微环境中S100A6直接和间接促进结直肠癌LoVo细胞迁移  

S100A6 in the microenvironment directly and indirectly promotes migration of colorectal carcinoma LoVo cells

作　　者：赵佳丽[1] 谢佳卿 谷月[1] 李爱芳[1] 陈露 黄茂 彭琪 曾宗跃[1] 周兰[1] ZHAO Jiali;XlEJiaqing;GU Yue;LI Aifang;CHEN Lu;HUANG Mao;PENG Qi;ZENG Zongyue;ZHOU Lan(Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, Chongqing Medical University, Chongqing 400016, China)

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《肿瘤》2018年第6期553-561,共9页Tumor

基　　金：重庆市渝中区科技计划项目(基础与前沿研究)(编号:20160106)~~

摘　　要：目的:深入探讨微环境中S100A6对结直肠癌LoVo细胞迁移的作用及其可能的分子机制。方法:制备并鉴定重组蛋白谷胱甘肽S-转移酶(glutathione S-transferase,GST)和融合蛋白GST-人S100A6(GST-human S100A6,GST-hS100A6)。用GST-hS100A6(终质量浓度为30μg/mL)处理LoVo细胞后,采用划痕愈合实验检测细胞的迁移能力变化,采用蛋白质印迹法检测LoVo细胞中总蛋白激酶B(protein kinase B,PKB,又称Akt)和磷酸化Akt(phosphorylated Akt,p-Akt)的表达变化。分别用磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/Akt信号通路抑制剂LY294002和GST-hS100A6单独或联合处理LoVo细胞后,采用划痕愈合实验检测细胞迁移情况。用GST-hS100A6刺激LoVo细胞后的条件培养液(CM-A6-LoVo)处理巨噬细胞,然后采用实时荧光定量PCR法检测该巨噬细胞中M2型标志物CD206和M1型标志物诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)的表达水平;进一步将该巨噬细胞与LoVo细胞共培养后,采用划痕愈合实验检测LoVo细胞的迁移能力。结果:成功获得GST-hS100A6和GST蛋白(作为对照)。与GST组相比,GST-hS100A6处理后的LoVo细胞划痕愈合率明显升高(P<0.05),而且LoVo细胞中p-Akt蛋白表达明显上调(P<0.05)。与单独GST-hS100A6处理组相比,LY294002联合GST-hS100A6处理后LoVo细胞的划痕愈合率明显降低(P<0.05)。与GST-hS100A6组相比,CM-A6-LoVo条件培养液处理后的巨噬细胞中CD206表达水平升高(P<0.05),而iNOS表达无明显变化(P>0.05);并且该巨噬细胞与LoVo细胞共培养可明显提高LoVo细胞的划痕愈合率(P<0.05)。结论:微环境中S100A6可直接和间接地促进结直肠癌LoVo细胞迁移,该作用机制可能与调控PI3K/Akt信号通路以及诱导巨噬细胞向M2型分化有关。Objective： To deeply investigate the effect of S100A6 in the microenvironment on migration of colorectal carcinoma LoVo cells, and to explore the possible molecular mechanism.Methods： The recombinant protein glutathione S-transferase （GST） and the fusion protein GST-human S100A6 （GST-hS100A6） were prepared and identified. After the treatment with GST-hS100A6 （30 μg/mL）, the migration of LoVo cells was detected by wound healing assay, the expressions of total protein kinase B （PKB, also known as Akt） and phosphorylation of Akt （p-Akt） in LoVo cells were detected by Western blotting. After the treatment with GST- hS100A6 and phosphatidylinositol 3-kinase （PI3K）/Akt signaling pathway inhibitor LY294002 alone or in combination, the migration of LoVo cells was detected by wound healing assay. After the macrophages were treated with the conditioned medium of LoVo cells stimulated by GST-hS100A6 （named as CM-A6-LoVo）, the expression levels of M2 phenotype marker CD206 and M1 phenotype marker inducible nitric oxide synthase （iNOS） in the macrophages were detected by real-time fluorescent quantitative PCR, and the migration ability of LoVo cells co-cultured with the macrophages was detected by wound healing assay.Results： GST-hS100A6 and GST protein （as a control） were successfully prepared. Compared with GST group, the wound healing rate of LoVo cells treated with GST-hS100A6 was significantly elevated （P 〈 0.05）, and the level of p-Akt protein in LoVo cells was significantly increased （P 〈 0.05）. Compared with the GST-hS100A6 alone group, the migration ability of LoVo cells was notably reduced in LY294002＋GST-hS100A6 group （P 〈 0.05）. Compared with GST-hS100A6 group, the expression of CD206 in the macrophages treated with CM-A6- LoVo was increased （P 〈 0.05）, while the expression of iNOS was not changed significantly （P 〉 0.05）, and the macrophages co-cultured with LoVo cells could significantly promote the migration of LoVo cells （P 〈 0.05�

关 键 词：结直肠肿瘤 肿瘤微环境 细胞迁移分析 巨噬细胞活化 S100A6 PI3K/AKT信号通路 

分 类 号：R735.34[医药卫生—肿瘤]