pEZsiRNA6.1/hHif-1α稳定转染食管鳞癌EC-9706细胞系的建立  

Establishment of esophageal squamous cell carcinoma cell line EC-9706stably tratnsfected with pEZsiRNA6.1/hHif-1α

作　　者：汲翔[1] 曹博[1] 金硕[1] 李军[1] 李懿[2] 范丹丹[2] 郭欢[3] 汲振余[2] 范天黎[1] 

机构地区：[1]郑州大学基础医学院,郑州450001 [2]郑州大学医药科学研究院,郑州450052 [3]甘肃省肿瘤医院基因多态性研究中心,兰州730050

出　　处：《华中师范大学学报（自然科学版）》2012年第6期740-743,共4页Journal of Central China Normal University：Natural Sciences

基　　金：国家自然科学基金项目(81071723;30873002);郑州大学大学生创新创业训练计划项目(2011cxsy175)

摘　　要：利用基因工程技术将合成的HIF-1αshRNA与pEZsiRNA6.1空载体连接,构建pEZsiR-NA6.1/hHif-1α载体,PCR和测序结果表明所构建的pEZsiRNA6.1/hHif-1α重组载体正确.进一步将所构建的载体转染人食管鳞癌EC-9706细胞系,利用G418筛选HIF-1α稳定干扰的细胞系,荧光显微镜检测结果表明:稳定转染pEZsiRNA6.1/hHif-1α的EC-9706细胞均有绿色荧光蛋白示踪.经CoCl2诱导模拟缺氧4h,Western Blot结果显示pEZsiRNA6.1/hHif-1α能有效抑制EC-9706缺氧食管癌细胞HIF-1α的诱导表达.HIF-1αshRNA synthesized and empty vector pEZsiRNA6.1were ligated by gene engineering technology,and pEZsiRNA6.1/hHif-1αrecombinant expression vector was constructed,and PCR and sequencing confirmed the corrections of pEZsiRNA6.1/hHif-1αrecombinant expression vector.Further,pEZsiRNA 6.1/hHif-1αvector was transfected into human esophageal squamous cell carcinoma EC-9706cell line,and G418was utilized to select the cell line with HIF-1αstable interference,the detection result of fluorescence microscope demonstrated that EC-9706cells stably transfection with pEZsiRNA6.1/hHif-1αappeared the green fluorescence.After treatment with CoCl 2 for 4h,the result of Western Blot revealed HIF-1α protein levels after chemical induction were markedly reduced in the cells after transfection,while it remained unchanged in the cells transfected with negative plasmid.This study will support the basis for its further functional study in Photodynamic therapy（PDT）.

关 键 词：HIF-1Α 基因沉默 光动力学疗法 食管鳞癌 

分 类 号：Q813[生物学—生物工程]