CN113308434A - Construction method of endothelial cell and pericyte co-culture model for researching tube formation - Google Patents

Abstract

The invention discloses a method for constructing a co-culture model of endothelial cells and pericytes for researching tube formation. The method comprises the following steps: (1) digesting the umbilical vein endothelial cells by trypsin to obtain umbilical vein endothelial single cells; (2) digesting the pericytes into pericyte single cells by using trypsin, and then adding CFSE dye with the final concentration of 2-10 mu mol/L for dyeing to obtain dyed pericytes; (3) and (3) enabling the umbilical vein endothelial single cells and the stained pericytes to be 5-20: 1, culturing the mixture in a bronchial epithelial cell culture medium, and observing tube formation in a fluorescence microscope. The method can dynamically observe the influence of the pericytes on the formation of the endothelial cells through a fluorescence microscope, reflects the effects of the human tumor pericytes and the tumor blood vessels through in vitro experiments, and has good social and economic values.

Description

Construction method of endothelial cell and pericyte co-culture model for researching tube formation

Technical Field

The invention relates to the field of tumor biology, in particular to a method for constructing a co-culture model of endothelial cells and pericytes for researching tube formation.

Background

Over the past decades we have had a more extensive and intensive understanding of primary tumors. Despite substantial advances in the treatment of some tumors, tumor treatment remains a worldwide health problem. Although new chemotherapeutic drugs and means are continuously discovered, chemotherapeutic drug resistance is still a big obstacle to limit the effect of chemotherapy. Studies have found that blood vessels are critical for the delivery of nutrients, oxygen, and even chemotherapeutic drugs in solid tumors^[1]. Therefore, the method improves the tumor vascular growth and is particularly important in the study of chemotherapy drug resistance.

Formation of new tumor vessels, i.e., tumor angiogenesis, involves enhanced proliferation and migration of endothelial cells, development of vascular basement membrane, and subsequent recruitment of supporting parietal cells including pericytes^[2]. Pericytes, also known as parietal cells, play important regulatory roles in stabilizing vascular walls, controlling vasodilation, regulating vascular perfusion, and the like^[3-4]. However, the role of pericytes in current tumor therapy studies is rarely mentioned.

Just as tumor endothelial cells differ from endothelial cells in normal tissue, tumor-derived pericytes appear abnormal or dysfunctional compared to pericytes in normal tissue. It is not clear whether cancer cells promote tumor growth by causing changes in the properties of pericytes. Indeed, in many cancers, there is a defect in pericyte-endothelial cell interaction, characterized by impaired vascular integrity^[5-6]. For example, pericyte shedding or shedding from tumor vessels can often be observed in some cancers, which is considered to be a cause of promoting cancer metastasis^[7-8]. On the other hand, pericytes have been shown to protect endothelial cells from antiangiogenesisEffects of drugs (e.g. bevacizumab), therefore in some animal models, treatment in combination with PDGFR tyrosine kinase inhibitor (i.e. to exclude pericytes) and VEGF inhibition is more effective at preventing tumor angiogenesis than anti-VEGF treatment alone^[9-10]. But the treatment has failed clinically. The main reason is that the conventional in-vitro human tube formation model does not co-culture pericytes, which influences the effectiveness of screening the targeted vascular drugs. At the same time, the relevance of pericytes in controlling cancer growth and development remains unclear, and the molecular mechanisms behind their regulation of tumor progression remain largely unknown.

Therefore, the study on the regulation and control of the pericytes on tumor blood vessels can create favorable conditions for the future study on the growth and the development of tumors and the reversal of cancer drug resistance. To solve the above scientific and clinical problems, this suggests that we need to develop a model system for co-culturing endothelial cells and pericytes for in vitro studies of tube formation.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a method for constructing a co-culture model of endothelial cells and pericytes for researching tube formation.

