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目的构建大鼠CXCR4的腺病毒表达载体,并观察其转染真皮多能干细胞(dermalmultipotentstemcells,dMSCs)后的表达情况。方法扩增含有CXCR4的全长cDNA,然后亚克隆至穿梭质粒pAdTrackCMV中,再与pAdEasy1质粒在大肠杆菌BJ5183中进行同源重组产生含有CXCR4的骨架质粒(pAdEasy/CXCR4)。将验证正确的pAdEasy/CXCR4用PacⅠ酶切后转染293细胞,从而包装出CXCR4的腺病毒表达载体(AdvCXCR4)。用AdvCXCR4转染dMSCs,并采用RTPCR、免疫荧光细胞化学和荧光激活细胞分类计数的方法检测其表达情况。结果PCR、酶切和测序结果证明,成功地将CXCR4全长cDNA克隆到骨架质粒中,并包装出腺病毒表达载体。同时,AdvCXCR4转染dMSCs后可有效地增加dMSCs中CXCR4的表达。结论CXCR4的腺病毒表达载体的成功构建和其在dMSCs中的表达,为研究CXCR4在干细胞移植中的应用奠定基础。
Objective To construct the adenovirus expression vector of rat CXCR4 and observe the expression of CXCR4 in dermalmultipotent stem cells (dMSCs). Methods The full-length CXCR4-containing cDNA was amplified and subcloned into the shuttle plasmid pAdTrackCMV. The pAdEasy1 plasmid was homologously recombined with the plasmid pAdEasy1 in E. coli BJ5183 to generate a CXCR4-containing backbone plasmid (pAdEasy / CXCR4). The correct pAdEasy / CXCR4 was verified by PacI digestion and transfected into 293 cells, thereby packaging the CXCR4 adenovirus expression vector (AdvCXCR4). The dMSCs were transfected with AdvCXCR4 and detected by RTPCR, immunofluorescence cytochemistry and fluorescent activated cell sorting. Results PCR, restriction enzyme digestion and sequencing proved that CXCR4 full-length cDNA was successfully cloned into the backbone plasmid and packaged adenovirus expression vector. Meanwhile, AdvCXCR4 transfected dMSCs can effectively increase the expression of CXCR4 in dMSCs. Conclusion The successful construction of CXCR4 adenovirus expression vector and its expression in dMSCs lay the foundation for the study of the application of CXCR4 in stem cell transplantation.