关键词: 绿色荧光蛋白, 酪氨酸羟化酶, 骨髓基质细胞, 反转录病毒载体

Abstract: Objective To investigate the expression of enhanced green fluorescent protein(EGFP) and tyrosine hydroxylase(TH) genes in bone marrow stromal cells(bMSCs).Methods bMSCs were isolated by a density gradient(lymphocytes separation) from limbs.After being cultured for 8～10 d in primary culture,the bMSCs were transduced by retrovirus supernatants of EGFP and TH from PT67 cells in sequence.The transduced bMSCs were then selected by different antibiotics.After having been selected for 7～10 d,we could get the bMSCs with stably expressed EGFP and TH genes. Results In primary culture,bMSCs could reach 70%～80% confluence after 3～4 d.In the second day after being transduced by retrovirus supernatants of EGFP,the bMSCs could express weak green fluorescence.The EGFP transdced bMSCs were selected by G418 and reached confluence about 8～10 d.The positive cells were over 60% and the EGFP expression was stable in the following passages. The stable EGFP expressed bMSCs were then transduced by retrovirus supernatants of TH and selected by hygromycin for about 8～10 d.The TH positive bMSCs were also over 60%.Conclusion EGFP and TH genes could transduce BMSCs readily and efficiently and the expression of EGFP and TH genes were stable in the following passages.The EGFP and TH genes expressed bMSCs are useful and important in the study of the migration of bMSCs in vivo and gene therapy for Parkinson disease.

Key words: green fluorescent protein, tyrosine hydroxylase, bone marrow stromal cells, retrovirus vector