The purpose of the invention is realized by the following technical scheme:

a method for constructing a co-culture model of endothelial cells and pericytes for studying tube formation, comprising the following steps:

(1) digesting the umbilical vein endothelial cells by trypsin to obtain umbilical vein endothelial single cells;

(2) digesting the pericytes into pericyte single cells by using trypsin, and then adding CFSE dye with the final concentration of 2-10 mu mol/L for dyeing to obtain dyed pericytes;

(3) and (3) enabling the umbilical vein endothelial single cells and the stained pericytes to be 5-20: 1, culturing the mixture in bronchial epithelial cell culture medium (BEpicM), and observing tube formation in a fluorescence microscope.

The umbilical vein endothelial cells in step (1) are preferably Human Umbilical Vein Endothelial Cells (HUVEC).

The umbilical vein endothelial cells can be obtained by conventional methods in the art, such as by commercial purchase, or isolated and cultured from human umbilical vein; preferably obtained by the following method: digesting the umbilical vein endothelial cells by trypsin to obtain umbilical vein endothelial single cells; more preferably by the following method:

washing human umbilical vein with normal saline until colorless, then filling trypsin preheated at 37 ℃ into the human umbilical vein, sealing, incubating at 37 ℃ for 15min, blowing human umbilical vein endothelial cells into DMEM complete culture medium to stop digestion, centrifuging, and resuspending and dispersing with endothelial cell culture medium (ECM) to obtain umbilical vein endothelial single cells.

The centrifugation conditions are as follows: centrifuge at 300g for 5 min.

The culture conditions are as follows: culturing at 37 deg.C for more than 24 hr.

The endothelial cell culture medium (ECM) has the following formula: 500ml basal medium, 25ml bovine serum (FBS), 5ml endothelial cell growth factor (ECGS) and 5ml penicillin/streptomycin solution (P/S); preferred is endothelial cell culture medium (ECM) purchased from ScienCell corporation, cat 1001.

The digestion conditions in the step (1) are as follows: digesting for 10-20 min at 37 ℃; preferably, digestion is carried out at 37 ℃ for 15 min.

The pericyte described in step (2) is preferably a human Pericyte (PC).

The human pericytes comprise Tumor Pericytes (TPC) and paracancer normal tissue pericytes (NPC); preferably Tumor Pericytes (TPC).

The tumor comprises lung cancer, liver cancer and the like.

The pericytes can be obtained by the conventional method in the field, or can be obtained by separating and culturing tumor tissues or paracancerous normal tissues; preferably obtained by the following method:

(I) washing tumor tissue or normal tissue beside cancer with normal saline to colorless, cutting, adding enzymolysis solution for enzymolysis and digestion, centrifuging, and dispersing with pericyte culture medium (PM) to obtain pericyte single cell;

(II) placing the single cells of the pericytes in the coated culture plate to be cultured until the cells are attached to the wall, and obtaining the Tumor Pericytes (TPC) or the paracancer normal tissue pericytes (NPC).

The enzymolysis solution in the step (I) is a DMEM culture medium containing 2% (w/v) type I collagenase type1, 2% (w/v) type III collagenase type3 and 1% (w/v) DNase.

The formula of the pericyte culture medium (PM) described in step (I) is as follows: 500ml basal medium, 10ml Fetal Bovine Serum (FBS), 5ml peripheral cell growth supplement (PGS) and 5ml penicillin/streptomycin solution (P/S); percell culture medium (PM) from ScienCell, Inc., cat # 1201 is preferred.

The centrifugation conditions described in step (I) are: centrifuge at 300g for 5 min.

The digestion conditions in step (I) are: digesting for 1-2 hours at 37 ℃; preferably, the digestion is carried out at 37 ℃ for 1.5 hours.

The coated culture plate in the step (II) is obtained by the following method: adding collagen into a gelatin solution, and uniformly mixing to obtain a coating solution; and adding the coating solution into the culture plate, and culturing in an incubator at 37 ℃ for more than 30min to obtain the coated culture plate.

The mass ratio of the collagen to the gelatin is preferably 1: 5.

the culture plate is preferably a 6-well plate.

