胃癌细胞促进网膜脂肪干细胞分化的机制研究

Mechanisms of differentiation of omental-adipose stromal cells promoted by gastric cancer cells

目的:本实验主要研究在胃癌条件培养基(conditioned medium,CM)诱导下网膜脂肪干细胞(omental-adipose stromal cells,O-ASCs)是否能分化为癌相关成纤维细胞(carcinoma-associated fibroblasts,CAFs),及ERK信号通路在其中的作用。方法:通过诱导分化成骨、成脂及流式细胞鉴定O-ASCs,将O-ASCs与MGC803和SGC7901 CM共培养,通过RT-PCR和Western-blot检测O-ASCs细胞CAFs标志物α-SMA、FSP-1、vimentin,旁分泌因子VEGFA、TGFβ-1、FAP、SDF-1的表达水平。将O-ASCs分为对照组,SGC7901-CM实验组,SGC7901-CM+U0126处理组,12 h后收集细胞。Western blot检测O-ASCs细胞CAFs标志物α-SMA、FSP-1及ERK1/2、p-ERK1/2的表达水平。结果:经鉴定原代培养出的细胞为O-ASCs,在SGC7901 CM和MGC803 CM作用下,CAFs标志物α-SMA、FSP-1、vimentin及旁分泌因子SDF-1、VEGFA、TGFβ-1、FAP表达均有明显增加(P<0.05)。与对照组比较SGC7901-CM组α-SMA、FSP-1、p-ERK1/2表达明显增加(P<0.05),ERK表达未见明显变化(P>0.05)。SGC7901-CM+U0126组与SGC7901-CM组比较,α-SMA、FSP-1及p-ERK1/2的蛋白表达水平明显降低(P<0.05),ERK表达变化无统计学意义(P>0.05)。结论:O-ASCs通过分化为CAFs及旁分泌作用参与胃癌腹膜转移,ERK信号通路在该过程中发挥了重要作用。

Objective：To investigate whether the omental-adipose stromal cells （O-ASCs） exposing to gastric cancer-conditioned medi-um （CM） could be inducted to differentiate into carcinoma-associated fibroblasts （CAFs） and the effect of ERK signaling pathway in the process. Methods： We identified O-ASCs by examining their ability to differentiate osteogenic and adipogenic lineages and through flow cytometry. O-ASCs were co-cultured with MGC803 and SGC7901CM. The expression of CAFs markers （α-SMA, FSP-1, and vimentin） and paracrine factors （VEGFA, TGF-β1, FAP, and SDF-1） were evaluated by RT-PCR and Western blot. In vitro cultures of O-ASCs were divided into three groups：the control, SGC7901-CM, and SGC7901-CM＋U0126 groups. Cells were collected after 12 h. West-ern blot was performed to evaluate the expression ofα-SMA, FSP-1, ERK, and p-ERK1/2. Results：The primary cells were O-ASCs. The expression levels of CAFs markers （α-SMA, FSP-1, and vimentin） and O-ASC paracrine factors （VEGFA, TGF-β1, FAP, and SDF-1） clearly in-creased （P〈0.05）. In comparison with the control, the expression of ERK in SGC7901-CM group did not change （P〉0.05）, while the ex-pression of p-ERK1/2,α-SMA, and FSP-1 significantly improved （P〈0.05）. Comparison of SGC 7901-CM＋U0126 and SGC 7901-CM groups showed that the expression levels of ERK had no statistical difference （P〉0.05）, while the expression levels of p-ERK1/2,α-SMA, and FSP-1 decreased （P〈0.05）. Conclusion：O-ASCs participate in the peritoneal metastasis of gastric cancer through differentiation by CAF and paracrine factors. The ERK signaling pathway is important in the differentiation of O-ASCs towards CAFs.