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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(MOI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。MOI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 study the feasibility of adenovirus-mediated green fluorescent protein (GFP) gene labeling of human adipose-derived stem cells (ADSCs). Methods Human ADSCs were obtained by enzymatic digestion. Surface markers were detected by flow cytometry and induced by adipogenic differentiation. AD-GFP was transfected into ADSCs with different multiplicity of infection (MOI of 25, 50, 100 respectively), and the transfection efficiency of each group was observed to find the most suitable transfection conditions. Observe the decay rate of GFP-labeled ADSCs over time and determine the duration of the labeling effect. Results High expression of CD49d, CD90 and CD105, no expression of CD34, CD45 and CD106 were observed in adipocytes. Lipid droplets were induced by adipogenesis for 2 weeks and reddish with oil red O, which was consistent with various characteristics of ADSCs. The transfection efficiencies were (77.3 ± 4.2)%, (94.7 ± 3.2)%, (96.3 ± 3.0)% and MOI = 50 respectively. After labeling GFP with ADSCs, the labeling rate could still reach as high as 55.0% within 2 weeks and then to 17.0% after 4 weeks. Conclusion The present study suggests that adenovirus can efficiently transduce GFP gene into ADSCs and rapidly express protein product for 2 to 4 weeks.