机构地区: 中山大学附属第二医院
出 处: 《中华神经医学杂志》 2007年第1期2-4,共3页
摘 要: 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.
领 域: [生物学]