抑癌基因PTEN真核表达载体的构建及其在人舌鳞癌SCC-4细胞系中的表达  

Construction of PTEN gene eukaryotic expressing vector and its expression in tongue squamous cell carcinoma SCC-4 cell line

作　　者：董肖婷[1] 张斌[1] 刘硕硕[1] 郭婷婷[1] 潘良朋[1] 贾妮[1] 

机构地区：[1]辽宁医学院附属第一医院口腔科,辽宁锦州121001

出　　处：《吉林大学学报（医学版）》2013年第2期282-285,428,共4页Journal of Jilin University：Medicine Edition

基　　金：辽宁省教育厅高等学校科学研究项目资助课题(L2010315)

摘　　要：目的:构建抑癌基因PTEN真核表达载体pEGFP-PTEN,并观察其在人舌鳞癌scc-4细胞系中的表达,为舌鳞癌的基因治疗提供理论依据。方法:提取人HeLa细胞总RNA,采用RT-PCR法获取PTEN全基因,克隆至pMD18-T载体,经EcoRⅠ和XhoⅠ双酶切后与真核表达载体pEGFP-N1连接,构建pEGFP-PTEN重组质粒。双酶切及测序鉴定后,经脂质体介导转染至体外培养的人舌鳞癌SCC-4细胞系,荧光显微镜观察绿色荧光蛋白在细胞内的表达;Western blotting法检测细胞内PTEN蛋白的表达。结果:pEGFP-PTEN重组质粒双酶切,克隆的目的基因片段约为1 200bp,测序结果与GenBank上PTEN序列完全一致;荧光显微镜下观察到pEGFP-PTEN在SCC-4细胞系中成功表达;Western blotting结果,转染组PTEN蛋白表达强度为1.07±0.15,与空转染组(0.62±0.11)和未转染组(0.57±0.08)比较差异有统计学意义(P<0.05)。结论:成功构建pEGFP-PTEN重组真核表达载体,该载体能在人舌鳞癌SCC-4细胞系中表达。Objective To construct the eukaryotic expressing vector of PTEN gene and to express the gene in SCC-4 cell line and to provide theoretical foundation for gene therapy of tongue squamous cell carcinoma.Methods Human total length of PTEN gene was obtained from HeLa cell line by RT-PCR and was cloned into pMD18-T to analyze the sequence.The correct PTEN was inserted into pEGFP-N1 vector.pEGFP-PTEN was identified by digestion with EcoRⅠ and XhoⅠ.Scc-4 cell line was transfeted by pEGFP-PTEN and the expression of PTEN was observed by fluorescence microscope and Western blotting.Results A 1 200 bp fragment had been cloned into pEGFP-N1 vector which was further identified by sequencing and NCBI BLAST analysis.pEGFP-PTEN was expressed successfully in SCC-4 cell line.Western blotting showed the expression of PTEN protein in pEGFP-PTEN group was 1.07±0.15,there were significant differences compared with pEGFP-N1 group（0.62±0.11） and blank control group（0.57±0.08）（P0.05）.Conclusion The eukaryotic recombinant vector pEGFP-PTEN is constructed successfully and could be overexpressed in SCC-4 cell line.

关 键 词：PTEN基因 质粒 SCC-4细胞系 基因转染 

分 类 号：R739.86[医药卫生—肿瘤]