5和5/F35型腺病毒载体对人骨髓间充质干细胞转染效率的比较

背景：在以人骨髓间充质干细胞为基础的基因治疗中，提高腺病毒载体对人骨髓间充质干细胞的转染效率尤为重要。目的：对比观察5和5/F35型腺病毒载体对体外培养的人骨髓间充质干细胞的转染效率。设计、时间及地点：对比观察，于2008-03/10在解放军北京军区总医院血液科实验室完成。材料：骨髓来源于解放军北京军区总医院血液科异基因造血干细胞移植中健康供者，均无造血系统疾病。方法：采用密度梯度离心和贴壁筛选的方法体外分离培养人骨髓间充质干细胞，传至第3代后用流式细胞仪检测人骨髓间充质干细胞表型；按1×104/孔密度接种于24孔板，细胞贴壁后分别换用成骨、成脂诱导液培养，以碱性磷酸酶、油红O染色检测其分化特性。用不同滴度编码增强型绿色荧光蛋白基因的腺病毒载体Ad5增强型绿色荧光蛋白和Ad5/F35增强型绿色荧光蛋白按5，20，100，400 感染复数转染体外培养的人骨髓间充质干细胞，荧光显微镜下观察。主要观察指标：流式细胞仪检测增强型绿色荧光蛋白阳性表达率，并用锥虫蓝拒染法检测不同病毒滴度感染后人骨髓间充质干细胞活细胞比例。结果：人骨髓间充质干细胞表面CD166、CD29、CD73、CD31、CD45、CD34和CD14阳性率分别为95.08%、99.53%、72.26%，1.50%，2.02%，3.80%和4.94%。人骨髓间充质干细胞成骨诱导后14 d碱性磷酸酶染色为阳性，成脂诱导后14 d油红O染色均为阳性。Ad5增强型绿色荧光蛋白和Ad5/F35增强型绿色荧光蛋白按5，20，100，400 感染复数分别感染人骨髓间充质干细胞，2 d后前者增强型绿色荧光蛋白阳性率分别为(0.72±0.14)%，(4.97±0.46)%，(9.80±3.43)%和(45.53±6.32)%；后者增强型绿色荧光蛋白阳性率分别为(24.31±10.55)%，(55.19±13.73)%，(87.68±9.5)%和(96.57±5.64)%。在5，20，100的感染复数，各组细胞存活率均在95%以上。在400的感染复数，细胞存活率明显降低，在90%以下。结论：Ad5/F35对体外培养的人骨髓间充质干细胞的转染效率明显优于Ad5载体。

Comparison transfection efficacy of type 5 with 5/F35 adenoviral vectors in human mesenchymal stem cells

BACKGROUND: Enhancing transfection efficacy of adenovirus vectors in human bone marrow mesenchymal stem cells (hBMSCs) is important for application in hBMSC-based gene therapy.OBJECTIVE: To compare transfection efficacy of type 5 with type 5/F35 adenovirus vectors in hBMSCs in vitro. DESIGN, TIME AND SETTING: The controlled study was conducted at the Laboratory of Department of Haematology of General Hospital of Beijing Military Area Command of Chinese PLA from March to October 2008. MATERIALS: Bone marrow was obtained from healthy donors, who underwent allogenic hematopoietic stem cell transplantation at the Department of Haematology of General Hospital of Beijing Military Area Command of Chinese PLA. None of these donors developed disease of hematopoietic system. METHODS: The hBMSCs were isolated and cultured by density gradient centrifugation and wall-adhesion method. Phenotypes of the cells were identified by flow cytometry. The third passage of hBMSCs was inoculated into a 24-well plate at 1×104 cells/per well and were induced with the adipocyte and osteoblast inductor after the adhesion of the cells. Alkaline phosphatase and Oil-Red-O staining were used to verify the differentiation of hBMSCs into adipocytes and osteoblasts, respectively. HBMSCs were transfected in vitro with Ad5 enhanced green fluorescent protein (EGFP) or Ad 5/F35 EGFP at various multiplicity of infection (5, 20, 100, 400), and then observed under the fluorescence microscope. MAIN OUTCOME MEASURES: Transfection efficacy of EGFP was detected by flow cytometry. The rates of living cells were detected by trypan blue dye exclusion at the differently viral titers. RESULTS: The positive rates of CD166, CD29, CD73, CD31, CD45, CD34, CD14 in hBMSCs were 95.08%, 99.53%, 72.26%, 1.50%, 2.02%, 3.80%, and 4.94%, respectively. Alkaline phosphatase staining and Oil-Red-O staining were positive 14 days after the differentiation of hBMSCs induced by corresponding inductors into osteoblasts and adipocytes, respectively. In addition, EGFP-positive cells in the hBMSCs infected by Ad5-EGFP were (0.72±0.14)%, (4.97±0.46)%, (9.80±3.43)% and (45.53±6.32)%, whereas the positive cells infected by Ad5/F35-EGFP were (24.31±10.55)%, (55.19±13.73)%, (87.68±9.5)%, and (96.57±5.64)% two days after transfection of hBMSCs with the adenoviral vectors at the multiplicity of infection of 5, 20, 100, and 400, respectively. The survival rate of HMSCs was more than 95% at the multiplicity of infection of 5, 20 and 100. However, the survival rate was significantly lower, lese than 90%, at the multiplicity of infection of 400. CONCLUSION: The transfection efficacy of Ad5/F35 EGFP in hBMSCs in vitro is significantly higher than that of Ad5 EGFP.