The Study Of IL-17promoting Proliferation Of Ovarian Cancer Stem Cell In Vitro

Objective:To isolate cancer stem cells in human ovarian cancer cell line A2780 and identify the expression of distinct markers of A2780 stem cells; we are planing to analyze the source of IL-17 and the relation between IL-17 and ovarian cancer stem cell in ovarian cancer tissue,then we are to identify the expression of IL-17R of A2780 stem cells and observe the effect of IL-17 promoting growth, proliferation and sphere of A2780 stem cells in vitro.Method: The A2780 cells wre cultured in serum-free medium with EGF,bFGF,insulin and BSA to induce tumor stem cells by continuous passage. Immunofluorescence and flow cytometry were applied to identify the expression of distinct markers CD133+ of A2780 stem cells;the immunohistochemistry was applied to identify the expression of IL-17 in ovarian cancer tissue and the Immunofluorescence was applied to identify the expression of CD4+,CD68+,CD133+ antigen and IL-17+ cells in ovarian cancer tissue;Then,the PCR and real-time PCR were applied to identify the expression of IL-17R and CD133+antigen in A2780 stem cells;Finally, the A2780 stem cells was stimulated by rhIL-17 in vitro and the MTT method was applied to the effect of IL-17 promoting growth,proliferation and sphere of A2780 stem cells.Results: The A2780 stem cells were successfully isolated from in cell line A2780.They formed typical neurospheres in serum-free medium and had strong self-remew capacity,the expression rate of CD133+antigen of A2780 stem cells was up to 90%;The results of immunohistochemistry illustrated that IL-17 were express in ovarian cancer tissue.The results of Immunofluorescence confirmed that CD4+ and CD68+ cells can produce IL-17 ,also confirmed that CD133+ antigen and IL-17 were co-expressed in A2780 stem cells.The results of PCR and real-time PCR reveal that A2780 stem cells express more IL-17R than A2780 cells, the results of flow cytometry reveal that IL-17R and CD133+antigen were co-expressed in A2780 stem cells.The results of MTT illustrated that the growth rate of A2780 stem cells was increased, the number of stem cells sphere became more and the volume of stem cells sphere increased.Conclusions: In summary,our studies provide strong evidence that the ovarian cancer stem cells could be isolated from ovarian cancer cells line, the CD133+antigen is the distinct markers of ovarian cancer stem cells.IL-17 is expressed in ovarian cancer tissue.CD4+ and CD68+ cells can produce IL-17.There are a lot of IL-17R expressed in ovarian cancer stem cells,and IL-17 can promote growth, proliferation and sphere of ovarian cancer stem cells.Thus,the study provide a solid foundation for further research of cancer stem cells and mechanism of effect of IL-17 on cancer stem cells.