腺病毒介导GFP基因标记人脂肪来源干细胞可行性研究

Feasibility study of Human adipose-derived stem cells marked with GFP gene mediated by adenovirus

目的研究腺病毒介导绿色荧光蛋白（GFP）基因标记人脂肪来源干细胞（ADSCs）的可行性。方法通过酶消化法获取人ADSCs，采用流式细胞术检测表面标记物以及成脂分化诱导进行鉴定。将Ad—GFP通过不同感染复数（M01分别为25、50、100）转染ADSCs，观察各组转染效率，找出最适合的转染条件。观察GFP标记ADSCs后随时间衰减速率，确定标记效果的维持时间。结果获取的细胞高表达CD49d、CD90、CD105，不表达CD34、CD45、CD106，经成脂诱导2周现脂滴，油红O染色成红色，符合ADSCs的多种特性。M01分别以25、50、100转染后，转染效率分别为（77．3±4．2）％、（94．7±3．2）％、（96．3±3．0）％，MOI=50为转染的适宜条件。GFP标记ADSCs后，在2周内的标记率仍可高达55．0％，至4周后则衰减至17．0％。结论本研究提示通过腺病毒能够高效地介导GFP基因导入ADSCs，快速表达出蛋白产物，维持2—4周的标记示踪效果。

Objective To research the feasibility of human adipose-derived stem cells （ADSCs） marked with green fluorescent protein （GFP） gene mediated by adenovirus. Methods The ADSCs were harvested from human liposuction fat by enzymatic digestion, then identified by detecting surface antigens and inducing adipogenic differentiation. Next, the Ad-GFP was transfected to ADSCs through different MOI （25, 50, and 100）. The transfec- tion efficiency of each group was also observed to determine the optimal transfection conditions. Finally, maintenance time was determined by observing the attenuation rate after GFP gene marked ADSCs. Results The strong expres- sion of the ceils were CD49d, CD90 and CD105, while the negative expression were CD34, CD45 and CD106. Lipid droplets were generated at 2 weeks after adipogenic differentiation, and oil red O staining was positive, which was in accordance with a variety of characteristics of ADSCs. Transfection efficiency after different 25, 50, and 100 MOI were （77.3±4.2） %, （94.7 ± 3.2） %, （96.3± 3.0） % respectively, indicating that MOI = 50 was the optimal condition. The transfection efficiencies of ADSCs marked by GFP were 55.0% at 2 weeks and 17.0% at 4 weeks. Conclusion This study suggests that adenovirus can efficiently mediate GFP gene into ADSCs and rapidly express the GFP gene, maintaining 2 to 4 weeks tracing effect to ADSCs.