稳定表达NLRC5基因肝癌细胞株的建立  

Construction of stable a human hepatoma cell line HepG2 with over-expression of NLRC5

作　　者：何莺华[1] 徐涛[1] 倪明明[1] 黄成[1] 李娟 孟晓明[1] 李俊[1] 

机构地区：[1]安徽医科大学药学院,合肥230032 [2]合肥瑶海区长淮街道社区卫生服务中心,合肥230011

出　　处：《安徽医科大学学报》2016年第1期5-8,共4页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81473268;81202978);安徽省自然科学基金(编号:1308085MH145)

摘　　要：目的建立起能够稳定表达NLRC5基因的肝癌Hep G2细胞株。方法设计遗传霉素(G418)的浓度梯度,通过对Hep G2细胞筛选最终确定筛选药物浓度。将构建好的人源p EGFP-C2-NLRC5重组质粒利用脂质体介导法转染至Hep G2肝癌细胞株中,用G418筛选出耐药阳性克隆。通过免疫荧光技术观察绿色荧光蛋白稳定表达情况,Western blot法验证NLRC5蛋白表达情况。结果 G418在14 d内使Hep G2细胞全部死亡的最小浓度是300 ng/ml。用G418筛选瞬转p EGFP-C2-NLRC5 14 d在荧光显微镜下可见耐药克隆的形成。Western blot法检测显示,稳定过表达组NLRC5的蛋白水平比空质粒转染组高(t=15.356,P<0.05)。结论成功构建稳定表达NLRC5基因的肝癌细胞株,为进一步研究NLRC5基因在肝癌的体内实验奠定了基础。Objective To establish a human hepatoma cell line HepG2 with over-expression of NLRCS. Methods Identified the suitable G418 concentration in the selection of stable transfection HepG2 cell. The eukaryotic ex- pression plasmid pEGFP-C2-NLRC5 was transfected into the HepG2 cells mediated by lipofectamine and then se- lected with G418. Immunofluorescence assay and Western blot were used to analyze the expression of NLRC5. Re- sults HepG2 cells were killed by G418 after 14 days with the minimum concentration of 300 ng/pd. Drug-resistant clones were formed after 14 days by selecting with G418. In the protein level, pEGFP-C2-NLRC5 HepG2 cell line was higher expression than pEGFP-C2 HepG2 cell line, the difference was statistically significant. Conclusion A hepatoma stable cell line over-expressing NLRC5 is successfully established, which may provide critical foundation for functional research of NLRC5 in liver cancer.

关 键 词：NLRC5 肝癌 稳定细胞株 HEPG2 

分 类 号：R735.7[医药卫生—肿瘤]