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Trastuzumab drug-resistant human breast cancer cell line and preparation method and application thereof

A technology of human breast cancer cells and trastuzumab, which is applied in the field of biomedicine, can solve the problems of inconspicuous drug resistance, and achieve the effects of small growth and proliferation speed, small decrease, and stable cell shape

Inactive Publication Date: 2015-07-08
ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, some researchers use the method of trastuzumab in vitro induction to establish BT-474 drug-resistant cell lines, and the general antibody treatment time is 3-9 months (see Konecny ​​GE, et al, (2006) Activity of the Dual Kinase Inhibitor Lapatinib (GW572016) against HER-2-Overexpressing and Trastuzumab-Treated Breast Cancer Cells, Cancer Res, 66:1630-1639; Yoshida R, et al, (2012) Mechanism of resistance to trastuzumab and molecular sensitization via ADCC activation by exogenous expression of HER2-extracellular domain in human cancer cells, Cancer Immunol Immunother, 61:1905–1916), but some studies suggest that the drug resistance of BT-474 drug-resistant cell lines established by in vitro induction method in mice Trastuzumab can still significantly inhibit tumor growth in vivo (see Kute TE, et al, (2009) Breast tumor cells isolated from in vitro resistance to trastuzumab remain sensitive to trastuzumab anti-tumor effects in vivo and to ADCC killing. Cancer Immunol Immunother, 58:1887–1896), so there are also studies using the method of trastuzumab in vivo induction to establish BT-474 drug-resistant cell lines, which also exhibit a certain degree of in vitro drug resistance (See Emerson EO, et al, (2005) In vivo selection and characterization of herceptin-resistance in the human breast adennocarcinoma model, BT-474, Proc Amer Assoc Cancer Res, Abstract 2646; Ritter CA, et al. (2007) Human Breast Canc er Cells Selected for Resistance to Trastuzumab In vivo Overexpress Epidermal Growth Factor Receptor and ErbB Ligands and Remain Dependent on the ErbB Receptor Network.Clin Cancer Res.13:4909-4919), however, about the BT-474 drug-resistant cell line to Trastuzumab The research results of the molecular mechanism of the relevant signal transduction of the drug resistance of zizumab are inconsistent. Specifically, when using the BT-474 drug-resistant cell line, Emerson EO et al. found that the expression of HER2 and EGFR receptors decreased, and the expression of IGF-IR receptors decreased. However, Ritter RA et al. found that the expression of HER2 receptor remained unchanged, and the expression of EGFR receptor and EGF growth factor increased. In addition, there are other literature reports using trastuzumab-resistant BT-47 human breast cancer cell line, but did not disclose the preparation process of the cell line

Method used

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  • Trastuzumab drug-resistant human breast cancer cell line and preparation method and application thereof
  • Trastuzumab drug-resistant human breast cancer cell line and preparation method and application thereof
  • Trastuzumab drug-resistant human breast cancer cell line and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: Construction of trastuzumab-resistant human breast cancer cell line BT-474 / HR

[0030] (1) resuscitate common human breast cancer cells BT-474 into T25 culture flasks, add RPMI 1640 medium containing 10% special-grade fetal bovine serum to cultivate, and the T25 culture flasks are placed in a 37°C, 5% CO2 incubator In the process, take the cells in the logarithmic growth phase, add trastuzumab (purchased from Roche, specification 320mg / bottle) to an effective concentration of 10ug / ml, and then replace the same concentration of trastuzumab every 3-4 days When the cells grow to 80% confluence, they are digested with 0.25% trypsin and subcultured into new T25 medium for culture, and the treatment is continued for 2 months;

[0031] (2) Gradually increase the effective concentration of trastuzumab in the culture medium for 3 times: increase the effective concentration of trastuzumab to 20ug / ml, and culture continuously for 2 months; increase the effective concent...

