摘要
目的:构建趋化因子CXC亚族CXCR4的慢病毒表达载体并观察其转染人脐带间充质干细胞后的表达。方法:用逆转录PCR方法获取CXCR4基因编码区片段,将构建的慢病毒载体质粒pLVTHM-EGFP-CXCR4与包装质粒psPAX2和包膜质粒pMD2.G共转染293T细胞,包装生产慢病毒。用相同滴度的慢病毒转导等量间充质干细胞(Mesenchymal Stem Cell,MSCs),后采用Real time PCR检测CXCR4 mRNA、Western Blot方法检测蛋白质的表达。结果:PCR、酶切和测序结果表明成功的构建了CXCR4基因重组慢病毒载体。同时用该慢病毒载体转染MSCs后可有效地增加MSCs中CXCR4的表达。结论:成功构建了CXCR4的慢病毒表达载体并能在MSCs中表达,为进一步研究其在干细胞移植中的应用奠定基础。
Objective： To construct the lentiviral expression vector of CXCR4 and study the expression in human umbilical cord mesenchymal stem cell （hUCMSCs）. Methods： A reverse transcription-polymerase chain reaction （RT-PCR） was performed to get CX- CR4 gene code fragment, 293T cells were co-transfected with lentiviral expression vector of CXCR4, psPAX2 packaging plasmid and pMD2.G. The plasmid was coated to pack lentivirus. Then lentiviral expression vector of CXCR4 were co-transfected with hUCMSCs and the expression of CXCR4 mRNA and protein expression level were detected by RT-PCR and western blot respectively. Results： By means of sequencing, restriction endonuelease analysis and PCR, lentiviral expression vector of CXCR4 has been successfully constructed. lentiviral expression vector of CXCR4 co-transfected with hUCMSCs can effectively increase the expression of CXCR4 in MSCs. Conclusions： Lentiviral expression vector of CXCR4 co-transfected with hUCMSCs has been successfully constructed, which can be applied to further research of MSCs.
出处
《现代生物医学进展》
CAS
2013年第28期5429-5432,共4页
Progress in Modern Biomedicine
关键词
慢病毒
人脐带间充质干细胞
基因重组
CXCR4
Lentiviral
Human umbilical cord mesenchymal stem cell
Gene construction
CXCR4
