In-vitro cell model for simulating revascularization after breast cancer radiotherapy and preparation method and application of in-vitro cell model

A breast cancer cell and cell model technology, which is applied in the field of in vitro cell models for simulating angiogenesis after breast cancer radiotherapy and its preparation, can solve the problem that the influence of blood vessels cannot be reflected, the influence of blood vessels is hindered, and the breast cancer cells and vascular endothelial cells cannot be reflected. Interaction and other problems, to achieve the effect of fast and convenient detection

Inactive Publication Date: 2021-09-28
PEOPLES HOSPITAL OF HENAN PROV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

First, this model only involves vascular endothelial cells and cannot reflect the interaction between breast cancer cells and vascular endothelial cells after radiotherapy
Second, even if the conditioned medium of breast cancer cells after radiotherapy is used to treat vascular endothelial cells, it cannot reflect the effect of cell-cell contact between breast cancer cells and vascular endothelial cells on angiogenesis. influences
However, the spatial isolation of tumor cells from vascular endothelial cells in an in vitro model using Transwell chambers hinders the effect of direct tumor cell-vascular endothelial cell contact on angiogenesis

Method used

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  • In-vitro cell model for simulating revascularization after breast cancer radiotherapy and preparation method and application of in-vitro cell model
  • In-vitro cell model for simulating revascularization after breast cancer radiotherapy and preparation method and application of in-vitro cell model
  • In-vitro cell model for simulating revascularization after breast cancer radiotherapy and preparation method and application of in-vitro cell model

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Construction of human umbilical vein endothelial cells (HUVEC-Fluc) stably transfected with fusion genes of firefly luciferase (Fluc) and green fluorescent protein (GFP)

[0041] 1. Recovery of HUVEC cells

[0042] The specific experimental operation of HUVEC cell recovery is as follows:

[0043] 1) Preheat the electric heating constant temperature water tank to 37°C.

[0044] 2) Take out the cryopreservation tube from the liquid nitrogen tank or -80°C ultra-low temperature refrigerator, quickly place it in a 37°C constant temperature water tank, and shake the cryopreservation tube gently to make the cells in it thaw quickly.

[0045] 3) After the cells in the cryopreservation tube are fully thawed, take out the cryopreservation tube and wipe the cryopreservation tube with 75% alcohol cotton ball. Open the cryopreservation tube in the ultra-clean workbench, transfer the cells in it to a 15 ml centrifuge tube, add 5 ml of 10% fetal bovine serum medium, and m...

Embodiment 2

[0106] Example 2: Discussion on the correlation between luciferase activity and cell number in HUVEC-Fluc cells

[0107] In the subsequent in vitro co-culture model, it is hoped that the proliferation of HUVEC-Fluc can be reflected by detecting the activity of HUVEC-Fluc firefly luciferase. Therefore, it is necessary for us to confirm whether there is a positive correlation between firefly luciferase activity and cell number in HUVEC-Fluc. We placed a specified number of HUVEC-Fluc in a 96-well plate, and after the cells adhered to the wall, the luciferase activity of HUVEC-Fluc in the 96-well plate was detected with the help of an in vivo biochemiluminescence imager.

[0108] The specific experimental operation is as follows:

[0109] The well-growing HUVEC-Fluc cells were digested to prepare a single cell suspension. Spread HUVEC-Fluc in 96-well plates at 100, 250, 500, 750, 1000, 2500, 5000, 7500, and 10,000 cells per well, respectively. After the cells were fully adhere...

Embodiment 3

[0113] Example 3: Construction of a cell model for simulating angiogenesis after radiotherapy for breast cancer

[0114] 1. Radiotherapy of breast cancer cell line MDA-MB-231 using a medical linear accelerator

[0115] The specific operation steps are as follows:

[0116] 1) Cell preparation: X-ray irradiation can be performed on the breast cancer cell line MDA-MB-231 grown in cell culture dishes of various specifications.

[0117] 2) Place the cells to be irradiated on a piece of transparent plexiglass with a thickness of 15 cm, and cover the cell culture dish with a thin transparent plastic plate to limit the irradiation field of view; the irradiation uses a medical linear accelerator that generates X-rays. The radiation dose rate was 3.6 Gy / min.

[0118] 2. Construction of a cell model for simulating angiogenesis after radiotherapy for breast cancer

[0119] In the present invention, MDA-MB-231 cells and HUVEC-Fluc cells are co-cultured after radiotherapy by a medical li...

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Abstract

The invention belongs to the technical field of medical biology, and discloses an in-vitro cell model for simulating revascularization after breast cancer radiotherapy. The in-vitro cell model is formed by co-culturing human umbilical vein endothelial cells and breast cancer cells subjected to radiotherapy irradiation, and the human umbilical vein endothelial cells are jointly marked by luciferase and green fluorescent protein. A preparation method of the in-vitro cell model comprises the following steps that breast cancer cells subjected to radiotherapy irradiation are inoculated into a culture medium, culturing is carried out at 37 DEG C for 12-48 hours, human umbilical vein vascular endothelial cells jointly marked by luciferase and green fluorescent protein are inoculated into the culture medium, and co-culturing is carried out. The in-vitro cell model can be used for simulating the process of promoting vascular endothelial cell regeneration by breast cancer cells after radiotherapy; and in addition, certain compounds or drugs are added into the model, and whether the substances have an inhibiting effect or not on revascularization after breast cancer radiotherapy is researched. Therefore, the in-vitro model can be used for screening drugs for inhibiting revascularization after breast cancer radiotherapy.

Description

technical field [0001] The invention belongs to the technical field of medicine and biology, and in particular relates to an in vitro cell model for simulating angiogenesis after radiotherapy for breast cancer, a preparation method and application thereof. Background technique [0002] According to the latest global cancer epidemiological study (GLOBOCAN), among all populations, there will be about 2.26 million new cases of female breast cancer in 2020, accounting for 11.7% of all cancers, and the incidence rate surpasses lung cancer, becoming the world's number one cancer; death The number of cases is about 685,000, ranking fifth. Breast cancer ranks first in both incidence and mortality among women worldwide. In China, the incidence of breast cancer ranks first among female cancers, accounting for about 15% of all female cancers. [0003] Radiation therapy (referred to as radiotherapy) is one of the important means of breast cancer treatment. Breast radiotherapy is reco...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N5/09C12Q1/02C12Q1/66
CPCC12N5/069C12N5/0693C12N5/0631C12N9/0069C07K14/43595C12Q1/66G01N33/5011C12N2502/30C12N2503/02G01N2500/10
Inventor 冯骁
Owner PEOPLES HOSPITAL OF HENAN PROV
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