脑源性神经营养因子基因重组腺病毒体外转染胚胎大鼠神经干细胞及其鉴定

Vitro transfection and its identification of neural stem cell with recombinant adenovirus containing BDNF gene in embryo rats

目的:以脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)基因重组腺病毒体外转染神经干细胞,建立能稳定表达BDNF的神经干细胞系,为下一步的动物实验提供材料。方法:(1)分离SD大鼠胎鼠的间脑,加入神经生长因子表皮生长因子(EGF)和碱性成纤维细胞生长因子(bFGF)在神经干细胞条件培养基中克隆培养。用免疫细胞化学方法鉴定分离的神经干细胞。(2)用BDNF基因重组腺病毒转染神经干细胞,并通过RT-PCR和Western-blot实验检测外源性BDNF基因的表达。结果:(1)分离培养出了大量具有不断增殖能力、表达神经巢蛋白的神经干细胞,并能经过诱导分化为神经元和神经胶质细胞。(2)60%～70%的神经干细胞感染BDNF基因重组腺病毒,Western-blot及RT-PCR实验显示BDNF大量表达。结论:本试验成功建立了神经干细胞的分离培养方法及能长期稳定表达BDNF的神经干细胞系。

Objective To establish in vitro the neural stem cell line that can express BDNF stably via the transfection of recombinant virus containing BDNF gene, Method Neural stem cells were isolated from diencephalons tissues of SD rat embryo, cultured in special culture medium containing bFGF and EGF, and then identified by immunocytochemistry, The cells were transfected with recombinant adenovirus containing BDNF gene segment and identified with RT-PCR and western-blot technique. Result The isolated and cultured neural stem cells had the ability to proliferate continuously. The cells could abundantly expressed nestin protein and were differentiated into neurons and glial cells by inducement. Sixty to seventy percent neural stem cells ＇were transfected by recombinant adenovirus containing BDNF gene segment successfully. RT-PCR and Western-blot showed that a great deal of BDNF were expressed in these stem cells. Conclusion The isolated and cultured methods of the neural stem cells from rat embryo and the stem cell line which can stably express BDNF were successfully established.