肿瘤相关性成纤维细胞分泌胞外体增强胃癌干细胞化疗耐药

Fibroblast-derived exosomes contribute to chemoresistance of cancer stem cells in gastric cancer

目的探讨肿瘤相关性成纤维细胞分泌胞外体增强胃癌干细胞化疗耐药。方法收集2019年1月新鲜胃癌组织,从中分离出新鲜胃癌组织中分离肿瘤相关成纤维细胞,收集其条件培养基,提取胞外体,分为条件培养基组、胞外体组和对照组,体外成球实验和体内成瘤实验观察加入条件培养基或胞外体条件下,5-氟尿嘧啶对胃癌干细胞成球能力、成瘤能力和肿瘤生长速度的影响。蛋白质印迹法(Western blot)检测Wnt信号通路的主要蛋白β-连环蛋白(β-catenin)和Wnt2。采用t检验。结果体外成球实验和体内成瘤实验显示,在加入条件培养基或胞外体条件下,5-氟尿嘧啶对胃癌干细胞成球率[(2.90±0.25)%、(1.40±0.21)%,t=8.318,P<0.05;(3.40±0.34)%、(1.39±0.16)%,t=3.324,P<0.05]、成瘤能力[(483.36±39.15)、(185.25±12.31)mg,t=14.713,P<0.05;(572.62±47.39)、(188.36±13.19)mg,t=18.412,P<0.05]和肿瘤生长速度[(622.45±51.06)、(235.32±28.25)mm3,t=14.168,P<0.05;(702.45±65.31)、(255.39±20.47)mm3,t=13.308,P<0.05]均明显高于对照组,差异均有统计学意义。体外实验中加入胞外体后β-catenin和Wnt2表达明显增强,而胞外体抑制剂GW4869处理后β-catenin和Wnt2明显减弱(0.61±0.07、0.28±0.06;0.58±0.05、0.24±0.03),差异有统计学意义(t=5.284、6.380,P<0.05)。结论肿瘤相关成纤维细胞可通过胞外体增强胃癌干细胞的耐药性,机制可能与增强Wnt信号通路有关。

Objective To study the enhanced chemoresistance of cancer stem cells in gastric cancer through fibroblast-derived exosomes.Methods Exosomes were extracted from conditioned medium of carcinoma-associated fibroblasts isolated from fresh gastric cancer tissues collected in January,2019.There were conditioned medium group,exosome group and control group in the experiment.In the presence of conditioned medium or exosome,effects of 5-Fu on balling capacity,tumor growing capacity,tumor growth rate of gastric cancer stem cells were observed by cells balling capacity and tumor growth experiments in vitro and in vivo.Key proteinβ-catenin and Wnt2 in Wnt signal pathway were tested by Western blotting in vitro experiment with exosome or GW4869.Results Balling capacity[(2.90±0.25)%,(1.40±0.21)%,t=8.318,P<0.05;(3.40±0.34)%,(1.39±0.16)%,t=3.324,P<0.05],tumor growing capacity[(483.36±39.15),(185.25±12.31)mg,t=14.713,P<0.05;(572.62±47.39),(188.36±13.19)mg,t=18.412,P<0.05],tumor growth rate[(622.45±51.06),(235.32±28.25)mm3,t=14.168,P<0.05;(702.45±65.31),(255.39±20.47)mm3,t=13.308,P<0.05]of gastric cancer stem cells treated with 5-Fu in the presence of conditioned medium or exosome were significantly increased as compared with those in control group.The expression ofβ-catenine and wnt2 in exosome group was significantly stronger than that in control group,and that in GW4869 group was weaker(0.61±0.07,0.28±0.06,t=5.284;0.58±0.05,0.24±0.03,t=5.284,6.380,P<0.05).Conclusion Fibroblast-derived exosomes contribute to the enhanced chemoresistance of cancer stem cells in gastric cancer,which might be related to Wnt/β-catenin signal pathway.