GDNF基因转染诱导神经干细胞向神经元分化的作用

Gene transfer of GDNF plasmid/liposome complexes in rat neural stem cells inducing NSC differentiate into neurons

目的探索转染GDNF基因到大鼠神经干细胞(neural stemcell,NSC)的方法,并研究其诱导大鼠神经干细胞向神经元和多巴胺能神经元分化的作用。方法体外扩增GDNF基因,并构建带有绿色荧光蛋白(GFP)的表达载体PcDNA3-GDNF-GFP质粒。悬浮培养大鼠胚胎神经干细胞,脂质体介导PcDNA3-GDNF-GFP质粒转染神经干细胞,荧光显微镜观察转染及表达情况;免疫细胞化学鉴定β-微管蛋白Ⅲ(β-Tubulin III)和酪氨酸羟化酶(tyrosinehydroxylase,TH),以确定神经干细胞的分化为神经元和多巴胺能神经元情况。结果转染后72 h,荧光蛋白大量表达。脂质体介导PcDNA3-GDNF-GFP质粒转染神经干细胞后,神经干细胞分化为神经元和多巴胺神经元的比例明显增高。结论脂质体介导GDNF基因转染神经干细胞的方法简单、有效,是一较为理想的基因转染方法。GDNF基因能明显促进神经干细胞分化为神经元和多巴胺神经元的比例。

Objective To explore transfering GDNF gene in rat neural stem cells （NSCs） with green fluorescent protein expression Vector, to detect rat NSCs differentiating into neurons and dopaminergic neurons. Methods GDNF gene was amplified by PCR in vitro. GDNF was constructed into pcDNA3 plasmid linked with GFP. A recombinant plasmid PcDNA3-GDNF-GFP was constructed which could express GDNF gene. GDNF gene was then transferred to rat NSCs by using lipofectamine techinque. Transfection and expression of GFP were observed under fluorescencemicroscope. After seven days , β-Tubulin Ⅲ and TH were detected by immunocytofluorescence respectively to quantitate the degree of differentiation of neurons and dopaminergic neurons from NSCs. Results Fluorescence proteins were mostly expressed within 72h. The rates of inducing differentiated neurons and dopaminergic neurons increased significantly as compared with that of control after were transferred rat NSCs by plasmid PcDNA3-GDNF-GFP. Conclusion Transfecting NSCs by lipofectamine techinque is simple and efficient, and is an ideal method for gene transfection. GDNF gene can increase greatly the percentage of neurons and dopaminergic neurons in the course of inducing the differentiation of rat NSCs.