density of 70-90%, puromycin was added to screen for successfully infected cells.

4. The method for preparing an angiogenesis model according to claim 1, characterized in that: The addition ratio of the cancer-associated fibroblasts in the oral squamous cell carcinoma tissue to the human umbilical vein endothelial cells stably expressing the m-Cherry red fluorescent protein is (4-6):1 according to the number of cells.

5. The method for preparing an angiogenesis model according to claim 1, characterized in that: The culture medium used for the co-culture is DMEM / HG culture medium and ECM culture medium in a volume ratio of (4-6):

1.

6. An angiogenesis model obtained by the method according to any one of claims 1 to 5.

7. Use of the angiogenesis model according to claim 6 in screening drugs for inhibiting and promoting angiogenesis.

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