The culture medium used in the culture in the step (II) is pericyte culture medium (PM), and the formula is as follows: 500ml basal medium, 10ml Fetal Bovine Serum (FBS), 5ml peripheral cell growth supplement (PGS) and 5ml penicillin/streptomycin solution (P/S); percell culture medium (PM) from ScienCell, Inc., cat # 1201 is preferred.

The culture conditions in step (II) are as follows: culturing at 37 deg.C for more than 24 hr.

The CFSE dye is preferably used in the step (2) in an amount calculated by adding it at a final concentration of 5. mu. mol/L in the reaction system (i.e., working concentration of 5. mu.M).

The ratio of the number of the umbilical vein endothelial single cells to the number of the stained pericytes in the step (3) is preferably 10: 1.

the formula of the bronchial epithelial cell culture medium (BEpicM) in the step (3) is as follows: 500ml of basal medium, 5ml of bronchial epithelial cell growth supplement (BEpicGS) and 5ml of penicillin/streptomycin solution (P/S), without serum; preferred is bronchial epithelial cell culture medium (BEpicM) purchased from ScienCell corporation, cat # 3211.

The culturing in the step (3) is carried out on a culture plate previously coated with Matrigel.

The culture plate is preferably a 96-well plate.

The culture conditions in the step (3) are as follows: culturing at 37 ℃ for 4-48 hours.

Any one of the endothelial cell and pericyte co-culture models constructed by the construction method for researching the tube-formed endothelial cell and pericyte co-culture model.

Compared with the prior art, the invention has the following advantages and effects:

1. the invention discloses a method for in vitro co-culturing human endothelial cells and pericytes to research the influence of tumor inner peripheral cells on tumor blood vessels, which is characterized in that Human Umbilical Vein Endothelial Cells (HUVEC) are mixed with pericytes and bronchial epithelial cell culture medium (BEpicM) which are stained with CFSE in advance, the mixture is added into a 96-well plate which is coated with matrigel in advance for culturing, and the tube forming condition is observed by a fluorescence microscope.

2. The invention co-cultures the human endothelial cells and the pericytes for the first time, can dynamically observe the influence of the pericytes on the formation of the endothelial cells through a fluorescence microscope, reflects the action of the human tumor inner pericytes and the tumor blood vessels through in vitro experiments, and further discloses a molecular mechanism behind the regulation and control of tumor progress of the pericytes.

3. The co-culture method can be used for researching the regulation and control of Tumor Pericytes (TPC) and paracancer normal tissue pericytes (NPC) on angiogenesis, can improve the function of tumor blood vessels by regulating the action of the pericytes, and provides a new idea for the research of regulating the tumor blood vessels and promoting the delivery of antitumor drugs in the future.

4. The co-culture method provided by the invention provides a normal or tumor angiogenesis model closer to the normal physiological condition, can more accurately test the preclinical effectiveness of the drug for targeting regulation and control of tumor angiogenesis and functions, reduces unnecessary animal models and clinical experimental tests, and has good social and economic values.

Drawings

FIG. 1 is a fluorescent microscope photograph of the growth of HUVEC and PC coculture along with statistics of their vascular length, number of branches and number; wherein A is the growth condition of HUVEC, NPC + HUVEC and TPC + HUVEC groups shot by a fluorescence microscope (green fluorescence is PC, and white light is a co-culture picture of HUVEC and PC); b is the statistics of the vessel length, branch number and number of HUVEC, NPC + HUVEC and TPC + HUVEC groups.

FIG. 2 is a graph of the co-culture growth of HUVEC and TPC taken by fluorescence microscopy in comparative examples 1-3; wherein; a is the co-culture growth condition of the HUVEC and the TPC in the human endothelial serum-free culture medium in the comparative example 1; b is the ratio of HUVEC and TPC in comparative example 2 is 1: 1, co-culture growth conditions; c is the ratio of HUVEC and TPC in comparative example 2 is 50: 1, co-culture growth conditions; d is the growth observed in the co-culture of HUVEC and TPC in comparative example 3 at a CFSE concentration of 20 μm; e is the growth observed in the co-culture of HUVEC and TPC in comparative example 3 at a CFSE concentration of 0.5m μm.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

The endothelial cell culture medium (ECM) (ScienCell, 1001) referred to in the examples of the present invention comprises 500ml basal medium, 25ml bovine serum (FBS, cat. No.0025), 5ml endothelial cell growth factor (ECGS, cat. No.1052), 5ml penicillin/streptomycin solution (P/S, cat. No. 0503).

