腺病毒与慢病毒载体对人脂肪间充质干细胞转导效率及基因表达的差异

Transduction of human adipose tissue-derived stromal cells using lentiviral vector and adenoviral vector:a comparative study

目的:比较重组腺病毒、慢病毒载体介导增强型绿色荧光蛋白对体外培养的人脂肪来源干细胞(hADSCs)的转导效率和外源基因表达差异。方法:原代分离培养hADSCs并鉴定,应用Ad5F35-EGFP及LV-EGFP以MOI=0、25、50、100、200、400、800感染hADSCs,在1、3、7、14、21天应用荧光显微镜、流式细胞仪观察检测表达EGFP细胞阳性率。MTT法检测转导对hADSCs增殖的影响。VonKossa染色、油红O染色检测体外诱导转导EGFP的hADSCs向成骨及成脂方向分化能力。结果:hADSCs细胞表面标记CD31、CD34、CD45、CD106和HLA-DR阴性,CD29、CD44、CD49d、CD105、CD166阳性。荧光显微镜观察Ad5F35-EGFP感染hADSCs后(1～7天)可见EGFP高表达,此后逐渐减弱;LV-EGFP感染hADSCs后21天,仍可见EGFP高表达。流式细胞仪检测Ad5F35-EGFP、LV-EGFP对hADSCs转导效率与MOI值呈正相关。MTT显示Ad5F35-EGFP在高MOI值(800)检测A值与对照组相比差异显著(P<0.01)。转导EGFP的hADSCs经体外诱导14天可向成骨、成脂方向分化。结论:与腺病毒相比,慢病毒能够更为安全、有效地感染体外培养hADSCs,并长期表达外源基因,是研究基因修饰hADSCs的理想载体。

Objective To compare the efficiency of human adipose tissue-derived stromal cells （hADSCs） transduced by adenoviral vector and lentiviral vector. Methods Adipose tissues were isolated to obtain hADSCs. Ad5F35-EGFP and LV -EGFP were constructed and transduced hADSCs at multiple of infection MOI ranging from 0,25,50,100,200,400,800. At 1,3,7,14,21d post transduction,EGFP positive was detected by fluorescence microscopy and flow cytometry （FCM）, which was followed by cell proliferation determination assessed with MTT assay at different MOIs. After induction of hADSCs modified with EGFP, von Kossa staining and red oil O staining were performed to test the formation of calcium and oil concentration. Results hADSCs＇s marker CD31,CD34,CD45,CD106 and HLA-DR were negative,CD29,CD44,CD49d,CD105,CD166 were positive. Fluorescence microscopy detected high EGFP. signal with hADSCs at ld to 7d after infection by Ad5F35-EGFP and high EGFP signal with hADSCs at 21d after infection by LV- EGFP.FCM results showed that MOI of Ad5F35-EGFP and LV-EGFP enhanced transduce efficiency of hADSCs in a dose-dependent manner. MTT assay showed that Ad5F35-EGFP （MOI=800） A had great contrast with control group （P〈0.01）. ADSCs encoding EGFP after differentiation into adipogenic and osteogenic lineages after lentiviral and adenoviral transduction. Conclusion Lentivirual vectors applied at high MOIs can result in integration and long-term gene expression.