中华临床医师杂志（电子版）
中華臨床醫師雜誌（電子版）
중화림상의사잡지（전자판）
CHINESE JOURNAL OF CLINICIANS(ELECTRONIC VERSION)
2013年
24期
11476-11480
,共5页
绿色荧光蛋白质类%慢病毒属%间质干细胞
綠色熒光蛋白質類%慢病毒屬%間質榦細胞
록색형광단백질류%만병독속%간질간세포
Green fluorescent proteins%Lentivirus%Mesenchymal stem cells
目的：通过对携带增强绿色荧光蛋白（enhanced-green fluorescent protein，eGFP）基因的慢病毒载体系统的构建，感染兔滑膜间充质干细胞（synovial mesenchymal stem cells，SMSC），以获得稳定表达GFP的兔SMSC。方法在体外分离培养、纯化兔SMSC，通过Lipofectamine2000转染法将第三代慢病毒载体四质粒共转染293FT细胞产生慢病毒，收集、浓缩后进行滴度测定，对培养好的兔SMSC进行慢病毒感染，在荧光显微镜下观察GFP基因表达情况。结果第三代慢病毒四载体转染293FT细胞72 h可见到很强的GFP表达，收集、浓缩后测得病毒滴度为4.0×108 TU/ml，感染48 h后在荧光显微镜下可见约90%的兔SMSC标记上GFP基因。结论通过Lipofectamine2000转染法成功构建了第三代慢病毒高效率载体系统，且对兔SMSC感染效果良好，为以后兔SMSC的体内示踪标记奠定了基础。
目的：通過對攜帶增彊綠色熒光蛋白（enhanced-green fluorescent protein，eGFP）基因的慢病毒載體繫統的構建，感染兔滑膜間充質榦細胞（synovial mesenchymal stem cells，SMSC），以穫得穩定錶達GFP的兔SMSC。方法在體外分離培養、純化兔SMSC，通過Lipofectamine2000轉染法將第三代慢病毒載體四質粒共轉染293FT細胞產生慢病毒，收集、濃縮後進行滴度測定，對培養好的兔SMSC進行慢病毒感染，在熒光顯微鏡下觀察GFP基因錶達情況。結果第三代慢病毒四載體轉染293FT細胞72 h可見到很彊的GFP錶達，收集、濃縮後測得病毒滴度為4.0×108 TU/ml，感染48 h後在熒光顯微鏡下可見約90%的兔SMSC標記上GFP基因。結論通過Lipofectamine2000轉染法成功構建瞭第三代慢病毒高效率載體繫統，且對兔SMSC感染效果良好，為以後兔SMSC的體內示蹤標記奠定瞭基礎。
목적：통과대휴대증강록색형광단백（enhanced-green fluorescent protein，eGFP）기인적만병독재체계통적구건，감염토활막간충질간세포（synovial mesenchymal stem cells，SMSC），이획득은정표체GFP적토SMSC。방법재체외분리배양、순화토SMSC，통과Lipofectamine2000전염법장제삼대만병독재체사질립공전염293FT세포산생만병독，수집、농축후진행적도측정，대배양호적토SMSC진행만병독감염，재형광현미경하관찰GFP기인표체정황。결과제삼대만병독사재체전염293FT세포72 h가견도흔강적GFP표체，수집、농축후측득병독적도위4.0×108 TU/ml，감염48 h후재형광현미경하가견약90%적토SMSC표기상GFP기인。결론통과Lipofectamine2000전염법성공구건료제삼대만병독고효솔재체계통，차대토SMSC감염효과량호，위이후토SMSC적체내시종표기전정료기출。
Objective To study the construction Method of a lentiviral vector containing Enhanced-green fluorescent protein gene and the infection ability of rabbit synovial mesenchymal stem cells (SMSC), in order to obtain a stable GFP expressing rabbit SMSC. Methods Isolated, cultured and purification rabbit SMSC in vitro, four lentiviral vectors were co-transfected 293FT cells to produce lentiviruses by Lipofectamine 2000 transfection method, and infection rabbit SMSC with Lentiviral after collecting, enrichment and titer determination. Results After 72 h transfection with lentivirus four carriers, a strong expression of GFP could be found in 293FT cells. The viral titer was 4.0×108 TU/ml after collecting and enrichment, GFP expression was observed in about 90%of the rabbit SMSC. Conclusion The third generation lentiviral vector containing eGFP gene was successfully established by Lipofectamine 2000 transfection method and it could infect rabbit SMSC efficiently, which will provide a basis for Rabbit SMSC tracing mark in vivo.