The enzymolysis solution is a DMEM culture medium containing 1-2.5% (w/v) type I collagenase 1(Worthington, LS004194), 1-2.5% (w/v) type III collagenase 3(Worthington, LS004182) and 0.5-1.5% (w/v) DNase (Roche, 10104159001); the enzymatic hydrolysate referred to in the examples is preferably a DMEM medium containing 2% (w/v) collagenase type I1, 2% (w/v) collagenase type III 3, 1% (w/v) DNase.

The pericyte culture medium (PM) (ScienCell, 1201) referred to in the examples of the invention comprises 500ml of basal medium, 10ml of fetal bovine serum (FBS, Cat. No.0010),5ml of a peripheral cell growth supplement (PGS, Cat. No.1252), and 5ml of a penicillin/streptomycin solution (P/S, Cat. No. 0503).

The bronchial epithelial cell culture medium (BEpicM) (ScienCell, 3211) referred to in the examples of the invention contained 500ml of basal medium, 5ml of bronchial epithelial cell growth supplement (BEpicGS, Cat. No.3262) and 5ml of penicillin/streptomycin solution (P/S, Cat. No.0503), without serum.

Example 1

This example provides a method for in vitro co-culturing human endothelial cells and pericytes for studying tube formation, all isolation and culture operations being performed in a biosafety cabinet, comprising the following steps:

(1) washing human umbilical vein (from Guangzhou hospital) with normal saline to colorless, injecting trypsin preheated at 37 deg.C into human umbilical vein with syringe, sealing, incubating at 37 deg.C for 15min, and blowing DMEM complete medium into human umbilical vein endothelial cells to stop digestion.

(2) Centrifuging the cells at room temperature of 300g for 5min, resuspending the cells into single cells with endothelial cell culture medium (ECM), placing the cells in 6-well plate at 37 deg.C and 5% CO₂The culture was carried out for 24 hours. And (4) sucking off the suspension cells and replacing with a new culture medium, wherein the anchorage-dependent growth cells are Human Umbilical Vein Endothelial Cells (HUVEC).

(3) Washing lung cancer tissue and paracancer normal tissue (both from Guangzhou hospital) with normal saline until colorless, and respectively cutting into pieces; then adding the enzymolysis liquid into the cut tissues respectively, and digesting for 1.5 hours at 37 ℃; wherein the enzymolysis solution is DMEM culture medium containing 2% (w/v) collagenase type I1, 2% (w/v) collagenase type III 3 and 1% (w/v) DNase.

(4) Digesting tissues and preparing a culture plate coating solution, adding 320 mu L of 30 mu G/mL collagen (Sigma, C7661-25MG) solution into 48mL of 0.1% (w/V) gelatin (Sigma, V900863-100G) solution, and uniformly mixing; the coating solution was then added to 6-well plates and incubated in an incubator at 37 ℃ for over 30 min. Excess coating solution was aspirated off before adding the cells.

(5) Centrifuging the digested cells in step (3) at room temperature of 300g for 5min, resuspending the cells in pericyte culture medium (PM), dispersing the cells into single cells, placing the single cells on the coated 6-well plate obtained in step (4), and treating the single cells at 37 ℃ with 5% CO₂The culture was carried out for 24 hours. And (4) sucking off the suspension cells and replacing with a new culture medium, wherein the adherent growth cells are Tumor Pericytes (TPC) and paracancer normal tissue pericytes (NPC).

(6) The HUVEC cells obtained in step (2), and TPC and NPC cells obtained in step (5) were observed under a microscope, and tube formation experiments were conducted when the cells were grown to 80% (confluency).

(7) The 96-well plate was placed on ice, 50. mu.L of Matrigel (R & D, 3432-010-01) was added to each well, and after leveling, incubation was carried out at 37 ℃ for 1 hour or more.

