过刊浏览

杆状病毒转导不同哺乳动物骨髓来源间充质干细胞

刘正山, 张成, 卢锡林, 李勇, 许勇峰, 熊符, 冯善伟, 李玲

中山大学附属第一医院神经内科.广东,广州 510080；中山大学附属第一医院干细胞与组织工程研究中心.广东,广州 510080；南方医科大学遗传学教研室.广东,广州 510515

摘要

杆状病毒作为一种新型基因载体，若能有效转导不同哺乳动物骨髓来源间充质干细胞(bone marrow--derived mesenchymal stem cells, BMSCs)，将会成为干细胞基因修饰研究领域中更理想的一种基因载体。该文探讨了重组杆状病毒(Bac--CMV--EGFP)对不同哺乳动物BMSCs的转导效率。体外原代培养小鼠、大鼠、猪、恒河猴及人的BMSCs。用培养3代以后的哺乳动物BMSCs进行病毒转导实验，转导2 d后用倒置荧光显微镜观察绿色荧光蛋白在不同哺乳动物BMSCs中的表达情况，并用流式细胞仪检测重组杆状病毒对不同哺乳动物BMSCs的转导效率。结果显示：原代培养的小鼠、大鼠、猪、恒河猴及人的BMSCs于体外传代3次以上后，细胞呈现较均一的梭形，漩涡状生长；倒置荧光显微镜观察显示，与小鼠、大鼠、猪的BMSCs相比，恒河猴及人有更多BMSCs表达绿色荧光蛋白，且荧光强度较强；杆状病毒对小鼠、大鼠、猪、恒河猴及人的BMSCs的平均转导效率分别为(21.21±3.02)%﹑(22.51±4.48)%﹑(39.13±5.79)%﹑(71.16±5.36)%及(70.67±3.74)%。上述结果表明，重组杆状病毒对不同哺乳动物BMSCs的转导效率不一样，对恒河猴及人的BMSCs转导效率较高，说明重组杆状病毒可作为人或灵长类动物BMSCs基因修饰研究领域中更理想的基因载体。

关键词： 骨髓来源间充质干细胞; 杆状病毒; 转导; 绿色荧光蛋白

Transduction of various mammalian bone marrow--derived mesenchymal stem cells by baculovirus

Liu Zhengshan, Zhang Cheng, Lu Xilin, Li Yong, Xu Yongfeng, Xiong Fu, Feng Shanwei, Li Ling

Department of Neurology,the First Affiliated Hospital,Sun Yat-Sen University.Guangzhou 510080,Guangdong；China；Department of Medical Genetics,Southern Medical University.Guangzhou 510515,Guangdong

Abstract

The use of stem cells will lead to novel treatments for a wide range of diseases due to the properties of self-renewing, pluripotent, and undifferentiated state, and the stem cells are usually genetically modified for cell and gene therapy. If the baculovirus, as a new gene vector, can be effectively transduced into various mammalian bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, it will be a better gene vector to genetically modify the stem cells. The aim of the present study was to investigate the transduction efficiency of baculovirus into various mammalian BMSCs, which expressed a reporter gene encoding enhanced green fluorescent protein (EGFP) under a cytomegalovirus immediate early (CMV-IE) promoter. The BMSCs in mouse, rat, porcine, rhesus, and human were cultured primarily in vitro. After more than three passages, the mammalian BMSCs were seeded into dishes and cultured in a humidified incubator at 37 ℃ with 5% CO_(2). When the cells reached about 80% confluence, the complete medium was removed by aspiration. The cells were transduced with baculovirus at a multiplicity of infection (MOI) of 200 vector genomes/cell with 500 #mu#L of PBS at 25 ℃ for 4 h. At the end of baculovirus transduction, cells were washed and incubated with 2 mL complete medium, and baculovirus-transduced mammalian BMSCs were cultured in a humidified incubator for 2 d. Then, the invert fluorescent microscope was used to observe GFP expression in different mammalian BMSCs, flow cytometry was used to detect the transduction efficiency of baculovirus in various mammalian BMSCs. After more than three passages, the BMSCs in mouse, rat, porcine, rhesus, and human showed a homogeneous spindle-shaped morphology. Compared with the BMSCs in mouse, rat and porcine, the invert fluorescent microscope observations showed that there were more BMSCs in rhesus and human expressing GFP, which were transduced with baculovirus, and greater mean fluorescence intensity in rhesus and human BMSCs was observed. The baculovirus could efficiently transduce the BMSCs in mouse, rat, porcine, rhesus, and human, and the transduction efficiency of baculovirus into BMSCs in mouse, rat, porcine, rhesus, and human was (20.21±3.02)%, (22.51±4.48)%, (39.13±5.79)%, (71.16±5.36)% and (70.67±3.74)%, respectively. In conclusion, baculovirus displays different transduction efficiency into various mammalian BMSCs. Due to the high transduction efficiency for primate and human BMSCs, baculovirus is possibly a more suitable gene vector to genetically modify BMSCs in human and primates.

Key words: bone marrow-derived mesenchymal stem cells;baculovirus;transduction;green fluorescent protein

收稿日期：　　录用日期：

通讯作者：　　E-mail:

引用本文：

刘正山, 张成, 卢锡林, 李勇, 许勇峰, 熊符, 冯善伟, 李玲. 杆状病毒转导不同哺乳动物骨髓来源间充质干细胞[J]. 生理学报 2008; 60 (3): 431-436.

Liu Zhengshan, Zhang Cheng, Lu Xilin, Li Yong, Xu Yongfeng, Xiong Fu, Feng Shanwei, Li Ling. Transduction of various mammalian bone marrow--derived mesenchymal stem cells by baculovirus. Acta Physiol Sin 2008; 60 (3): 431-436 (in Chinese with English abstract).