ABSTRACT
AIM: To observe the enhanced green fluorescent protein (EGFP) gene expression and cytotoxicity in human adipose derived adult stem cells (hADSCs) by pEGFP-N1, Ad5-EGFP and rAAV-2/1-EGFP, and investigate the suitable gene-transferred vehicle. METHODS: The experiment was conducted at Chinese National Human Genome Center and the Third Hospital of Peking University from January to July 2006. ①After the patients and their relative were informed consent, the subcutaneous adipose tissue was obtained from the patients undergoing routine total hip joint replacement in Department of Orthopaedics, Third Hospital of Peking University. pEGFP-N1 was provided by Clotech Company, Ad5-EGFP and rAAV-2/1-EGFP by Vector Gene Technology Company. ②hADSCs were cultured in vitro after isolated from the adipose tissue after dissected and digested with type I collagenase. ③hADSCs of passage 3 were infected with pEGFP-N1, Ad5-EGFP and rAAV-2/1-EGFP and the EGFP expression and the cell toxicity were observed. ④Twenty-four hours after being transfected, 5?104 cells were reseeded in a 24-well plate and the solution was changed three times every week. The growth curves of each group were drawn. Normal non-transfected cells served as control. The influence of different transfection ways on the growth of hADSCs was observed. RESULTS: ①Comparison of transfected efficiency with different ways: pEGFP-N1 transfection showed a higher cytotoxicity and lower efficiency of 10.5%; Ad5-EGFP could efficiently transfect hADSCs (multiplicity of infection=5?102, 82.5%); when MOI was 0.05); however, the growth capability of hADSCs was decreased significantly in the pEGFP-N1 transfection group compared with the control, and the differences were significant at day 3-10 after transfection (P