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| Feasibility of transfection of rat adipose-derived stem cells with recombinant adenovirus Ad-DDAH2 |
| LI Xuefeng1, ZHOU Shihao1, WANG Xinhua1, PANG Yiwei1, WANG Yanlin1, LI Qingchun1, DIAO Xinghua1, LIU Haiyan2,* |
1 Department of Reproductive Medicine, Binzhou Medical University Hospital, Binzhou 256603, Shandong, P.R. China;
2 Department of Gastroenterology Medicine, Binzhou Medical University Hospital, Binzhou 256603, Shandong, P.R. China |
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Abstract 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.Results Ad-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 (95.83 0.53)%, (91.32 0.27)% and (1.59 0.06)%, 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.
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Received: 11 September 2020
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