【摘要】 目的:探讨肿瘤相关成纤维细胞(CAFs)在调控胃癌细胞发生上皮间质转化(EMT)及迁移能力中的作用。方法:培养胃癌细胞系(SNU-1和MKN45)和原代成纤维细胞,分离和鉴定原代成纤维细胞;Western blot和实时定量PCR技术检测细胞内胸腺细胞分化抗原(Thy-1)的表达情况;并且建立胃癌细胞和CAFs共培养体系,将细胞分为胃癌细胞组、共培养体系组(空白对照组)、共培养体系+同型Ig G抗体组(阴性对照组)、共培养体系+抗Thy-1抗体组。Transwell实验检测Thy-1对共培养体系中胃癌细胞的迁移能力的影响;Western blot技术检测Thy-1对共培养体系胃癌细胞株的上皮(E-cad)及间质(N-cad和ZEB2)表型标记蛋白表达的影响。结果:成功分离CAFs,与正常成纤维细胞的形态并无明显差异,均出现纺锤形态,发现CAFs中α-平滑肌肌动蛋白(α-SMA)的表达比配对NFs细胞的表达量显著增加,提示分离出的CAFs可以作为后续相关实验研究;Western blot和实时定量PCR技术检测提示,CAFs中Thy-1的mRNA和蛋白质高表达,这与配对的NFs相比具有明显的统计学意义,且Thy-1在胃癌细胞系中低表达;CAFs与胃癌细胞共培养后,与胃癌细胞组相比,胃癌细胞系的迁移能力明显增强,且E-cad的表达显著降低,N-cad和ZEB2的表达明显升高,但共培养体系中CAFs预先孵育Thy-1抗体后,发现与阴性对照组相比,胃癌细胞系的迁移能力和EMT效应明显减弱。结论:CAFs细胞内过表达的Thy-1增强体外共培养体系中胃癌细胞的迁移能力和EMT效应。更多还原

【Abstract】 Objective:To explore the effects of CAFs on the migration and EMT of human gastric cancer cells. Methods:In vitro culture human gastric cancer cells and primary CAFs. Then separation and identification of primary CAFs were performed;the mRNA and protein expression of Thy-1 were analyzed by Western blot and quantitiative Real-time RT-PCR. After establishing co-cultured system,the cells were divided into four groups human gastric cancer cells group;co-culture system group( blank control group);co-culture system+ Ig G antibody group( negative control group) and co-culture system + Thy-1 antibody group,then the migration of human gastric cancer cells was detected by Transwell experiment,and the protein expression of E-cad,N-cad and ZEB2 of gastric cancer were observed by Western blot. Results:Primary gastric CAFs were successfully separated. Besides,CAFs and NFs had the similar feature,displaying spindle-like shape. The expression of α-SMA in the separated CAFs was significantly higher than the expression in the matched NFs,which indicated the CAFs were identified and could be used for the further study. The detection of Thy-1 in gastric CAFs were over-expressed,which was much higher than that in matched NFs group. The expression of Thy-1 in gastric cancer cells was very low. Co-cultured with CAFs,E-cadherin expression was decreased,while expression of N-cad and ZEB2 were enhanced compared to gastric cancer cells group;migration abilities of co-cultured with CAFs were also enhanced. Notably,the migration and EMT were reversed in the co-cultured cells compared to negative control group when Thy-1 was weaken. Conclusion:Over expression of Thy-1 of CAFs enhances mediate the migration ability and the effects of EMT of gastric cancer cell in co-culture system.更多还原