摘要
目的构建Tiam1基因的真核表达载体,评估其转染人胃癌细胞株MKN45细胞后对胃癌细胞功能的影响。方法实时荧光定量PCR分析Tiam1基因在胃癌细胞中的表达;将外源性重组真核表达载体Tiam1基因(N1/Tiam1)转染到人胃癌细胞株MKN45内,经G418筛选并建立稳定表达Tiam1基因的胃癌MKN45细胞株,为:N1/Tiam1组,转染空质粒细胞(N1)组和未处理细胞(MKN45)组的两个对照组分别为:N1组和MKN45组;通过细胞增殖、划痕和侵袭实验分别观察上调Tiam1基因表达后的胃癌细胞功能。结果与N1组相比,N1/Tiam1组中Tiam1蛋白表达明显上调[(0.36±0.05)vs.(2.67±0.14),P<0.01];与对照组相比较,N1/Tiam1实验组较N1对照组细胞增殖数量明显增加(P=0.000);与转染N1空载体的MKN45细胞对照组相比,转染了N1/Tiam1高表达质粒的MKN45细胞促进胃癌细胞的迁移和侵袭。结论 Tiam1基因真核表达载体的筛选成功,为继续深入的研究Tiam1基因在胃癌中的功能奠定了基础。
Objective To investigate the expression and function of Tiam1 gene in human gastric cancer MKN45 cell line by constructing the eukaryotic expression vector of theTiam1 gene and to explore its potential role in gastric carcinogenesis. Methods The expression levels of the Tiam1 gene were detected by real-time PCR in human gastric cancer MKN45 cell lines. A recombinant plasmid carrying specific for Tiam1 gene expression vector was constructed and transfected into MKN45 cells by the lipofectin-mediated method. Cells stably expressing the Tiam1 gene were selected using G418. MKN45 cells untransfected and those transfected with empty N1 plasmid were used as controls. The expression levels of the Tiam1 gene were detected by real-time PCR and Western blotting in stably transfected-MKN45 cells, biological function of the Tiam1 gene was analyzed by cell growth activity, scratch healing and invasion assays. Results Compared with N1 empty vector, Compared with N1 empty vector, the expression of theTiam1 protein was significantly increased （0.36±0.05 vs. 2.67±0.14, P0.01）. Cell growth activity were was significantly increased in in MKN45 cell line highly expressed N1/ Tiam1 vector compared to that in the control grouup （P=0.000 ）. Scratch healing and invasion assays of MKN45 cells also showed that migration and invasion were promoted in the Tiam1 group compared with the control group. Conclusion The eukaryotic expression vector of the Tiam1 gene was constructed successfully, which might contribute to probe the functions of Tiam1 in human gastric cancer in depth.[
出处
《中华普外科手术学杂志（电子版）》
2012年第3期24-26,43,共4页
Chinese Journal of Operative Procedures of General Surgery(Electronic Edition)
基金
山东省自然基金面上项目(ZR2011HM041
Y2007C102
ZR2011HM076)
山东省科技发展计划专项基金(2011GGB14158
2007H2071)
国家自然基金青年基金项目(81101858)
关键词
胃肿瘤
癌基因
遗传载体
细胞系
肿瘤
Gastric carcinoma
Oncogenes
Genetic vector
Cells line
tumor