(8) Digesting the HUVEC, TPC and NPC cells obtained in the step (6) with trypsin respectively; wherein, HUVEC cells were dispersed at 4X 10 after counting with bronchial epithelial cell culture medium (BEpicM)⁵Each/ml, storing on ice; TPC and NPC cells were washed with 2mL of PBS buffer, and then resuspended in 1X 10 of PBS buffer⁶Per ml;

(9) adding the TPC and NPC cells obtained in the step (8) into CFSE dye (Tonbo Biosciences,13-0850-U500) (https:// Tonbo. com/products/CFSE) to the concentration of 5 mu M, and incubating for 15min at room temperature in the dark; then adding 5 times volume of RPMI-1640(GIBCO, C11875500BT) medium containing 10% (v/v) FBS in cold condition to stop staining, and incubating on ice for 5 min; after centrifugation, the cells were washed once with PBS buffer, and the appropriate number of cells were resuspended in BEpicM medium to 4X 10⁴Obtaining dyed TPC and NPC cell suspension liquid;

(10) the experimental groups were as follows: HUVEC, B.HUVEC + NPC, C.HUVEC + TPC three groups, then the HUVEC cells obtained in step (8) (4X 10)⁵Pieces/ml) were dispersed with BEpICM and then mixed with an equal volume of BEpICM medium, NPC cells (4X 10) dispersed with BEpICM after staining in step (9), respectively⁴One/ml) and TPC cells (4X 10)⁴Pieces/ml) were mixed and placed in a 96-well plate previously coated with matrigel in step (7), and cultured in an incubator at 37 ℃.

(11) HUVEC and PC cell growth were observed with a fluorescence microscope at 4, 12, 24, 48 hours plating, respectively: white light can be used to observe the extent of tube formation and green fluorescence can be used to observe the interaction of PC and HUVEC cells.

(12) The experimental results are shown in fig. 1, and after the white light picture and the fluorescence picture are superimposed, it can be seen that the pericytes are all adsorbed to the periphery of the blood vessels. The statistical result shows that the length, the branch number and the number of the blood vessels are HUVEC > PC > TPC, which proves that the pericytes can pull the blood vessels, so that the blood vessels become smaller and less, and the peripheral cell pulling effect of the tumor tissue is stronger than that of the normal tissue. The results suggest that the pericytes are taken as the treatment targets to improve the tumor drug delivery and provide a new idea for tumor treatment.

Example 2

The remaining steps in the tube formation process were the same as in example 1, except that: in the step (10), the ratio of Human Umbilical Vein Endothelial Cells (HUVEC) to TPC is 5: 1. 20: 1. after co-culturing HUVEC and TPC cells, reticuloendothelial structure was similarly observed under a fluorescence microscope (the experimental results were similar to those of example 1, and no additional figure is given).

Example 3

The remaining steps in the tube formation process were the same as in example 1, except that: the final concentration of the CFSE dye in step (9) was 2. mu.M, 10. mu.M. After co-culturing HUVEC and TPC cells, reticuloendothelial structure was similarly observed under a fluorescence microscope (the experimental results were similar to those of example 1, and no additional figure is given).

Comparative example 1

The remaining step reagents during the tube formation experiment were the same as in example 1, except that: the medium used in the co-culture in step (10) was changed from BEpICM to human endothelial serum-free medium (SFM, Gibco11111044) supplemented with recombinant human fibroblast growth factor (bFGF; 20ng/mL), epidermal growth factor (EGF; 10ng/mL), human plasma fibronectin (10. mu.g/mL) and 1% (v/v) penicillin/streptomycin solution for 48 hours (the pericytes used in the co-culture here were TPC cells).

As a result, as shown in FIG. 2A, co-culturing HUVEC and TPC cells after medium replacement did not form a significant reticuloendothelial structure, and it was confirmed that BEpicM medium used in example 1 was the optimal condition for tube formation.

