Liu Zheng-Shan, Zhang Cheng, Lu Xi-Lin, Li Yong, Xu Yong-Feng, Xiong Fu, Feng Shan-Wei, Li Ling
Department of Neurology, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China.
Sheng Li Xue Bao. 2008 Jun 25;60(3):431-6.
The use of stem cells will lead to novel treatments for a wide range of diseases due to their 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 is to investigate the transduction efficiency of recombinant baculovirus (BacV-CMV-EGFP), which expressed a reporter gene encoding enhanced green fluorescent protein (EGFP) under a cytomegalovirus immediate early (CMV-IE) promoter, into various mammalian BMSCs. The BMSCs of 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 °C with 5% CO(2). When the cells reached about 80% confluence, the complete medium was removed by aspiration. The cells were transduced with recombinant baculovirus at a multiplicity of infection (MOI) of 200 vector genomes/cell with 500 μL PBS at 25 °C 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 inverted fluorescent microscope was used to observe GFP expressions in different mammalian BMSCs, and flow cytometry was used to detect the transduction efficiency of baculovirus in various mammalian BMSCs. After more than three passages, the BMSCs of mouse, rat, porcine, rhesus, and human showed a homogeneous spindle-shaped morphology. Compared with the BMSCs of mouse, rat and porcine, the inverted fluorescent microscope observations showed that there were more BMSCs expressing GFP and greater mean fluorescence intensity in rhesus and human transduced with baculovirus. The baculovirus could efficiently transduce into the BMSCs of mouse, rat, porcine, rhesus and human, and the transduction efficiency 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 of human and primates.
由于干细胞具有自我更新、多能性和未分化状态的特性，其应用将为多种疾病带来新的治疗方法，并且干细胞通常经过基因改造用于细胞和基因治疗。如果杆状病毒作为一种新型基因载体，能够在体外有效地转导到各种哺乳动物骨髓来源的间充质干细胞（BMSCs）中，那么它将是一种用于对干细胞进行基因改造的更好的基因载体。本研究的目的是研究重组杆状病毒（BacV-CMV-EGFP）的转导效率，该病毒在巨细胞病毒立即早期（CMV-IE）启动子的控制下表达编码增强型绿色荧光蛋白（EGFP）的报告基因，转导对象为各种哺乳动物的BMSCs。小鼠、大鼠、猪、恒河猴和人的BMSCs首先在体外进行原代培养。经过三代以上培养后，将哺乳动物BMSCs接种到培养皿中，置于37℃、5% CO₂的湿润培养箱中培养。当细胞达到约80%汇合度时，通过吸管吸出完全培养基。在25℃下，用500 μL PBS将重组杆状病毒以200个载体基因组/细胞的感染复数（MOI）转导到细胞中4小时。杆状病毒转导结束时，洗涤细胞并加入2 mL完全培养基，将经杆状病毒转导的哺乳动物BMSCs在湿润培养箱中培养2天。然后，使用倒置荧光显微镜观察不同哺乳动物BMSCs中GFP的表达情况，并使用流式细胞术检测杆状病毒在各种哺乳动物BMSCs中的转导效率。经过三代以上培养后，小鼠、大鼠、猪、恒河猴和人的BMSCs呈现出均匀的纺锤形形态。与小鼠、大鼠和猪的BMSCs相比，倒置荧光显微镜观察显示，经杆状病毒转导的恒河猴和人的BMSCs中，表达GFP的细胞更多，平均荧光强度更高。杆状病毒能够有效地转导到小鼠、大鼠、猪、恒河猴和人的BMSCs中，转导效率分别为（20.21±3.02）%、（22.51±4.48）%、（39.13±5.79）%、（71.16±5.36）%和（70.67±3.74）%。总之，杆状病毒对各种哺乳动物BMSCs的转导效率不同。由于对灵长类和人类BMSCs具有较高的转导效率，杆状病毒可能是一种更适合对人类和灵长类BMSCs进行基因改造的基因载体。
