重组腺病毒Ad-DDAH2转染大鼠脂肪干细胞的可行性

Feasibility of transfection of rat adipose-derived stem cells with recombinant adenovirus Ad-DDAH2

目的探讨腺病毒载体(Ad-DDAH2)转染大鼠脂肪干细胞(ADSCs)的可行性。方法构建Ad-DDAH2-GFP质粒。原代培养大鼠ADSCs,流式细胞术检测免疫表型CD29、CD45、CD90表达情况。以不同滴度的Ad-DDAH2-GFP转染ADSCs,用荧光显微镜计数转染效率及细胞形态。通过RT-PCR及Western Blot检测DDAH2在ADSCs中的表达情况。结果Ad-DDAH2-GFP重组腺病毒载体构建、包装成功,且病毒滴度达到2E+10PFU/ML。第4代ADSCs生长曲线呈现“S”形。细胞免疫表型CD29、CD90阳性率分别为(95.83±0.53)%、(91.32±0.27)%,CD45阳性率为(1.59±0.06)%,符合ADSCs免疫表型特征。当病毒感染复数值为80、100时,转染效率均可达到90%以上。但病毒感染复数值为100时,ADSCs出现明显细胞病理现象,因此病毒感染复数值为80被视为最佳感染复数,RT-PCR及Western Blot鉴定可见阳性扩增条带。结论Ad-DDAH2-GFP重组腺病毒载体构建成功,可以高效转染大鼠ADSCs。DDAH2在mRNA及蛋白稳定长期表达,为后续体内实验奠定了实验基础。

Objective To explore the feasibility of transfection of rat adipose-derived stem cells with adenovirus vector(Ad-DDAH2).Methods Ad-DDAH2-GFP plasmid was constructed.Adipose-derived stem cells of the original generation in rats were cultured.The expression of immunophenotype CD29,CD45 and CD90 was detected by flow cytometry.Adipose stem cells were transfected with Ad-DDAH2-GFP with different titers.The transfection efficiency and cell morphology were counted by fluorescence microscope.The expression of DDAH2 in adipose-derived stem cells was detected by RT-PCR and Western blot.ResultsAd-DDAH2-GFP recombinant adenovirus vector was successfully constructed and packaged,and the virus titer reached 2E+10PFU/ML.The growth curve of the 4th generation adipose-derived stem cells is S-shaped.The results of flow cytometry showed that the positive rates of CD29,CD90 and CD45 were(95.830.53)%,(91.320.27)%and(1.590.06)%,respectively,which accorded with the immunophenotype characteristics of adipose-derived stem cells.When the virus infection complex values are 80 and 100,the transfection efficiency can reach more than 90%.However,when the virus infection complex value is 100,the adipose-derived stem cells have obvious cytopathological phenomena,so the virus multiplicity of infection 80 is regarded as the best multiplicity of infection,and positive amplification bands can be seen by RT-PCR and Western blot.Conclusion Ad-DDAH2-GFP recombinant adenovirus vector was successfully constructed,which can efficiently transfect rat adipose-derived stem cells.The long-term stable expression of DDAH2 at mRNA and protein levels has laid the experimental foundation for subsequent vivo experiments.