【摘 要】
：
目的:探讨胶质细胞源性神经营养因子(GDNF)慢病毒载体(lentivirus vector)体外转染嗅鞘细胞(OEcs)并能稳定表达目的蛋白的最佳感染复数(MOI),为修复脊髓损伤提供了新的方法和路径。方法:构建GDNF-lentivirus,体外转染293T细胞,获得高滴度的慢病毒浓缩液,测定病毒滴度达2x108 TU/ml.不同MOI的情况F.GDNF-lentivirus体外转染OEC,.
【机 构】
：
5l0630 广州,南方医科大学第三附属医院骨科 南方医科大学南方医院骨科
论文部分内容阅读
目的:探讨胶质细胞源性神经营养因子(GDNF)慢病毒载体(lentivirus vector)体外转染嗅鞘细胞(OEcs)并能稳定表达目的蛋白的最佳感染复数(MOI),为修复脊髓损伤提供了新的方法和路径。
方法:构建GDNF-lentivirus,体外转染293T细胞,获得高滴度的慢病毒浓缩液,测定病毒滴度达2x108 TU/ml.不同MOI的情况F.GDNF-lentivirus体外转染OEC,.Western blot检测GDNF的表达.
结果:相差显微镜明视野下动态观寨转染后的OECs生长状态良好,荧光显微镜下见大量绿色荧光蛋白(GFP)标记的转染成功的OECs.MOI=I0时.OECs的转染事最高,约为75％,MOI=50时约为醴％.MOI=10和MOI=1时分别为25％和13％.阴性对照组未见有绿色荧光表达.转染后第5天,收集OECs进行Western blot检测,MOI=100和MOI=50时GDNF表达最强,两者差异无统计学意义(P＞O.05).MOI=IO表达较少,而MOI=1表达最少,对照组无GDNF表达.
结论:GDNF-lentivinos在体外既能高效转染OECs又不影响细胞活性,且转染后的OECs可以长期稳定表达GDNF的适宜MOI为50-100之间.
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