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[目的]研究癌相关成纤维细胞的条件培养基对卵巢癌细胞侵袭能力的影响及作用机理。[方法]原代培养人卵巢正常成纤维细胞(NOF)和癌相关成纤维细胞(CAF),并收取条件培养基NOFcm和CAFcm。用无血清的培养基DMEM、NOFcm和CAFcm分别处理SKOV3细胞,诱导SKOV3细胞发生上皮间质转化(EMT),通过形态观察和Western blot验证。形态上发生上皮间质转化(EMT)的SKOV3细胞,用transwell小室及三维培养实验检测细胞的侵袭能力的改变。[结果]CAF的条件培养基能在体外诱导卵巢癌细胞SKOV3在形态上发生改变:细胞连接疏松,部分细胞呈梭形,且明显下调E-cadherin蛋白表达量,明显上调Vimentin蛋白表达量。Transwell侵袭实验显示CAFcm处理后的SKOV3穿透matrigel胶的细胞数(284.00±32.86)相比于无血清的DMEM组(60.60±11.72)和NOFcm组(116.60±14.22),数量明显增多(P=0.000374和P=0.001258);三维培养实验中只有CAFcm组和NOFcm组能形成克隆球,而在无血清的DMEM组不能形成克隆球,CAFcm组(37.75±1.70)相比于NOFcm组(14.5+±1.29),形成的克隆球明显增大,向外侵袭能力增强,数量增多(P<0.001)。[结论]卵巢癌CAF通过诱导卵巢癌细胞发生EMT,进而促进卵巢癌细胞的侵袭能力。
[Objective] To investigate the effect of conditioned media of cancer-associated fibroblasts on invasion ability of ovarian cancer cells and its mechanism of action. [Method] Primary cultured human ovarian normal fibroblasts (NOFs) and cancer-associated fibroblasts (CAFs) were collected, and conditioned media (NOFcm and CAFcm) were collected. SKOV3 cells were treated with serum-free medium DMEM, NOFcm and CAFcm, respectively, to induce epithelial-mesenchymal transition (EMT) in SKOV3 cells. Morphological observation and Western blot were used to verify the results. SKOV3 cells were morphologically epithelial-to-mesenchymal transition (EMT). Transwell chamber and three-dimensional culture experiments were used to detect the changes of cell invasiveness. [Result] The conditioned medium of CAF induced the morphological changes of ovarian cancer SKOV3 cells in vitro: the cells were loosely connected and some cells were fusiform, and the expression of E-cadherin protein was significantly down-regulated and the expression of Vimentin protein was significantly increased. Transwell invasion assay showed that the number of SKOV3-penetrating matrigel after CAFcm treatment (284.00 ± 32.86) was significantly higher than that of serum-free DMEM (60.60 ± 11.72) and NOFcm (116.60 ± 14.22) (P = 0.000374 And P = 0.001258). In the three-dimensional culture experiment, only the CAFcm group and the NOFcm group could form the cloned spheres, while the cloned spheres could not be formed in the serum-free DMEM group. Compared with the NOFcm group (37.75 ± 1.70 vs 14.5 ± 1.29 ), The formation of a significant increase in the sphere of cloning, invasive ability increased, the number increased (P <0.001). [Conclusion] CAF of ovarian cancer induces EMT in ovarian cancer cells and then promotes invasiveness of ovarian cancer cells.