An Experimental Study On The Construction And Biological Characterization Of HADSCs Cell Lines Overexpressed With HGH And HT?4

OBJECTIVE This study was designed to transfect human adipose-derived mesenchymal stem cells with human growth hormone(hGH)and human Thymosin beta 4(hT?4)eukaryotic overexpression vectors by lentiviral transfection.(hADSCs),and their expression in hADSCs and their biological properties were examined to explore new seed sources for tissue engineered skin.Methods The specimens of normal adipose tissue were collected from the surgical patients in our hospital.The primary hADSCs were isolated and cultured,and the cells were identified and passaged.hGHSC and hT?4 overexpressing hADSCs cell lines were prepared by lentiviral transfection.The viral vector carries the puromysin resistance gene,and the cells containing the Puromysin are used to screen the infected cells to stabilize the strain.The transfected cells were tested for biological characteristics.RT-PCR was used to detect the change of hADSCs at the gene level,and Elisa and Weston-blot were used to detect the changes of hADSCs at the protein level.Results The surface markers of hADSCs were positive for CD90 and CD44,while CD34 was negative.The results of RT-PCR showed that hHDSCs transfected with hGH increased the expression of hGH by 1.78 times compared with untransfected hADSCs.The expression of hADSCs transfected with hT?4 was 1.65 times higher than that of untransfected hADSCs.The gray-scale analysis data of Weston-Blot showed that hGHSCs overexpressing hGH had higher hGH expression than hADSCs;hADSCs overexpressing hT?4 had higher hT?4 expression than hADSCs;hADSCs overexpressing hGH and hADSCs overexpressing hT?4 had higher ERK expression.In hADSCs,hADSCs overexpressing hGH and hADSCs overexpressing hT?4 expressed higher levels of p-ERK than hADSCs.Elisa results showed that the supernatant protein content of hGH-hADSCs was significantly higher than that of hADSCs(*P<0.01);the cytoplasmic protein content of hGH-hADSCs was significantly higher than that of hADSCs(**P<0.01);hT?4-hADSCs The protein content of supernatant was significantly higher than that of hADSCs(*P<0.01).The cytoplasmic protein content of hT?4-hADSCs was significantly higher than that of hADSCs(**P<0.01).Conclusion(1)Lentiviral transfection can successfully construct hADSCsoverexpressing hGH and hT?4 cell lines.(2)The expression of hGH and hT?4 in hADSCs overexpressing hGH and hT?4 cell lines was significantly higher than that in hADSCs cell lines.