Establishment Of Ovarian Cancer Drug Resistance Cell Line A2780-CP And Functional Verification Of CD44~+ Tumor Stem Cells

Objective:Establishment of cisplatin resistant strain A2780 of ovarian cancer epithelial cell line A2780-CP;Differences in biological characteristics between A2780-CP and A2780;Biological characteristics of CD44~+tumor stem cells in A2780-CP.To find the relationship between cell resistance of ovarian cancer and the biological characteristics of tumor stem cells.Methods:1.The drug-resistant ovarian cancer cell line A2780-CP was obtained by small dose of continuous cisplatin stimulation;2.The difference of drug sensitivity between A2780-CP and A2780 were studied by MTT;3.The difference between A2780-CP and A2780 in the cycle and the difference in the ability of anti apoptosis were studied by flow cytometry;4.Flow cytometry was used to analyze the differences of two common tumor stem cell markers:CD44,CD24 in drug-resistant and normal cell lines;5.CD44~+cell sorting by flow cytometry in A2780-CP cells;6.CD44~+cell function of stem cells research:1)the difference of Transwell cell migration assay of CD44~+cells and negative cells in migration ability;2)the difference of Transwell cell invasion assay of CD44~+cells in invasion ability;3)under serum-free conditions of CD44~+cells into the ball ability difference;4)nude mice xenograft experiment CD44~+and CD44~-cells;7.Immunohistochemical analysis of the expression of CD44 markers in ovarian cancer tissue samples.Results:1.The difference between A2780-CP and A2780 cell phenotype change;the proportion of cells in G2 phase decreased the cell cycle;2.The IC50 value of A2780-CP is 114.7μm,nearly three times higher than the resistance of the A2780 cell lines that have not been screened for stress;3.A2780-CP’s anti-apoptotic ability has no significant advantage over the A2780;4.A2780-CP cell surface tumor stem cell marker CD44 expression rate is much higher than A2780;In vitro migration,proliferation and invasion of CD44~+tumor stem cells were higher than CD44~-cells.CD44~+cells will allow the nude mouse to become a tumor in vitro in2 weeks or so;5.Immunohistochemical results showed that CD44 stem cell markers were highly expressed in ovarian cancer tissues with high grade malignancy,and no expression was found in normal ovarian tissue.Conclusions:.1.Obtain Ovarian cancer resistant cell line A2780-CP by increasing the dose of cisplatin;2.By comparing the cell surface markers of A2780-CP cell lines and A2780,we found that CD44 is indeed a tumor stem cell marker associated with tumor resistance;3.The ability of CD44~+A2780-CP cell migration,proliferation and invasion is consistent with the characteristics of tumor stem cells,and is verified by tumorigenicity experiments in nude mice.The comparison of clinical ovarian cancer tissue samples with normal samples also indicated a link between CD44 tumor markers and ovarian cancer and its pathological grading.