稳定表达siRNA-STAT3基因Hep-2细胞系的建立  

Construction of stable expression of siRNA-STAT3 gene in Hep-2 cell line

作　　者：孙红村 王俊阁 皮丽宏[2] 高昆[3] 

机构地区：[1]煤炭总医院耳鼻咽喉科,北京100028 [2]河北省人民医院耳鼻咽喉科,河北石家庄050051 [3]河北省人民医院麻醉科,河北石家庄050051

出　　处：《中国耳鼻咽喉头颈外科》2010年第8期399-402,共4页Chinese Archives of Otolaryngology-Head and Neck Surgery

摘　　要：目的建立稳定表达siRNA-STAT3基因的Hep-2细胞系,为进一步探讨STAT3在喉癌中的作用机制提供基础。方法产生针对STAT3的小发卡状RNA寡核苷酸,连接到PGPU6/GFP/Neo载体,双酶切和质粒测序鉴定真核表达质粒PGPU6/GFP/Neo-siRNA-STAT3,G418筛选稳定转染细胞系,蛋白印迹法测定p-STAT3的表达。分别用MTT法绘制生长曲线和平板克隆形成实验测定单细胞增殖能力。结果构建了重组干扰质粒并筛选出了阳性克隆,蛋白印迹法鉴定STAT3被抑制后p-STAT3表达也明显下降。生长曲线显示siRNA-STAT3组3天后抑制效应才较为明显(P=0.001),5d后抑制效应更加突出(P=0.000),其抑制效应呈时间-效应关系。另外,siRNA-STAT3组单克隆形成率也明显低于对照组(P=0.000)。结论本研究筛选了可稳定、较高水平表达siRNA-STAT3的Hep-2细胞系。干扰STAT3表达对细胞增殖抑制的作用较为明显。OBJECTIVE To construct Hep-2 cell line with stable transfection of siRNA-STAT3 gene, and to explore the relationship between STAT3 expressionand the pathogenesis of laryngeal cancer. METHODS Specific STAT3 oligonucleotides were designed and synthesized. These oligonucleotides were annealed form the double strands DNA fragments. Then the fragments were cloned into pGPU6/GFP/Neo vector. The recombinant PGPU6/GFP/Neo-siRNA-STAT3 plasmid was confirmed by enzyme digestion and sequence analysis at the same time. Positive clones were selected out by G418 and p-STAT3 expression was identified by Western-blot analysis. The growth curve of the Hep-2 cells was drawn based on MTT assay and the growth ability of single Hep-2 cell was measured according to the plate colony formation assay respectively. RESULTS PGPU6/GFP/NEO-siRNA-STAT3 expression vector was successfully constructed. Western-blot analysis identified that p-STAT3 expression declined obviously in siRNA- STAT3 group compared with that in negative control and blank group. The growth curve explained that the Hep-2 cells of siRNA-STAT3 group began to show obvious inhibitory effect until they were inoculated in the culture plate for 3 days（P =0.001）.There were pronounced inhibitory effect after 5 days（P=0.000）. The results also showed time-effect relationship of the inhibition. Furthermore, the cell colony formation rate of siRNA- STAT3 group was less than the negative control and blank group（P =0.000）. CONCLUSION We successfully selected out Hep-2 cell line expressed siRNA-STAT3 gene stably and efficiently in this study. Down regulation of STAT3 correlated with the inhibition of growth of Hep-2 cells.

关 键 词：喉肿瘤 癌 鳞状细胞 转录激活因子3 转染 细胞增殖 

分 类 号：R739.65[医药卫生—肿瘤]