慢病毒介导的Isll基因在人骨髓间充质干细胞中的表达  

Expression of lentivirus-mediated Isll gene in human mesenchymal stem cells

作　　者：王银芬[1] 李伟强[2] 方榕[1] 刘佳[1] 杨达雅[1] 胡承恒[1] 伍贵富[1] 项鹏[2] 

机构地区：[1]中山大学附属第一医院心血管研究所卫生部辅助循环重点实验室,广州510080 [2]中山大学干细胞与组织工程研究中心

出　　处：《中华生物医学工程杂志》2011年第3期220-224,共5页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金面上项目（30770896）;国家自然科学基金青年基金（81000150）;广东省科技计划（2009b080701018）

摘　　要：目的研究慢病毒介导的Isll基因在人骨髓间充质干细胞（hMsc）中的表达情况，并检测其下游基因Nkx2．5和Flk一1的表达。方法多位点Gateway载体构建技术构建2K7puro／EF1α-Isll慢病毒表达载体（由EF1α启动子启动Isll基因表达）。包装细胞293FT细胞获得慢病毒，再转染至hMSC。利用嘌呤霉素抗性筛选出Isll阳性细胞。应用RT．PCR、Westernblotting检测hMSC中Isll基因在转染后1-6周的mRNA和1—4周的蛋白表达情况。结果成功构建了慢病毒载体EF1α—Isll，转染后hMSC中检测出Isll基因mRNA和蛋白的表达，其mRNA表达随着培养时间延长而上调，第3周表达量（0．65±0．14）较1、2周增多（0．36±0．09，0．37±0．05，均P〈0．05），3周后表达趋于稳定。转染后在无任何诱导剂的情况下，检测到Isll下游基因Nkx2．5的表达，但尚未见Flk-1的表达。结论通过慢病毒载体将Isll基因转染hMSC后，可获得长期稳定的表达，并在无任何诱导剂的情况下，能促进下游基因Nkx2．5的表达。Objective To explore the expression of lentivirns- mediated Isll gene in human mesenchymal stem cells （hMSC） and to detect the expressions of its downstream genes Nkx2.5 and Flk-1. Methods Lentiviral expression vector 2K7puro/EF1α-Isll was constructed with multi-site Gateway vector construction technique （promotor EF1α driving Isll gene expression）. After packaging in 293FT cells, the lentivirus was then transfected into hMSC. The Isll-positive hMSC were screened by resistance to puromycin. Reverse transcription polymerase chain reaction （RT-PCR） and Western blotting were then used to detect the Isll mRNA expression in hMSC within 1-6 weeks and protein expression within 1-4 weeks after transfection, respectively. Results Lentiviral vector EF1α-Isll was successfully constructed, and mRNA and protein expressions of Isll gene in hMSC were detected after transfection. Notably, the mRNA expression of Isll increased over time, which was higher at week 3 （0.65＋0.14） compared to weeks 1 and 2 （0.36：1：0.09 and 0.37 ＋0.05, both P〈0.05） and remained stable after week 3. In the absence of inducer, expression of Isll downstream gene Nkx2.5 but not Flk-1 was detected in Isll-positive hMSC. Conclusion Long-term stable expression profile of Isll genes may be obtained by transfeetion into hMSC through lentiviral vectors, which can also result in expression of downstream Nkx2.5 gene in absence of inducer.

关 键 词：慢病毒属 Isl1基因 入骨髓间充质干细胞 转染 遗传载体 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]