Separation And Identification With Breast Cancer Stem Cells And Study On Proliferation And Drug-resistance Of Breast Cancer Stem Cells

Objective: To know how to change about self-renewal and the proportion of breast cancer stem cells in diverse environment,and how to evade from chemotherapy about breast cancer stem cells. Methods: 1.Study on the patients with breast cancer that gained therapy in Department of Breast and Thyroid Center,Union Hospital during January,2004～ June,2005. To obtain mammary cancer tissue after operation,and prepare single cell suspensions by collagenase,then gain breast cancer stem cells by flow cytometry.Finally we identitified breast cancer cells by model of SOD/SCID mice with reconstruction tumor.2. To separate breast cancer cells with expressing CD44~+ CD24～ and other breast cancer cells from MCF-7 by flow cytometry. Then to mensurate the activity of telomerase of different cells,and observate their ability of self-renewal and clone in vitro,and their ability of reconstruction tumor in vivo.3. To mensurate the expression of CD44 and CD24 of human breast cancer primary cells and MCF-7 breast cancer cell line in diverse incubation circumstance,and observate the proportion of breast cancer stem cell in diverse environment.4. To mensurate the expression of MDR1 of different breast cancer cells from human breast cancer primary cells and MCF-7 breast cancer cell line,and the kill effect of chemotherapy drug with different breast cancer cells in vitro,and the change of the proporation and the expression of MDR1 of breast cancer stem cells after chemotherapy.Results:We gain excellent effect of separation with breast cancer tissues,we could obtain 0.5—1×10~7 cells from 1cm~3 breast cancer tissues, breast cancer primary cells mainly include expressing Lin~-CD44~+CD24～- breast cancer cells and expressing Lin~-CD44~-breast cancer cells, expressing Lin~-CD44~+CD24~- breast cancer cells could reconstruct tumor by transplanting SOD/SCID mice.MCF-7 cells mainly include expressing CD44~+CD24~-breast cancer cells and expressing CD44~+CD24~+ breast cancer cells,although they allhave ability of reconstruction tumor by transplanting SOD/SCID mice, the ability of expressing CD44~+CD24~- breast cancer cells is more stronger. From normal,benign to malignant mammary tissue,the proporation of CD44+CD24-/low mammary cell gradually rise,the proporation of stem cell gradually decrease with the number of MCF-7 gradually increasing in the process of culture,and the proporation of breast cancer stem cells from human breast cancer primary cells and MCF-7 breast cancer cell line markedly increases after chemotherapy. Although the expression of MDR1 of breast cancer stem cells is higher than expressing CD44~ breast cancer cells from human breast cancer primary cells,it is not different in different breast cancer cells from MCF - 7,and the kill effect of chemotherapy drug for different breast cancer cells from MCF - 7 is similar. After chemotherapy, the proporation and the expression of MDR1 of breast cancer stem cells rise all. Conclusion:Expressing Lin~-CD44~+CD24~- breast cancer cells from human breast cancer primary cells are breast cancer stem cells,and Expressing CD44~+CD24~+ breast cancer cells from MCF-7 maybe are breast cancer progenitor cells. The proporation of mammary stem cell that is modulated by diverse factors in microenvironment could reflect property of mammary tissue and self- renewal speed of mammary cell. The role of breast cancer stem cell is very important in chemotherapy.Breast cancer stem cells more easily escape from chemotherapy than other breast cancer cells,and obtain ability of drug resistance by mutation of gene. Speedup of breast cancer stem cells self-renewal after chemotherapy will promote relapse of breast cancer.