摘要
背景:细胞移植前,将目的基因转染干细胞可增强细胞治疗效果。但要实现目的基因的高效转移并适度表达,载体系统的选择是关键。目的:将携带绿色荧光蛋白的慢病毒载体感染人脂肪源性干细胞,检测病毒感染效率及感染后的细胞活力。方法:利用脂质体转染法获取慢病毒原液。设定不同感染复数(1,5,10,15,20),将带有增强绿色荧光蛋白的慢病毒载体感染人脂肪源性干细胞。荧光显微镜下观察病毒转染后人脂肪源性干细胞内增强绿色荧光蛋白的表达,流式细胞仪测定病毒感染效率。茜素红染色及油红 O 染色鉴定病毒转染后人脂肪源性干细胞的多向分化潜能。MTT 法检测转染后人脂肪源性干细胞的增殖情况。结果与结论:随病毒感染时间及感染复数值的增加,人脂肪源性干细胞内绿色荧光表达明显增强,感染后第 3 天,感染复数为 20 时人脂肪源性干细胞内可见明显绿色荧光表达,病毒感染效率为 60%;第 5～7 天,病毒感染效率可达 90%～95%。转染后人脂肪源性干细胞经成骨及成脂诱导液培养后,茜素红染色及油红 O 染色阳性,表明病毒感染未影响人脂肪源性干细胞的多向分化能力。病毒感染后,人脂肪源性干细胞的增殖能力未受明显影响。说明携带增强绿色荧光蛋白的慢病毒载体可以有效感染人脂肪源性干细胞。
BACKGROUND：Before transplantation, the ability of stem cells was obviously increased by the transfection of target gene.The key of gene therapy is that target gene was transferred effectively and was expressed appropriately in stem cells.The choice of carrier system will affect the treatment effectiveness.OBJECTIVE：To observe the transfection efficiency of lentiviral vector transfecting the human adipose-derived stem cells （hASCs） and to detect the vitality of hASCs transfected.METHODS：The recombinant lentiviral supernatant was obtained by standard lipofectamine reagent.The hASCs were transfected by lentiviral vector containing enhanced-green fluorescent protein （EGFP） with different multiplicity of infection （MOI=1, 5, 10, 15, 20）.Transfection efficiency was assessed by flow cytometer for EGFP expression under fluorescence microscope.The multi-directional differentiation of hASCs after transfection was evaluated by alizarin red staining and oil red O staining.The proliferation of hASCs after transfection was detected by MTT assay.RESULTS AND CONCLUSION：Transfection efficiency was 60% at 3 day and 90%-95% at 5-7 day after infection under MOI=20.The alizarin red staining and oil red O staining showed hASCs after transfected hASCs were induced to osteogenic and adipogenic lineages, which suggested that viral infection could not affect multi-directional differentiation of hASCs.The proliferation of transfected hASCs had no marked change.This showed that hASCs transfected by lentiviral vectors carrying EGFP can effectively infect hASCs.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第36期6695-6698,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
上海市青年科技启明星计划基金(09QA1407500)项目
上海市自然科学基金(08ZR1423000)项目~~
