神经干细胞转染酪氨酸羟化酶基因后的分化

目的 探讨神经干细胞（NSCs）转梁酪氨酸羟化酶（TH）基因后的分化。方法 从胚胎16天Wistar大鼠室管膜前下周围区分离、增殖、鉴定NSCs；将TH基因和缺陷性逆转录病毒载体N2A构建成真核表达质粒，以电穿孔将其转入PA317包装细胞内；收集PA317包装产生的逆转录病毒颗粒，感染体外培养的NSCs，经G418筛选，获得成功转染TH基因的NSCs克隆；分别以0.4ng/ml bFGF和5％胎牛血清诱导转染及未转染TH基因的NSCs分化，比较TH基因转染及不同诱导方式对NSCs分化的影响，同时在分化细胞内检测TH的表达。结果 以0.4ng/ml bFGF诱导可使95％以上的NSCs分化为神经元，而5％FBS诱导则大多分化为神经胶质细胞，无论是否转染TH基因，神经元及神经胶质细胞的分化比例不成生改变；TH基因转染后能在神经干细胞的子代细胞内高效、稳定表达。结论 TH基因转染不影响NSCs的分化潜能，TH能在神经干细胞的子代细胞中有效表达0.4ng/ml bFGF诱导可以促使NSCs分化为神经元。

The differentiation of neural stem cells after tyrosine hydroxylase gene transferring

Objective The effects of tyrosine hydroxylase (TH) gene transference and low concentration of bFGF on differentiation of neural stem cells (NSCs) were investigated. Methods NSCs from the subependyma zone of embryonic day 16 (E16) Wistar rats were isolated, characterized and proliferated in vitro; TH N 2A expressing plasmids constructed with THcDNA and deficient recombined retroviral vectors N 2A were transferred into PA 317 packaging cells by electroporation; The retrovirus produced by the PA 317 packaging cells were harvested and used to infect NSCs in vitro. NSCs clones with TH gene were successfully selected by G 418 . NSCs with TH N 2A plasmids or not were induced by 0 4ng/ml bFGF or 5%FBS. The numbers of neurons and glial cells were counted and the expression of TH in differentiated cells was detected. Results More than 95% NSCs differentiated into neurons when induced by 0 4ng/ml bFGF, but majorities of NSCs differentiated into glial cells as induced by 5% FBS, The ratios of neurons and glia differentiated from NSCs would not change while TH gene was introduced into NSCs. Compared to control group, the ratios of differentiated positive TH cells significantly increased after TH gene being transferred. Conclusions TH gene transference does not influence the neuronal and glial differentiation of NSCs, but it can be efficiently expressed in NSCs. The medium with 0 4ng/ml bFGF can increase the numbers of neurons differentiated from NSCs.