Embodiment 2

[0034] Embodiment 2: Morphological observation of cell lines

[0035] The trastuzumab-resistant human breast cancer cells BT-474 / HR in the sample group and the common human breast cancer cells BT-474 in the control group were digested with 0.25% trypsin, and then seeded in 6-well cell culture plates, each well containing For 5×105 cells, add RPMI 1640 medium containing 10% special grade fetal bovine serum, culture in an incubator at 37°C and 5% CO2 for 24 hours, take the cells in the logarithmic growth phase, and replace them with 10ug / ml trastuzumab The medium of monoclonal antibody was cultured in an incubator at 37°C and 5% CO2 for 72 hours, and photographed under an inverted phase-contrast microscope to observe the morphology of living cells. The results were as follows: figure 1 As shown, in the absence of trastuzumab, trastuzumab-resistant human breast cancer cells BT-474 / HR (hereinafter referred to as cells BT-474 / HR) and common human breast cancer cells BT- The morpho...

Embodiment 3

[0036] Example 3: In vitro proliferation of cell lines and their sensitivity to trastuzumab

[0037] Cells BT-474 / HR in the sample group and BT-474 cells in the control group were digested with 0.25% trypsin, and then inoculated in seven 96-well cell culture plates, among which No. 1-7 culture plates were used for culturing plus trastuzumab The above two kinds of cells on the 1st, 2nd, 3rd, 4th, 5th, 6th and 7th day of monoclonal antibody treatment, each cell was inoculated into 12 wells, each well contained 10,000 cells, and 100 μL containing 10% special grade fetal bovine serum was added The RPMI 1640 culture medium was placed in an incubator at 37°C and 5% CO2 for 24 hours, and then replaced with fresh medium (the fresh medium of 6 culture plates inoculated with cells BT-474 / HR and BT-474 was added with 10ug / ml of trastuzumab), and the time at this time was recorded as 0, placed at 37 ° C, 5% CO2 After cultivating in an incubator for 24 hours, according to the CCK-8 kit (pu...

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Abstract

The invention discloses a trastuzumab drug-resistant human breast cancer cell line BT-474 / HR. The human breast cancer cell line has the collection number of CCTCC NO:C2014245 in China Center for Type Culture Collection, and the collection date of the human breast cancer cell line is 14, January in 2015. The invention also discloses a method for constructing the trastuzumab drug-resistant human breast cancer cell line BT-474 / HR. The method comprises the following step of preparing a human breast cancer cell line BT-474 / HR with excellent in-vitro and in-vivo trastuzumab drug resistance by utilizing a human breast cancer cell line BT-474 and combining two means of in-vitro induction and in-vivo induction of trastuzumab. According to the cell line, the HER2 overexpression characteristics of a parental cell line are remained, and the cell line has excellent in-vitro and in-vivo drug resistance on an HER2 targeted antibody drug. Therefore, the human breast cancer cell line can be directly applied to development and research of novel anti-tumor treatment drugs.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a trastuzumab-resistant human breast cancer cell and a preparation method and application thereof. Background technique [0002] HER2, also known as ErbB2, is one of the epidermal growth factor receptor (EGFR) family members, which is a transmembrane glycoprotein with receptor tyrosine kinase activity encoded by the HER2 gene. As an important tumor marker molecule, HER2 has gene amplification or protein overexpression in various malignant tumors such as breast cancer, gastric cancer, ovarian cancer, lung cancer, and prostate cancer, and is clinically called HER2-positive tumors. According to statistics, the proportion of HER2 overexpression in breast cancer patients is as high as 20-30%, and the clinical manifestations of HER2 positive tumors are high malignancy, insensitive to radiotherapy and chemotherapy, easy to relapse and metastasis after treatment, and their potential ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09A01K67/027C12Q1/02
Inventor 宋礼华胡思怡张志辉
Owner ANHUI ANKE BIOTECHNOLOGY (GRP) CO LTD
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