Comparative example 2

The remaining steps in the tube formation process were the same as in example 1, except that: the ratio of Human Umbilical Vein Endothelial Cells (HUVEC) and TPC cells in step (10) is 1: 1. 50: 1.

the results are shown in FIGS. 2B and 2C: when the ratio of HUVEC to pericytes is 1: at 1, the HUVEC + PC group was consistently unable to form a reticuloendothelial structure due to too strong pericyte pulling (FIG. 2B). Whereas the ratio of HUVEC to pericytes was set at 50: 1, results pericytes had little effect on the vessel pull (FIG. 2C).

Comparative example 3

The remaining steps in the tube formation process were the same as in example 1, except that: the final concentrations of CFSE dye in step (9) were 20. mu.M and 0.5. mu.M.

The results are shown in FIGS. 2D and 2E: the final dye concentration of 20 μ M reduced the PC activity, failing to expand into normal adherent morphology, with no stretching effect (FIG. 2D). At a CFSE concentration of 0.5. mu.M, the fluorescence intensity was too weak, and the images were essentially quenched without completing the photographing at the two time points (FIG. 2E).

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Reference to the literature

[1]Hanahan,D.,and Weinberg,R.A.(2011).Hallmarks of cancer:the next generation.Cell144,646–674.

[2]Chung,A.S.,Lee,J.,and Ferrara,N.(2010).Targeting the tumour vasculature:insights from physiological angiogenesis.Nat.Rev.Cancer 10,505–514.

[3]Armulik,A.,Genove,G.&Betsholtz,C.Pericytes:developmental,physiological,and pathological perspectives,problems,and promises.Dev Cell 21,193-215,doi:10.1016/j.devcel.2011.07.001(2011).

[4]Lindahl,P.,Johansson,B.R.,Leveen,P.&Betsholtz,C.Pericyte loss and microaneurysm formation in PDGF-B-deficient mice.Science 277,242-245,doi:10.1126/science.277.5323.242(1997).

[5]Geevarghese,A.&Herman,I.M.Pericyte-endothelial crosstalk:implications and opportunities for advanced cellular therapies.Transl Res 163,296-306,doi:10.1016/j.trsl.2014.01.011(2014).

[6]Bergers,G.&Song,S.The role of pericytes in blood-vessel formation and maintenance.Neuro Oncol 7,452-464,doi:10.1215/S1152851705000232(2005)

[7]Xian,X.et al.Pericytes limit tumor cell metastasis.J Clin Invest 116,642-651,doi:10.1172/JCI25705(2006).

[8]Ribeiro,A.L.&Okamoto,O.K.Combined effects of pericytes in the tumormicroenvironment.Stem Cells Int 2015,868475,doi:10.1155/2015/868475(2015).

[9]Benjamin,L.E.,Golijanin,D.,Itin,A.,Pode,D.&Keshet,E.Selective ablation ofimmature blood vessels in established human tumors follows vascular endothelial growth factorwithdrawal.J Clin Invest 103,159-165,doi:10.1172/JCI5028(1999).

[10]Chen,Z.,Xu,X.H.&Hu,J.Role of pericytes in angiogenesis:focus on cancerangiogenesis and anti-angiogenic therapy.Neoplasma 63,173-182,doi:10.4149/201_150704N369(2016).

Claims (10)

1. A method for constructing a co-culture model of endothelial cells and pericytes for studying tube formation, comprising the steps of:

(1) digesting the umbilical vein endothelial cells by trypsin to obtain umbilical vein endothelial single cells;

(2) digesting the pericytes into pericyte single cells by using trypsin, and then adding CFSE dye with the final concentration of 2-10 mu mol/L for dyeing to obtain dyed pericytes;

(3) and (3) enabling the umbilical vein endothelial single cells and the stained pericytes to be 5-20: 1, culturing the mixture in a bronchial epithelial cell culture medium, and observing tube formation in a fluorescence microscope.

2. The method of claim 1 for constructing a model for studying the co-culture of endothelial cells and pericytes during tube formation, wherein:

the amount of the CFSE dye in the step (2) is calculated according to the addition of the CFSE dye in the reaction system with the final concentration of 5 mu mol/L;

the umbilical vein endothelial single cell and the stained pericyte in the step (3) are 10: 1.

