人β-catenin-EGFP融合表达慢病毒载体构建及在毛囊干细胞中的表达  

Construction of lentivirus vector containing human β-catenin-EGFP and its expression in human hair follicle stem cells

作　　者：杨鹏高[1] 胡孝辉[1] 高丰厚[2] 俞为荣[1] 徐鹏[1] 方勇[1] 

机构地区：[1]上海交通大学医学院第三人民医院烧伤整形科,上海201900 [2]上海交通大学医学院第三人民医院实验中心,上海201900

出　　处：《上海交通大学学报（医学版）》2009年第9期1030-1034,共5页Journal of Shanghai Jiao tong University:Medical Science

基　　金：上海市自然科学基金(06ZR14138)~~

摘　　要：目的构建人β-连环蛋白（β-catenin）与增强型绿色荧光蛋白（EGFP）融合表达的慢病毒载体,感染人毛囊干细胞并予以鉴定。方法提取人血管内皮细胞总RNA,RT-PCR扩增获得β-catenin基因全部序列,采用TA克隆技术获取基因亚克隆pUCm-T-β-catenin,AgeⅠ酶切连接pGC-FU载体构建β-catenin融合EGFP共表达慢病毒载体质粒pGC-FU-β-catenin-EGFP。质粒转化感受态细菌,筛选阳性克隆,经RT-PCR及测序鉴定正确后转染FT293细胞,Western blotting分析鉴定。质粒再转染FT293细胞进行慢病毒质粒包装,荧光显微镜观察,Real-time PCR测定病毒滴度。包装后慢病毒质粒感染体外分离和培养经免疫荧光染色鉴定的人毛囊干细胞,RT-PCR鉴定,荧光显微镜观察感染效率。结果RT-PCR及测序鉴定证实目的基因正确克隆至慢病毒载体中。在转染pGC-FU-β-catenin-EGFP的FT293细胞,Western blotting证实细胞表达β-catenin;质粒包装后荧光显微镜观察FT293细胞见大量绿色荧光时测得病毒滴度为2.0×108TU/mL。在感染慢病毒质粒的人毛囊干细胞,当感染复数为10时,感染后48h的感染效率可达80%以上,感染后3周的感染效率仍维持在80%-90%。结论成功构建β-catenin-EGFP融合表达慢病毒载体,可高效感染人毛囊干细胞且表达稳定、持久。Objective To construct the lentivirus carrying human β-catenin-EGFP（enhanced green fluorescent protein）and observe its expression in human follicle stem cells.Methods The β-catenin gene sequence was amplified by RT-PCR from extraction of total RNA of human vascular endothelial cells.TA cloning technique was utilized to acquire gene subcloned pUCm-T-β-catenin.After transformation reaction,candidate clone was further analyzed by PCR and gene sequencing.Then the plasmid was transfected into FT293 cells.After identification by Western blotting,the plasmid was transfected into FT293 cells again for packaging.Infection titer was monitored by green EGFP expression.The expression of β-catenin-lentivirus in human follicle stem cells were observed under inverted fluorescence microscope.Results The β-catenin gene was cloned into the lentivirus successfully.The high expression of green fluorescence protein in FT293 cell line was found under fluorescent microscope.Viral titer checked by real-time PCR was about 2.0×108 TU/mL.When the multiplicity of infection（MOI）was 10,the infection efficiency of β-catenin-lentivirus in human follicle stem cells was nearly 80% after infection 48 h around.After 3 weeks of continuous observation,we found the infection efficiency still keeping in the range of 80%-90%.Conclusion The lentivirus expression vector for β-catenin was successfully constructed.It can steadily infect human follicle stem cells and the infection efficiency is considerable high.

关 键 词：慢病毒 Β-连环蛋白 增强型绿色荧光蛋白 人毛囊干细胞 

分 类 号：R346[医药卫生—基础医学]