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Building of in-vitro three-dimensional cell model of breast cancer and application of in-vitro three-dimensional cell model in research of drug resistance mechanism and reversal agents screening

A cell model, human breast cancer technology, applied in tumor/cancer cells, animal cells, vertebrate cells, etc., can solve problems such as drug screening and prediction failure

Inactive Publication Date: 2014-12-24
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0002] As the current mainstream in vitro research model, the monolayer cell model is quite different from solid tumors in vivo, which directly leads to the failure of drug screening and prediction

Method used

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  • Building of in-vitro three-dimensional cell model of breast cancer and application of in-vitro three-dimensional cell model in research of drug resistance mechanism and reversal agents screening
  • Building of in-vitro three-dimensional cell model of breast cancer and application of in-vitro three-dimensional cell model in research of drug resistance mechanism and reversal agents screening
  • Building of in-vitro three-dimensional cell model of breast cancer and application of in-vitro three-dimensional cell model in research of drug resistance mechanism and reversal agents screening

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: Construction of three-dimensional multilayer cell transport model of breast cancer MCF-7

[0012] Experimental Materials:

[0013] Human breast cancer cells MCF-7 cells were provided by Tianjin Institute of Hematology, at 37°C, 5% CO 2 Under normal conditions, the culture medium is RPMI1640 (Gibeo) containing 10% calf serum (PAA). Fetal bovine serum (Fetal bovine serum) was purchased from Hyclone (Logan, Utah, USA); trypsin (Trypsin) was purchased from Amersco (Solon, Ohio, USA); penicillin and streptomycin were purchased from Nanjing Shengxing Bioengineering Company (Jiangsu ); Matrigel was purchased from BD Biosciences (USA), Millicell cell culture chamber was purchased from Millipore (Merck Millipore, USA). Doxorubicin hydrochloride powder (purity > 99%) was purchased from Nanjing Shenglin Chemical Industry (Jiangsu); LY335979 was purchased from Selleck Chemicals (USA); Ginsenoside 20(S)-Rh2 (purity>98%) was purchased from the Department of Organic Chem...

Embodiment 2

[0016] Example 2: Construction of breast cancer MCF-7 monolayer cell transport model

[0017] Experimental material: same as embodiment one

[0018] experimental method:

[0019] MCF-7 cells were digested and counted, and the cells were resuspended with 10% serum-containing RPMI1640 medium. with 2*10 5 The density of cells / well and the volume of 400ul were inoculated in the chamber of 24-well Millicell cell culture chamber. Add 600 ul of 10% serum-containing RPMI1640 medium to the outer chamber to continue culturing until the cells form a dense monolayer, and change the medium every other day.

Embodiment 3

[0020] Example 3: Transport Kinetics and Uptake Experiments

[0021] Experimental material: same as embodiment one

[0022] experimental method:

[0023] 1. MCF-7 multilayer cell transport model and single layer cell transport model construction methods are the same as the experimental methods in Example 1 and Example 2

[0024] 2. Rotational dynamics experiment

[0025] After the MCF-7 multilayer cells and monolayer cells assisted in the Millicell cell culture chamber were cultured and matured, the cell culture chamber was washed twice with PBS. Add 400ul and 600ul Hank's Balanced Salt Solution (HBSS) to the upper and lower chambers, respectively, to equilibrate at 37°C for 10min. Divided into three groups for pre-administration: 0.1% DMSO group; LY335979 (1 μM) group and 20(S)-Rh2 (10 μM), incubated at 37° C. for 1 hour. After the pre-administration solution in the upper chamber was removed, the upper chamber was divided into three groups for administration: ADR (10 μM)...

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Abstract

The invention relates to building of an in-vitro three-dimensional cell model of a breast cancer and an application of the in-vitro three-dimensional cell model in research of a drug resistance mechanism and reversal agents screening. A certain amount of human breast cancer MCF-7 cells are planted on a Millicell cell culture chamber cell by applying a'3D no base and embedded' method; and a three-dimensional multi-layer cell model of a human breast cancer MCF-7 cell, of which the thickness is about 40 microns, is formed by cultivation and the number of layers is 10-12. Transport kinetics and consuming behaviors of chemotherapy adriacin doxorubicin are investigated on the MCF-7 multi-layer cell model by single use and combination of P-GP inhibitors LY335979 to prove the drug resistance in the culture process; and the in-vitro three-dimensional cell model is applied to screening of an adriamycin resistance-reversal agent ginsenoside 20(S)-Rh2.

Description

technical field [0001] The invention relates to the technical field of tumor in vitro models, in particular to the establishment of an in vitro three-dimensional cell model of breast cancer and its application in the research of drug resistance mechanism and reversal agent screening. Background technique [0002] As the current mainstream in vitro research model, the monolayer cell model is quite different from solid tumors in vivo, which directly leads to the failure of drug screening and prediction. Three-dimensional cell culture technology is a technology between monolayer cell culture and animal experiments, which can be used to study the interaction between cells, cells and cell matrix, and the effect of tumor microenvironment on cell differentiation, proliferation, apoptosis, etc. effects on death and gene expression. Compared with the traditional two-dimensional planar cell culture, it can more realistically simulate the structure of solid tumors, making up for the d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12Q1/02
Inventor 王广基张经纬鲁萌周芳
Owner CHINA PHARM UNIV
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