3. the method of claim 1 for constructing a model for studying the co-culture of endothelial cells and pericytes during tube formation, wherein:

the formula of the bronchial epithelial cell culture medium in the step (3) is as follows: 500ml basal medium, 5ml bronchial epithelial cell growth supplement and 5ml penicillin/streptomycin solution, without serum.

4. The method of claim 1 for constructing a model for studying the co-culture of endothelial cells and pericytes during tube formation, wherein:

the umbilical vein endothelial cells in the step (1) are human umbilical vein endothelial cells;

the pericytes in the step (2) are human pericytes.

5. The method of claim 4 for constructing a model for studying the co-culture of endothelial cells and pericytes during tube formation, wherein:

the umbilical vein endothelial cells in the step (1) are obtained by the following method: washing human umbilical vein with normal saline until colorless, then pouring trypsin preheated at 37 ℃ into human umbilical vein, sealing, incubating at 37 ℃ for 15min, blowing human umbilical vein endothelial cells into DMEM complete culture medium to stop digestion, centrifuging, and suspending and dispersing with endothelial cell culture medium to obtain umbilical vein endothelial single cells.

6. The method of claim 5 for constructing a model for studying the co-culture of endothelial cells and pericytes during tube formation, wherein:

the centrifugation conditions are as follows: centrifuging at 300g for 5 min;

the culture conditions are as follows: culturing at 37 deg.C for more than 24 hr;

the formula of the endothelial cell culture medium is as follows: 500ml basal medium, 25ml bovine serum, 5ml endothelial cell growth factor and 5ml penicillin/streptomycin solution;

the digestion conditions in the step (1) are as follows: digesting for 10-20 min at 37 ℃.

7. The method of claim 4 for constructing a model for studying the co-culture of endothelial cells and pericytes during tube formation, wherein:

the pericytes in the step (2) are obtained by the following method:

(I) washing tumor tissue or normal tissue beside cancer with normal saline to colorless, shearing, adding enzymolysis solution for enzymolysis and digestion, centrifuging, and dispersing with pericyte culture medium to obtain pericyte single cell;

(II) placing the single cells of the pericytes in a coated culture plate to be cultured until the single cells adhere to the wall, so as to obtain pericytes of the tumor or the paracancer normal tissue pericytes;

the enzymolysis solution in the step (I) is a DMEM culture medium containing 2% (w/v) type I collagenase type1, 2% (w/v) type III collagenase type3 and 1% (w/v) DNase.

8. The method of claim 7 for constructing a model for studying the co-culture of endothelial cells and pericytes during tube formation, wherein:

the centrifugation conditions described in step (I) are: centrifuging at 300g for 5 min;

the digestion conditions in step (I) are: digesting for 1-2 hours at 37 ℃;

the culture medium used for culturing in the step (II) is a pericyte culture medium;

the formula of the pericyte culture medium is as follows: 500ml of basal medium, 10ml of fetal bovine serum, 5ml of peripheral cell growth supplement and 5ml of penicillin/streptomycin solution;

the coated culture plate in the step (II) is obtained by the following method: adding collagen into a gelatin solution, and uniformly mixing to obtain a coating solution; then adding the coating solution into the culture plate, and placing the culture plate in an incubator at 37 ℃ for culturing for more than 30min to obtain a coated culture plate;

the mass ratio of the collagen to the gelatin is 1: 5;

the culture conditions in step (II) are as follows: culturing at 37 deg.C for more than 24 hr.

9. The method of claim 1 for constructing a model for studying the co-culture of endothelial cells and pericytes during tube formation, wherein:

the culture in the step (3) is carried out on a culture plate coated with Matrigel in advance;

the culture conditions in the step (3) are as follows: culturing at 37 ℃ for 4-48 hours.

10. The endothelial cell and pericyte co-culture model constructed by the method for constructing the endothelial cell and pericyte co-culture model for researching tube formation according to any one of claims 1 to 9.

Images