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目的检测卵巢癌患者腹水中肿瘤相关成纤维细胞(CAF)含量,并探讨其对卵巢癌肿瘤细胞侵袭能力的影响。方法从20例卵巢癌腹水中分离细胞后,流式检测其中α-SMA细胞阳性率并分为高低两群。在TCGA数据库557例卵巢癌标本数据库中验证α-SMA与上皮间质转化(EMT)相关的转录因子(SNAI1、TWIST1、ZEB1)的相关性;在mRNA和蛋白水平比较以上两群中肿瘤细胞上皮间质转化程度;磁珠分选成纤维细胞贴壁后α-SMA免疫荧光观察其形态;Western Blot验证SKOV3细胞系与人胚肺成纤维细胞系MRC5体外悬浮共培养后EMT程度的改变,Transwell和系膜清除试验验证在悬浮共培养状态下MRC5对SKOV3侵袭能力的影响。结果卵巢癌腹水中α-SMA阳性细胞占腹水细胞(34.40±18.67)%;TCGA数据库中α-SMA与上皮间质转化相关的转录因子显著正相关(r值分别为0.347 0、0.526 2和0.700 2,P<0.01);实时定量PCR(qRT-PCR)及Western Blot验证α-SMA-H组中肿瘤细胞EMT程度较α-SMA-L组更高;腹水中成纤维细胞贴壁免疫荧光成高表达α-SMA的间质样细胞形态;体外悬浮共培养后,MRC5促进SKOV3的EMT和系膜清除能力。结论卵巢癌腹水中存在CAF并可以促进卵巢癌腹水中肿瘤细胞EMT和侵袭能力。
Objective To detect the content of tumor-associated fibroblasts (CAF) in ascites of patients with ovarian cancer and to investigate its effect on the invasiveness of ovarian cancer cells. Methods After the cells were isolated from ascites of 20 patients with ovarian cancer, the positive rate of α-SMA cells was detected by flow cytometry and divided into two groups. The correlation between α-SMA and epithelial-to-mesenchymal transition (EMT) -related transcription factors (SNAI1, TWIST1 and ZEB1) was verified in 557 ovarian cancer specimen databases of TCGA database. The mRNA and protein levels of tumor cell epithelium The morphological changes of α-SMA were observed by immunofluorescence with α-SMA after the fibroblasts adhered to each other. The changes of EMT in SKOV3 cell line and human embryo lung fibroblast cell line MRC5 after in vitro co-culture were observed by Western Blot. Transwell And mesangial clearance test to verify the effect of MRC5 on SKOV3 invasion in suspension co-culture. Results α-SMA positive cells in ascites of ovarian ascites cells were (34.40 ± 18.67)%. There was a significant positive correlation betweenα-SMA and transcription factors related to epithelial-mesenchymal transition in TCGA database (r = 0.347 0, 0.526 2 and 0.700 2, P <0.01). Real-time quantitative PCR (qRT-PCR) and Western Blot showed that the level of EMT in α-SMA-H group was higher than that in α-SMA-L group MRC5 promoted the EMT and mesangial scavenging ability of SKOV3 after co-culture in vitro. Conclusions The presence of CAF in the ascites of ovarian cancer can promote the EMT and invasiveness of tumor cells in ascites of ovarian cancer.