DEC1基因shRNA慢病毒表达载体的构建及其稳转胶质瘤细胞系的建立  

Construction of shRNA lentiviral expression vector of DEC1 gene and establishment of its stable transfection glioma cell line

作　　者：魏学辉[1] 李晓明[1] 张耀[1] 茹懿[2] 王秦豪[2] 李霞[2] 林伟[1] WEI Xuehui LI Xiaoming ZHANG Yao RU Yi WANG Qinhao LI Xia LIN Wei(Department of Neurosurgery, Xijing Hospital Department of Biochemistry and Molecular Biology, School of Basic Medicine, Fourth Military Medical University, Xihn 710032, China)

机构地区：[1]第四军医大学西京医院神经外科,陕西西安710032 [2]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710032

出　　处：《中华神经外科疾病研究杂志》2017年第1期15-19,共5页Chinese Journal of Neurosurgical Disease Research

基　　金：国家自然科学基金资助项目(81572469)

摘　　要：目的构建人分化型胚胎软骨发育基因1(DEC1)的短发卡RNA(shRNA)慢病毒表达载体并建立稳定敲减DEC1表达的人脑胶质瘤细胞株T98G。方法根据Gen Bank中DEC1基因c DNA序列设计合成两条特异性shRNA序列,同时设计1条非特异性序列作为阴性对照,分别克隆到PsiLVRU6MP质粒载体内,经酶切电泳、DNA测序鉴定后包装成慢病毒颗粒。再以3组慢病毒感染胶质瘤细胞系T98G,用嘌呤霉素筛选后,荧光显微镜下观察m Cherry的表达,Western Blot检测各组DEC1蛋白的表达水平。结果 DEC1基因shRNA慢病毒表达载体经酶切、测序鉴定证实克隆正确。荧光显微镜检测显示各组细胞均已被慢病毒高效感染。Western Blot结果显示,两干扰组细胞各自DEC1蛋白表达水平明显低于未处理组和阴性对照组,分别占未处理组的39.47%±0.69%和41.17%±0.89%(P<0.05),但两干扰组之间无显著性差异(P>0.05);阴性对照组与未处理组相比亦无明显差异(P>0.05)。结论成功构建DEC1基因shRNA慢病毒表达载体,感染胶质瘤T98G细胞系获得成功。两干扰组慢病毒均能明显敲减目的基因DEC1的表达,为进一步研究DEC1在胶质瘤细胞系T98G中的生物学功能和作用机制提供了实验基础。Objective The differentiated embryochondrocyte expressed gene 1（DEC1）short hairpin RNA （shRNA） lentiviral expression vector and stably knock down DEC1 in human glioma cell line T98G were constructed.MethodsAccording to the cDNA sequence of the DEC1 gene provided by the GenBank, two specific shRNA sequences were designed and synthesized. A nonspecific sequence was designed as negative control. They were cloned into the PsiLVRU6MP plasmid vector. After the recombinant vectors were identified by enzyme digestion and DNA sequencing, they were packaged to obtain the lentiviral particles. Then T98G glioma cell line was infected with the three groups of lentivirus. After screening with puromycin, the expression of mCherry was observed under the fluorescence microscope, and the expression level of DEC1 protein in each group was detected by Western Blot.ResultsshRNA lentiviral expression vector targeting DEC1 gene was confirmed by restriction endonuclease digestion and DNA sequencing. Fluorescence microscopy showed that each group of cells had been efficiently infected by shRNA lentivirus. Western Blot results showed that DEC1 protein levels in the two DEC1shRNA groups were significantly downregulated compared with the untreated group and the negative control group, accounting for 39.47%±0.69% and 41.17%±0.89% of the untreated group, respectively （P〈0.05）. But there were no significant differences either between two DEC1shRNA groups or between the negative control and the untreated group （P〉0.05）.ConclusionThe DEC1shRNA lentiviral expression vector was successfully constructed and stably infected into human glioma T98G cell line. Both groups of DEC1shRNA lentivirus could markedly suppressed the expression of target gene DEC1. The results provide the experimental basis for further study of DEC1 function and mechanism in the human glioma cell line T98G.

关 键 词：分化型胚胎软骨发育基因1 短发卡RNA 慢病毒表达载体 胶质瘤 

分 类 号：R739.41[医药卫生—肿瘤]