[1]苗莉,景涛,何国祥,等.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[J].第三军医大学学报,2008,30(05):417-420.
 MIAO Li,JING Tao,HE Guo-xiang,et al.Dual-stable expression of angiotensin II type 2 receptor gene medicated by doxycycline regulatable system of mesenchymal stem cells[J].J Third Mil Med Univ,2008,30(05):417-420.
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
30卷
期数:
2008年第05期
页码:
417-420
栏目:
论著
出版日期:
2008-03-15

文章信息/Info

Title:
Dual-stable expression of angiotensin II type 2 receptor gene medicated by doxycycline regulatable system of mesenchymal stem cells
作者:
苗莉景涛何国祥蒋清安刘建平冉擘力王海东
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,胸心外科
Author(s):
MIAO Li JING Tao HE Guo-xiang JIANG Qing-anLIU Jian-pingRAN Bo-liWANG Hai-dong
Department of Cardiology, Department of cardiothoracis surgery, Southwest Hospital, Third Military Medical University
关键词:
AT2R间充质干细胞强力霉素可调控表达
Keywords:
angiotensin II type 2 receptor mesenchymal stem cells doxycyclineregulatable expression
分类号:
R329.2; R394-33; R978.14
文献标志码:
A
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(0~104 ng/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48 h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。
Abstract:
Objective To construct mesenchymal stem cells(MSCs) of Dualstable expression of angiotensin Ⅱ type 2 receptor(AT2R) gene medicated by tetracycline regulatable system. 〖WTFZ〗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 reverse transcription polymerase chain reaction (RT-PCR) respectively. According to the expression levels regulated by doxycycline in different cell clones,the MSC line was filted 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 concentration-dependent 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 .

参考文献/References:

苗莉, 景涛, 何国祥,等. 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[J]. 第三军医大学学报,2008,30(5):417-420.

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更新日期/Last Update: 2008-04-22