胆囊癌相关成纤维细胞分泌IGFBP3促进淋巴内皮细胞的迁移

Gallbladder cancer-associated fibroblasts promote the migration of lymphatic endothelial cells via releasing IGFBP3

目的探讨胆囊癌相关成纤维细胞(CAFs)对淋巴内皮细胞(LECs)迁移能力的影响及相关分子机制。方法通过酶消化法提取胆囊癌的原代CAFs和正常胆囊的纤维细胞(NFs),收集相应细胞上清液(condition medium,CM),采用半定量蛋白因子芯片和ELISA实验筛查两者CM中IL-6、类胰岛素生长因子结合蛋白3(IGFBP3)等常见细胞因子水平。通过免疫组化检测胆囊癌和癌旁组织中α-SMA(CAFs标志物)和IGFBP3表达,分析其与患者临床病理特征的关系;培养LECs细胞,根据不同的处理方法将其分为无血清培养基组(Control组)、CAF-CM共培养组、NF-CM共培养组、IGFBP3组和CAF-CM+IGFBP3抑制剂(2-Deoxy-D-glucose)组,Transwell迁移实验和划痕实验检测各组中LECs迁移能力的变化;Western blotting检测不同处理条件下LECs的E-cadherin、N-cadherin和Vimentin蛋白的表达。结果蛋白因子芯片和ELISA检测发现CAF-CM较NF-CM中IGFBP3水平明显升高,免疫组化染色显示胆囊癌组织α-SMA高表达,且与淋巴结转移、TNM分期和IGFBP3高表达存在明显正相关。CAF-CM组中的IGFBP3可明显促进LEC迁移,并上调N-cadherin、Vimentin表达;下调E-cadherin表达;采用2-Deoxy-D-glucose处理后,可以逆转CAF-CM对LECs迁移和相关蛋白的表达变化。结论CAFs通过分泌IGFBP3影响EMT过程,从而促进LEC细胞迁移。

Objective To investigate the effects of gallbladder cancer-associated fibroblasts(CAFs)on the migration of lymphatic endothelial cells(LECs)so as to elucidate the molecular mechanisms involved.Methods The CAFs and normal fibroblasts(NFs)were extracted by enzymatic digestion,and the supernatant(CM)of CAFs and NFs was collected.The levels of IL-6,IGFBP3 and other related cytokines were detected by semi-quantitative protein factor microarray and ELISA.The expressions ofα-SMA(CAFs maker)and IGFBP3 in gallbladder cancer and para-cancer tissues were detected by immunohistochemistry,and the correlation ofα-SMA and IGFBP3 expressions with clinicopathological characteristics were analyzed.LECs were cultured and divided into serum-free medium group(control group),CAF-CM co-culture group,NF-CM co-culture group,IGFBP3 group,and CAF-CM+IGFBP3 inhibitor(2-Deoxy-D-glucose,2-DG)group according to different treatment.Transwell migration assays and wound healing assays were applied to analyze the migration ability of LECs under different treatment.The expressions of E-cadherin,N-cadherin and Vimentin were detected by Western blotting.Results Protein factor microarray and ELISA showed that the concentration of IGFBP3 in CAF-CM was significantly increased,and theexpression ofα-SMA was significantly related to lymph node metastasis,advanced TNM stage and expression of IGFBP3.IGFBP3 secreted from CAF-CM significantly promoted LECs migration,up-regulated the expression of N-cadherin and Vimentin,and down-regulated the expression of E-cadherin.Treatment with IGFBP3 inhibitor 2-DG could reverse the effect of CAF-CM on migration of LECs and related protein expressions.Conclusion Gallbladder CAFs promote the migration of LECs via releasing IGFBP3,which affects EMT transformation.