强力霉素可调控的双重稳定表达AT2R的骨髓间充质干细胞的建立  

Dual-stable expression of angiotensin Ⅱ type 2 receptor gene medicated by doxycycline regulatable system of mesenchymal stem cells

作　　者：苗莉[1] 景涛[1] 何国祥[1] 蒋清安[1] 刘建平[1] 冉擘力[1] 王海东[2] 

机构地区：[1]第三军医大学西南医院心血管内科,重庆市介入心脏病学研究所,重庆400038 [2]第三军医大学西南医院胸心外科,重庆400038

出　　处：《第三军医大学学报》2008年第5期417-420,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30400180);第三军医大学中青年基金(2004)~~

摘　　要：目的构建四环素类抗生素可调控的双重稳定表达AT2R的骨髓间充质干细胞。方法将四环素可调控系统（Tetracycline-on，Tet-on）的4种质粒用脂质体转染法连续两个回合转染体外培养的大鼠骨髓间充质干细胞并抗性筛选，分别采用发光计检测不同细胞克隆荧光素酶活性改变以及RT-PCR法检测AT2R目的基因表达情况，根据各个细胞克隆受强力霉素调控表达的程度，筛选出高诱导、低背景表达目的基因AT2R的骨髓间充质细胞系，并检测在不同浓度（0～10^4ng／ml）强力霉素干预下以及转染后不同时间（0～8周）目的基因的蛋白表达情况。结果连续两个回合的转染及筛选后获得的引入Tet-on系统的细胞系，高诱导低背景表达AT2R，在强力霉素诱导下48h内可以使AT2R表达显著增加，强力霉素在一定浓度范围内可以诱导AT2R的表达呈剂量依赖性的增加，且至少在8周内保持稳定。结论构建的含四环素调控系统的骨髓间充质干细胞系诱导活性可靠，使外源AT2R基因的表达处于有效的主动控制下。Objective To construct mesenchymal stem cells（MSCs） of Dual-stable expression of angiotensin Ⅱ type 2 receptor（AT2R） gene medicated by tetracycline regulatable system. Methods Rat MSCs were transfected twice successively with tetracycline induced regulatable system by DOTAP liposomal transfection reagent. The luciferase protein activity was detected in different cell clones by luminometer and the expression of AT2R mRNA was evaluated by cording to the expression levels reverse transcription polymerase chain reaction （RT-PCR） respectively. Acregulated by doxycycline in different cell clones, the MSC line was tilted with high induction and low background. The expression of AT2R in MSCs was evaluated in different concentration of doxycycline （0 - 104 ng/ml） and time after transfection （0 - 8 weeks） by western blot. Results The expression rate of AT2R in double stable MSCs was increasing significantly by addition of doxycycline in a concentrationdependent manner by western blot, and the high expression was detected at 48 h after induced by doxycycline and could keep up over 8 weeks. Conclusion The well established double stable MSC lines make the AT2R gene under the active control of doxycycline and can be used as a good model to study the biological effect of AT2R.

关 键 词：AT2R 间充质干细胞 强力霉素 可调控表达 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学] R394.33[医药卫生—基础医学]