TrkA基因修饰、诱导神经干细胞分化为胆碱能神经元

Study on neural stem cells differentiated into cholinergic neuron induced by TrkA gene transfection

目的研究外源性TrkaA基因修饰C17．2神经干细胞，对神经干细胞定向分化的影响。方法采用脂质体法将携带有TrkA基因的真核表达载体pcDNA3．1（＋）/TrkA转染C17．2神经干细胞，Western blot观察转染后基因表达情况；将正常生长的C17．2神经干细胞随机分成两组（A组和C组），将重组质粒转染阳性的C17．2神经干细胞随机分成两组（B组和D组），并用100ng／mg神经生长因子（NCF）分别作用于C组及D组，应用间接免疫荧光染色方法，观察各组细胞胆碱乙酰转移酶（CHAT）的表达。结果Western blot结果显示转染组TrkA蛋白表达明显高于非转染组，说明外源性TrkA基因导入靶细胞，并实现蛋白表达；间接免疫荧光染色显示，经NGF孵育的转染组细胞（D组）约有26％呈ChAT阳性，而非转染组（C组）约为9％，未经NGF孵育的A、B组未观察到CHAT阳性细胞。结论应用外源性TrkA基因修饰神经干细胞，造成TrkA基因过度表达，在NGF作用下，可以诱导更多的神经干细胞向胆碱能神经元分化。

Objective To study the effects of exogenous TrkA gene transfection on C 17.2 NSCs differentiation. Methods The recombinant eukaryotic expression vector pcDNA3.1 （＋）/TrkA were transfected into C 17.2 NSCs by lipofectamin method. The expression of TrkA gene in C 17.2 NSCs was assayed by Western blot; The normal C17.2 NSCs were divided into two groups（A and C） randomly, and the C17.2 NSCs which the recombinant plasmids were transfected into were divided into two groups（B and D）; The cells of C and D groups were incubated with 100 ng/mL NGF. The expression of ChAT was detected by indirect immunofluorescence staining. Results The result of Westem blot showed that TrkA gene was overexpressed after pcDNA3.1 （＋）/TrkA plasmids were transfected into C17.2 NSCs, indicating that the recombinants were expressed successfully in C17.2 NSCs; The result of indirect IF staining showed that incubated with 100 ng/mL NGF, the radio of ChAT positive cells of D group which the recombinant plasmids were transfected into was 26%, significantly increased compared with C group （9%）. While there were not ChAT positive cell detected in A and B groups without the treatment of NGF. Conclusion These results suggest that under the effect of NGF, the overexpression ofTrkA gene could induce more NSCs to differentiate into cholinergic neuron.