[1]王荥,王延洲,徐惠成.deltex-1腺病毒载体的构建及其在大鼠bMSCs中的表达[J].第三军医大学学报,2011,33(15):1555-1558.
 Wang Ying,Wang Yanzhou,Xu Huicheng.Expression of deltex-1 in rat bone marrow mesenchymal stem cells by adenovirus vector[J].J Third Mil Med Univ,2011,33(15):1555-1558.
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
33卷
期数:
2011年第15期
页码:
1555-1558
栏目:
论著
出版日期:
2011-08-15

文章信息/Info

Title:
Expression of deltex-1 in rat bone marrow mesenchymal stem cells by adenovirus vector
作者:
王荥王延洲徐惠成
第三军医大学西南医院妇产科
Author(s):
Wang Ying Wang Yanzhou Xu Huicheng
Department of Obstetrics and Gynaecology, Southwest Hospital, Third Military Medical University, Chongqing, 400038, China
关键词:
deltex-1基因克隆腺病毒载体骨髓间充质干细胞表达
Keywords:
deltex-1 clone adenovirus vector bone marrow mesenchymal stem cells expression
分类号:
R331.22; R394-33; R394.3
文献标志码:
A
摘要:
目的     构建deltex-1基因腺病毒载体并研究其在大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,bMSCs)中的表达。     方法      采用高保真酶PCR扩增deltex-1基因并测序,亚克隆至pAdTrack-CMV腺病毒穿梭载体后在BJ5183细菌中进行重组,挑选正确的重组子在FT293细胞中包装成病毒。以CsCl2方法纯化病毒,并进行滴度测定。将目的基因病毒感染bMSCs细胞后收集总RNA及蛋白,用PCR及Western blot方法检测目的基因表达水平。     结果      ①成功扩增了大小为1 883 bp的大鼠deltex-1,并成功构建了大小为4.5 kb的同源重组腺病毒穿梭载体,滴度为1.23×1011 IU/ml;②对培养的大鼠bMSCs进行流式检测,结果为CD90、CD44阳性表达,分别为99.57%和85.66%,而CD45表达阴性,阳性率为0.35%;③荧光显微镜观察deltex-1转染效率为60%~70%,RT-PCR及Western blot检测结果显示转染后deltex-1表达明显增强,说明转染成功。     结论      成功构建deltex-1基因腺病毒载体并能在bMSCs中高表达。
Abstract:
Objective       To construct an adenovirus vector of rat deltex-1 gene and then investigate its expression in rat bone marrow mesenchymal stem cells (bMSCs).      Methods      After deltex-1 gene was amplified by high-fidelity PCR, the product was subcloned to the pAdTrack-CMV adenovirus vector after sequencing, and recombined in the BJ5183 bacterium. The correct recombinant were picked and then packaged to virus in FT293 cells. Then the virus was purified with CsCl2 and its titer was determined. At last, the expression of deltex-1 gene was determined by detecting the contents of total RNA and protein of the bMSCs with the target gene by PCR and Western blotting respectively.      Results      Rat deltex-1 gene fragment of 1 883 bp was successfully amplify, and constructed in the reconstituted adenovirus shuttle vector of 4.5 kb, and the titer of the vector was 1.23×1011 IU/ml. Flow cytometric analysis showed cell-surface antigens for rat bMSCs were CD90 99.57%, CD44 85.66% and CD45 0.35%. The efficiency of transfection was 60% to 70% under fluorescent microscope. RT-PCR and Western blotting suggested that the expression of deltex-1 of transfected MSCs was obviously higher than controls. All of these reveal that the transfection of deltex-1 into rat bMSCs was successful.      Conclusion      The adenovirus vector of deltex-1 is successfully constructed and the bMSCs infected by the virus express the target gene at a high level.

参考文献/References:

王荥, 王延洲, 徐惠成. deltex-1腺病毒载体的构建及其在大鼠bMSCs中的表达[J].第三军医大学学报,2011,33(15):1555-1558.

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更新日期/Last Update: 2011-07-21