作　　者： ; ; ; ; ;

机构地区： 广东省人民医院

出　　处： 《中国病理生理杂志》 2007年第7期1402-1407,共6页

摘　　要： 目的:构建人miR-133a重组腺病毒,研究其在人骨髓间充质干细胞(hMSCs)中的表达。方法:从人基因组DNA中扩增包含miR-133a的PCR产物,并定向插入腺病毒穿梭载体pAdTrack-CMV。将经pmeⅠ线性化的重组穿梭载体与腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌BJ5183,通过同源重组获得重组腺病毒质粒rAd-mir-133a。将rAd-mir-133a在人胚肾293细胞(HEK293)中包装并扩增出miR-133a的重组腺病毒。将重组腺病毒感染hMSCs,利用RT-PCR和实时定量PCR分别检测初级miR-133a和成熟miR-133a的表达。结果:限制性内切酶分析和DNA测序结果显示,重组腺病毒穿梭载体pAdTrack-CMV-miR-133a构建正确。在HEK293细胞中成功包装并扩增出重组腺病毒rAd-miR-133a。rAd-miR-133a感染的hMSCs中初级miR-133a转录子和成熟miR-133a表达显著增强。结论:成功构建了人miR-133a重组腺病毒,并在hMSCs中高效表达出miR-133a,为进行miR-133a的功能研究创造条件。 AIM： To construct recombinant adenovirus vector carrying human miR - 133a and study its expression in human mesenehymal stem cells （hMSCs）. METHODS： The PCR product containing miR - 133a was amplified from human genomic DNA and inserted into the adenoviral shuttle vector pAdTrack - CMV. Then the recombinant shuttle plasmid linearized by prne I was eotransformed into competent E. coli. BJ5183 with the adenoviral backbone plasmid pAdEasy - 1 to generate the recombinant adenovirus vector rAd - mir - 133a. tAd - mir - 133a was then packaged and amplified in human embryonic kidney 293 （ HEK293 ） cells. The purified rAd - miR - 133a was used to infect the hMSCs and the expression of miR - 133a was detected by non - quantitative RT - PCR and real - time PCR. RESULTS： The recombinant adenovirus shuttle vector pAdTrack - CMV - miR - 133a was constructed and verified by restriction endonuclease analysis and DNA sequence analysis, rAd - miR - 133a was successfully packaged and amplified in HEK293 cells. The tran- scriptions of primary miR - 133a and mature miR - 133a were over - expressed in the hMSCs infected with rAd - miR - 133a. CONCLUSION： The recombinant adenovirus vector carrying human miR- 133a is successfully constructed, which lay a foundation for miR - 133a function study.

关 键 词： 微小 载体构建 骨髓间质干细胞 腺病毒

领　　域： [生物学]