摘要
目的制备人微小RNA-16(miR-16)重组腺病毒,研究其在人骨髓间充质干细胞(hMSCs)中的表达及对细胞周期素依赖蛋白激酶6(CDK6)表达的影响。方法从人基因组DNA中扩增miR-16前体的DNA,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pAdTrack-CMV-miR-16与腺病毒骨架质粒pAdEasy-1共转化感受态大肠杆菌BJ5183,在细菌内同源重组获得重组腺病毒质粒rAdTrack-miR-16。将经PacⅠ线性化的rAdTrack-miR-16在人胚肾293细胞(HEK293)中包装并扩增出miR-16的重组腺病毒rAd-miR-16。将重组腺病毒感染hMSCs,分别检测miR-16前体和miR-16靶基因CDK6的表达。结果 pAdTrack-CMV-miR-16构建正确,在HEK293细胞中成功包装并扩增出重组腺病毒rAd-miR-16。rAd-miR-16感染的hMSCs中miR-16前体水平显著升高(P<0.05),CDK6mRNA的表达降低不明显,CDK6蛋白表达水平显著降低(P<0.01)。结论成功制备了表达人miR-16的重组腺病毒,并能有效介导miR-16在hMSCs中的表达,为进行miR-16的功能研究创造了条件。
Objective To prepare recombinant adenovirus encoding human miR-16 precursor and evaluate miR-16 expression mediated by adenovirus in human bone marrow-derived mesenchymal stem cells（hMSCs）.Methods miR-16 precursor was PCR-amplified from human genomic DNA and cloned into the adenoviral shuttle vector pAdTrack-CMV..The recombinant shuttle plasmid pAdTrack-CMV-miR-16 was then linearized by PmeⅠand followed by co-transformation into chemical competent E.coli.BJ5183,with the adenoviral backbone plasmid pAdEasy-1,was used to generate the recombinant adenovirus vector rAdTrack-miR-16..rAdTrack-miR-16 was linearized by PacⅠand transfected into human embryonic kidney 293（HEK293）cells for the packaging of the recombinant adenovirus rAd-miR-16..Concentrated rAd-miR-16 was used to infect hMSCs and the expression of miR-16 precursor in hMSCs was determined by RT-qPCR assay.CDK6 mRNA and protein was detected by RT-qPCR and Western blotting assay,respectively.Results Recombinant adenovirus shuttle vector pAdTrack-CMV-miR-16 was constructed and confirmed by restriction endonuclease analysis and DNA sequencing..rAd-miR-16 was successfully amplified in HEK293 cells.Human miR-16 precursor was significantly increased in hMSCs infected with rAd-miR-16（P0.05）..The level of CDK6 mRNA was decreased but the degree of reduction was not significant..However,the protein expression level of CDK6 was significantly reduced in hMSCs infected with rAd-miR-16（P0.01）.Conclusion Recombinant adenovirus encoding human miR-16 precursor was successfully prepared..The recombinant adenovirus is useful in the functional study of miR-16.
出处
《热带医学杂志》
CAS
2011年第4期365-368,407,共5页
Journal of Tropical Medicine
基金
国家自然科学基金(30672077
81070102)
关键词
miR-16
载体构建
腺病毒
骨髓间充质干细胞
表达
miR-16
vector construction
adenovirus
bone marrow mesenchymal stem cells
expression
