摘要
目的探讨慢病毒载体介导人脑源性神经营养因子（hBDNF）和绿色荧光蛋白fG踊基因转染大鼠神经干细胞州SCs）后hBDNF的表达及其生物学特性的变化。方法构建hBDNF和GFP基因共表达的慢病毒载体并转染NSCs（hBDNF—GFP-NSCs组）．同时设GFP转染NSCs组（GFP-NSCs组）和未转染的NSCs组（NSCs组）。应用RT-PCR和Western blot法分别检测3组细胞中hBDNF mRNA和蛋白的表达：ELISA检测hBDNF—GFP-NSCs组细胞转染前后培养液中hBDNF含量的变化；使用上述3组细胞的上清液培养背根神经节（DRG）与NSCs，观察DRG的生长情况并应用流式细胞法检测NSCs分化为神经元的比例。结果RT—PCR、Western blot结果显示转染后7dhBDNF—GFP-NSCs组hBDNF mRNA和蛋白的表达均明显强于GFP—NSCs组和NSCs组；ELISA检测显示hBDNF—GFP转染NSCs后上清中hBDNF含量增加，第5天分泌达最高峰，与转染前比较差异均有统计学意义（P〈0．05）；使用hBDNF—GFP—NSCs组上清液培养DRG和NSCs，4d后DRG很快伸出突起，流式细胞法检测显示NSCs分化为神经元的比例高于其他两组。结论NSCs可作为基因转染载体，被hBDNF-GFP基因重组慢病毒转染后仍可保持原有生物学特性，并稳定表达和分泌有生物学活性的hBDNF和GFP。
Objective To explore the expression of human brain-derived neurotrophic factor （hBDNF） and green fluorescent protein （GFP） in hBDNF-GFP gene-transfected rat neural stem cells （NSCs） and the changes in the biological characteristics of the transfected cells. Methods NSCs were transfected with a lentiviral vector carrying hBDNF and GFP genes （hBDNF-GFP-NSCs） or GFP gene only （GFP-NSCs）, with normal NSCs as the control. The expression levels of hBDNF mRNA and hBDNF protein in all the 3 groups were detected by reverse transcriptase-polymerase chain reaction （RT-PCR） and Western blot, respectively. Enzyme-linked immunosorbent assay （ELISA） was performed to detect hBDNF level in the cell culture medium before and after hBDNF-GFP gene transfection. Dorsal root ganglion （DRG） neurons and NSCs were cultured with the supernatants of the transfected NSCs and normal NSCs, and the growth status of the DRG neurons was observed and the proportion of NSCs differentiating into neurons determined. Results Compared with GFP-NSCs and normal NSCs, hBDNF-GFP-NSCs showed obvious hBDNF overexpression at both mRNA and protein levels 7 days after the transfection, hBDNF content in the supernatant of hBDNF-GFP-NSCs culture increased significantly with time and peaked 5 days after the transfection （P〈0.05）. Four days after culture in hBDNF-GFP-NSCs supernatant, the DRG neurons and adherent NSCs extended cells processes, and the ratio of the NSCs differentiating into neurons was higher in cells cultured in hBDNF-GFP-NSCs supematant than in those culture in GFP-NSCs and normal NSCs supematants. Conclusion Lentivirus can be used as the vector for hBDNF and GFP gene transfection into NSCs, and hBDNF-GFP gene-transfected NSCs maintain the basic characteristics of NSCs and are capable of stable expression and secretion ofhBDNF and GFP.
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2009年第8期785-789,共5页
Chinese Journal of Neuromedicine
基金
上海市教委自然科学基金重点项目（04BB23、07ZZ42）
关键词
神经干细胞
脑源性神经营养因子
绿色荧光蛋白
基因转染
Stem cells
Brain derived neurotrophic factor
Green fluorescent protein
Gene transfection
