重组腺病毒CXCL12基因表达载体的构建及其转染间充质干细胞后对其Runx2基因表达的影响

Construction of Recombinant Adenovirus Vector Expressing the CXCL12 and its Effect on Expression of Runx2 After Transfecting the Mesenchymal Stem Cells

目的:构建趋化因子CXCL12的重组腺病毒表达载体,观察其对间充质干细胞(MSC)中成骨特异性转录因子Runx2表达的影响。方法:将目的基因CXCL12全长c DNA模板进行PCR扩增、酶切后与p HBAd-MCMV-GFP载体结合,获得p HBAd-MCMV-CXCL12-GFP重组载体;将重组载体和包装质粒共转染HEK293细胞进行包装、扩增;利用所携带的绿色荧光蛋白基因测定病毒滴度,观察基因转染效率,QPCR和Western印迹检测CXCL12重组腺病毒转染后MSC中CXCL12和Runx2的表达。结果:酶切、PCR及测序结果证实CXCL12重组腺病毒载体构建成功,转染MSC后CXCL12和Runx2的表达明显升高,AMD3100可以降低CXCL12基因转染后MSC中Runx2的表达。结论:趋化因子CXCL12和MSC表面的CXCR4结合对MSC中成骨特异性转录因子Runx2的表达具有促进作用。

Objective： To construct the recombinant adenovirus vector expressing the CXCL12, and detect the ex-pression level of the Runx2 after it infected mesenchymal stem cells（MSC）. Methods： The CXCL12 gene fragmentwas amplified by PCR according to the c DNA of CXCL12, and then the fragment was cloned into p HBAd-MCMV-GFP. The recombinant plasmid was co-transfected into 293 T cells with packing plasmids for packaging and ampli-fying. Infection titer and rate were monitored by green fluorescent protein expression. The expression of CXCL12 and Runx2 were detected by Western blot and QPCR after Ad-CXCL12 infected MSC. Results： The results of se-quence scan, restriction endonuclease, and PCR confirmed that CXCL12 was cloned into the adenovirus vector suc-cessfully. The protein and m RNA levels of CXCL12 were increased significantly after Ad-CXCL12 infected MSC,meanwhile the levels of Runx2 were increased significantly too. The expression level of Runx2 was decreased afterincubation with AMD3100. Conclusion： Pretreatment of MSC with Ad-CXCL12 could significantly affect the ex-pression level of Runx2.