重组SHH腺病毒纤维蛋白缓释支架对神经干细胞增殖与分化的影响

Effect of recombinant SHH adenovirus fibrin slow-release scaffold on proliferation and differentiation of neural stem cells

目的研究包含重组SHH腺病毒纤维蛋白缓释支架对神经干细胞(NSCs)增殖与分化的影响。方法实验分组:FG-SHH组(将NSCs种植于重组SHH腺病毒-支架组)和对照组(controls)[以培养板、重组SHH腺病毒转染组(SHH)、纤维蛋白胶支架组(FG)为对照组];体外分离培养并鉴定NSCs,构建重组SHH腺病毒纤维蛋白缓释支架,用免疫荧光和免疫印迹方法测定转染效率;MMT法检测细胞增殖活力;免疫荧光和免疫印迹法检测上述各组细胞表达神经细胞相关蛋白:β微管蛋白(β-tubulin Ⅲ)、髓磷脂碱性蛋白(MBP)和胶质纤维酸性蛋白(GFAP)的表达状态。结果体外培养的NSCs高表达nestin;转染重组SHH腺病毒后NSCs可稳定表达SHH;FG-SHH支架促NSCs增殖能力较强,并且FG-SHH组β-tubulin Ⅲ及MBP阳性细胞率和蛋白表达水平均高于对照组(P<0.05),FG-SHH组GFAP的阳性细胞率和蛋白表达水平低于对照组(P<0.05)。结论重组SHH腺病毒纤维蛋白缓释支架可以促进NSCs的增殖及其向神经元和少突胶质细胞分化,并抑制星形胶质细胞分化。

Objective To study the effects of fibrin scaffold that can release recombinant SHH adenovirus slowly on the proliferation and differentiation of neural stem cells(NSCs).Methods Experimental group:NSCs were planted in recombinant SHH adenovirus-scaffold group,culture plate,recombinant SHH adenovirus transfection group,fibrin scaffold group as control group(controls).The neural stem cells were isolated,cultured and identified.The fibrin scaffold that can release recombinant SHH adenovirus slowly was constructed.The efficiency of transfection was determined by immunofluorescence and Western blot.The cell proliferation was tested by MTT.Immunofluorescence and immunoblotting were used to detect the expression ofβ-tubulin Ⅲ,and MBP and GFAP in NSCs after inducing in each group.Results NSCs expresses nestin highly.NSCs stably expressed SHH after transfection with recombinant SHH adenovirus;FG-SHH scaffold promotes the proliferation of NSCs,and the positive rates and protein expression levels ofβ-tubulin Ⅲ and MBP in the FG-SHH group are increased significantly as compared with the controls.The positive rates and protein expression of GFAP in the FG-SHH group are significantly lower than that by the controls.Conclusions Recombinant SHH adenovirus fibrin scaffold can promote NSC proliferation and differentiation into neurons and oligodendrocytes and inhibit the differentiation of astrocytes.