The Study Of Cancer-associated Fibroblasts In Cisplatin Resistance Of Human Ovarian Cancer Cells

Part Ⅰ: Isolation,purification and Culture of Cancer-associated Fibroblasts in Human Epithelial Ovarian CancerObjective:To lay a foundation for further research on the role and mechanism of CAFs in the development and progression of ovarian cancer and cisplatin resistance,isolate and identify cancer-associated fibroblasts(CAFs)from human ovarian cancer tissues.Methods: Collected 42 cases of human epithelial ovarian cancer and 28 cases of normal ovarian epithelial tissues,isolated primary human normal fibroblasts(NFs)and cancer-associated fibroblasts(CAFs)from normal ovarian and epithelial ovarian cancer.Enzyme digestion time difference method was used to sublimate CAFs and NFs.A inverted phase-contrast optical microscope was used to observe cell morphology,and indirect immunofluorescence assay was used to identify CAFs.Result: CAFs were isolated from fresh human ovarian cancer tissue,with a spindle-like shape.Expressed Positively smooth muscle excitonin-α(α)and fibroblast activation protein(FAP)and other fibroblast markers in CAFs.Conclusions: Primary human epithelial ovarian cancer-associated fibroblasts(CAFs)are successfully isolated,sublimated and cultured.Part Ⅱ:Relationship between human epithelial ovarian cancer-associated fibroblasts and cisplatin resistance in ovarian cancer cellsObjective:To investigate the effect of cancer-associated fibroblasts(CAFs)on the cisplatin resistance of ovarian cancer cells,and to explore the possible mechanisms.Methods: Collected CAFs or NFs culture supernatants and established indirect co-culture system with ovarian cancer SKOV3 cells.CCK8 assay,scratch assay,flow cytometry assay,ELISA assay,real-time quantitative polymerase chain reaction(Real-time PCR)and immunoblot assay were used to detect cell proliferation activity and cisplatin toxicity,cell migration ability,apoptosis rate,OPN concentration in supernatant of CAFs cells,drug resistance-related gene YAP 、 CTGF and Cyr61 expression at the m RNA and protein levels before and after co-culture.Result: The success rates of CAFs and NFs separation and purification were 70% and60% respectively;The cell proliferation ability,cisplatin half inhibition concentration(IC50)and migration ability of CAFs group were significantly increased compared with those of SKOV3 group and NFs group(P<0.001,P<0.01,P<0.01);The apoptosis rate of cisplatin induced in CAFs group was also significantly lower than that in SKOV3 group and NFs group(P<0.001);After CAFs co-culture,the expression of resistance-related gene YAP,CTGF and Cyr61 at m RNA and protein levels was significantly increased compared with those of SKOV3 group and NFs group(P<0.01,P<0.05,P<0.01);The expression level of OPN in cell supernatant of CAFs group was significantly higher than that of SKOV3 group and NFs group(P<0.001).Conclusions: CAFs can promote the proliferation and migration of SKOV3 cells,make SKOV3 cells resist apoptosis,and thus reduce the sensitivity of SKOV3 cells to cisplatin.The mechanism may be related to the up-regulation of drug resistance gene YAP and CTGF、Cyr61 downstream gene expression in ovarian cancer cells.