卵巢癌相关成纤维细胞对上皮性卵巢癌细胞增殖、凋亡、迁移和侵袭活性的影响

The effect of ovarian carcinoma-associated fibroblasts on the proliferation,apoptosis,migration and invasion of epithelial ovarian carcinoma cell

目的:采用Transwell体外共培养体系,研究直接分离自卵巢癌的癌相关成纤维细胞CAFs对卵巢癌细胞增殖、凋亡、迁移和侵袭活性的影响,探讨CAFs在上皮性卵巢癌进展中的作用。方法:采用0.4μm孔径的Tranwell小室进行CAFs或者NFs与卵巢癌细胞CAOV3体外共培养,用RT-PCR的方法检测共培养之后的卵巢癌细胞CAOV3中增殖细胞核抗原(PC-NA)表达的改变,以了解卵巢癌细胞增殖的变化。②用流式细胞仪检测共培养之后CAOV3细胞的细胞周期和Annexin-V/PI检测CAOV3细胞的凋亡情况。③用8μm孔径的Transwell小室建立卵巢癌CAFs与CAOV3细胞的交互作用模型,观测CAFs对CA-OV3细胞迁移特性的影响。④以Matrigel模拟重建基底膜,用8μm孔径的Transwell小室建立卵巢癌CAFs与CAOV3细胞的交互作用模型,观测CAFs对CAOV3细胞侵袭特性的影响。结果:与CAFs共培养之后卵巢癌细胞CAOV3的PCNA基因表达显著增高,并且随着CAFs细胞量的增加,CAOV3细胞增殖活性显著增强。同时CAFs细胞的PCNA基因表达下降,随着CAOV3细胞数量的增加,PCNA表达明显减少(P=0.011)。②采用流式细胞仪检测细胞周期的方法可以发现,与CAFs共培养之后CA-OV3细胞的凋亡显著减少(P=0.008),而在与NFs共培养之后的CAOV3细胞中未发现这一现象。③同时用Annexin-V/PI的方法,用流式细胞仪进行检测,也可以证实与CAFs共培养之后CAOV3细胞的凋亡显著减少(P=0.011),而与NFs共培养之后,CAOV3的凋亡没有明显变化。④与对照组相比CAFs或者NFs对CAOV3作用8h后,CAOV3迁移明显增加,但CAFs组的增加有极显著性(P<0.01)。⑤侵袭实验中发现,与对照组相比CAFs或者NFs对CAOV3作用24h后,CAOV3侵袭明显增强,但CAFs组的增强有极显著性(P<0.01)。结论:CAFs能促进卵巢癌CAOV3细胞的增殖,减少其凋亡,并且两种成纤维细胞对卵巢癌细胞均有促迁移和侵袭的作用,但是CAFs的作用更加显著。这些发现提示,在CAFs与卵巢癌CAOV3细胞的交互作用中,产生的信号因子能够促进卵巢癌的进展。

Objective： To observe the effects of CAFs on the proliferation, apoptosis, migration and invasion of ovarian caicinoma cell line CAOV3 cells with the interaction coculture model between primary ovarian CAFs, NFs and ovarian carcinoma cell line CAOV3 established by Transwell chamber and to elucidate the role of CAFs in ovarian carcinoma progression. Methods： ①The interaction co - culture model between primary ovarian CAFs, NFs and ovarian carcinoma cell line CAOV3 was established by Transwell chamber of 0. 4 μm diameter. In order to find out the effect of proliferation of CAFs to CAOV3, the RT - PCR assay was used to observe the changing of PCNA mRNA expression. ②Cell cycle examination and Annexin - V/PI assays were used to verify the apoptosis of CAOV3 after the co - culture with CAFs or NFs with flow cytometry. ③The interaction model between primary ovarian CAFs, NFs and ovarian carcinoma cell line CAOV3 was established by Transwell chamber of 8 μm diameter to observe the effect of migration of CAOV3. ④ Matrigel was used to remodel the basement membrane, and the interaction model between primary ovarian CAFs, NFs and ovarian carcinoma cell line CAOV3 was established by Transwell chamber of 8 μm diameter to observe the effect of invasion of CAOV3. Results： ①The expression of PCNA mRNA in CAOV3 was significantly increased after the co - culture with CAFs, and with the number of CAFs increased, the motility of proliferation in CAOV3 was significantly increased （P = 0. 011 ）. ②By flow eytometry of the cell cycle, the apoptosis of CAOV3 was significantly decreased after the co - culture with CAFs （ P = 0. 008 ）, while it was not found in CAOV3 co - cultured with NFs.③Meanwhile, the decreasing apoptosis of CAOV3 was observed with flow cytometry in Annexin - V/PI assay （P = 0. 011 ）, but the apoptosis of CAOV3 did not change significant- ly after the co - culture with NFs.④The migration of CAOV3 was significantly increased after the co - culture with CAFs or NFs for 8 h, moreover, there was an extremely significant increase in CAFs group （P 〈0. 01 ）, when contrast with the control group.⑤In the invasion assay, the invasion of CAOV3 was significantly increased （P 〈0. 01 ）, especially in CAFs group, when CAOV3 was co -cultured with CAFs or NFs for 24 h. Conclusion： CAFs can promote the proliferation and decrease the apoptosis of ovarian carcinoma CAOV3 cells, both CAFs and NFs can promote the migration and invasion of CAOV3, but it is much more obvious in CAFs. During the interaction between CAFs and ovarian carcinoma CAOV3 cells, the signaling can promote the progression of ovarian carcinoma.