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

A technology of endothelial cells and construction methods, applied in the field of construction of endothelial cells and pericyte co-culture models, can solve the problems of unclear correlation of pericyte cancer, affecting the effectiveness of screening drugs targeting blood vessels, and unknown molecular mechanism, etc., to achieve Reduce the effect of animal models and clinical experimental tests, good social and economic value, and improve tumor vascular function

Active Publication Date: 2021-08-27
SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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Problems solved by technology

But the treatment failed clinically
The main reason is that the previous in vitro human tube formation model did not co-culture pericytes, which affected the effectiveness of screening drugs targeting blood...

Method used

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  • Construction method of endothelial cell and pericyte co-culture model for researching tubulation
  • Construction method of endothelial cell and pericyte co-culture model for researching tubulation

Examples

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Embodiment 1

[0054] This example provides a method for co-culturing human endothelial cells and pericytes in vitro to study tube formation. All isolation and culture operations are carried out in a biological safety cabinet, which specifically includes the following steps:

[0055] (1) Rinse the human umbilical cord vein (from a hospital in Guangzhou) with normal saline until it is colorless, pour 37°C preheated trypsin into the human umbilical cord vein with a syringe, seal it, and incubate at 37°C for 15 minutes. Endothelial cells were insufflated into complete DMEM medium to terminate digestion.

[0056] (2) Centrifuge the cells at room temperature at 300g for 5 minutes, resuspend and disperse them into single cells with endothelial cell medium (ECM), and then place the cells in a 6-well plate at 37°C and 5% CO 2 Incubate for 24 hours. The suspended cells were sucked away to replace the medium, and the adherent growth cells were human umbilical vein endothelial cells (HUVEC).

[0057]...

Embodiment 2

[0068] The reagents in other steps during the tube formation process were the same as those in Example 1, except that the ratios of human umbilical vein endothelial cells (HUVEC) and TPC in step (10) were 5:1 and 20:1. After co-cultivating HUVEC and TPC cells, the reticular vascular structure can also be observed under a fluorescence microscope (the experimental results are similar to those in Example 1, so no additional figures are shown).

Embodiment 3

[0070] The reagents in the remaining steps during 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 μM or 10 μM. After co-cultivating HUVEC and TPC cells, the reticular vascular structure can also be observed under a fluorescence microscope (the experimental results are similar to those in Example 1, so no additional figures are shown).

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Abstract

The invention discloses a construction method of an endothelial cell and pericyte co-culture model for researching tubulation. The method comprises the following steps: (1) digesting umbilical vein endothelial cells with trypsin to obtain umbilical vein endothelial single cells; (2) digesting pericytes into single cells of the pericytes by using trypsin, and then adding CFSE dye with the final concentration of 2-10 [mu]mol/L for staining to obtain stained pericytes; and (3) uniformly mixing the umbilical vein endothelial single cells and the stained pericytes according to the cell number ratio of (5-20):1, conducting culturing by using a bronchial epithelial cell culture medium, and observing the formation condition of the tube on a fluorescence microscope. According to the method disclosed by the invention, the influence of pericytes on the formation of endothelial cell tubes can be dynamically observed through a fluorescence microscope, the effect of human tumor pericytes on tumor blood vessels can be reflected through in-vitro experiments, and the method has good social and economic values.

Description

technical field [0001] 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 studying tube formation. Background technique [0002] Over the past few decades our understanding of primary tumors has expanded and deepened. Although the treatment of some tumors has undergone substantial development, tumor treatment is still a worldwide health problem. Although new chemotherapy drugs and methods are constantly being discovered, chemotherapy resistance is still a major obstacle that limits the efficacy of chemotherapy. In solid tumors, blood vessels are critical for delivery of nutrients, oxygen and even chemotherapy drugs, study finds [1] . Therefore, improving tumor angiogenesis is particularly important in the study of chemotherapy resistance. [0003] The formation of new tumor blood vessels, known as tumor angiogenesis, involves enhanced proliferation and migration of endo...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/09
CPCC12N5/069C12N5/0693C12N2509/00C12N2509/10C12N2502/28C12N2502/30C12N2533/54
Inventor 黄炳培江雪孔祥展赵新保徐秋萍丘晓奕孙瑞璞
Owner SUN YAT SEN MEMORIAL HOSPITAL SUN YAT SEN UNIV
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