SHH基因修饰的大鼠鼻黏膜外胚层间充质干细胞向神经样细胞的诱导分化

目的: 探讨音猬因子（sonic hedgehog,SHH）基因转染对大鼠鼻黏膜外胚层间充质干细胞（ectomesenchymal stem cell, EMSCs）分化为神经样细胞的影响。方法: 利用组织贴壁法分离、培养和纯化EMSCs，SHH基因重组腺病毒转染EMSCs（SHH-EMSCs），免疫荧光染色观察转染前后EMSCs的干细胞标志蛋白巢蛋白、波形蛋白和Connexin-43，蛋白质印迹检测SHH蛋白表达。诱导分化实验分为4组：SHH-EMSCs诱导组、SHH-EMSCs正常培养组、EMSCs诱导组、EMSCs正常培养组；倒置相差显微镜观察分化过程中细胞形态变化；免疫荧光染色和蛋白质印迹检测诱导后轴突膜蛋白（GAP-43）、 β-3微管蛋白（TUBB3）、胶质纤维酸性蛋白（GFAP）和髓鞘碱性蛋白（MBP）的表达。并将SHH-EMSCs悬液注入大鼠脊髓组织内，观察该细胞在脊髓内存活和分化情况。结果： 免疫荧光染色结果显示，经SHH基因转染后的EMSCs巢蛋白、波形蛋白和Connexin-43阳性率达90%；蛋白质印迹结果显示，SHH蛋白表达明显升高。SHH-EMSCs诱导组培养7 d后，细胞呈现双极、多极和锥形的典型神经细胞样形态；免疫荧光染色和蛋白质印迹结果表明SHH-EMSCs诱导组GAP-43、TUBB3、MBP和Smoothen的表达水平明显高于其他3组，差异有统计学意义；GFAP在4组细胞中均为弱表达，无显著差异。SHH EMSCs在大鼠脊髓内存活情况良好且表达TUBB3。 结论： SHH基因转染的EMSCs具有更高的向神经样细胞分化效率，并可在脊髓内存活；EMSCs可望成为移植脊修复髓损伤的一种新的种子细胞。

Induced differentiation of SHH gene modified rat ectomesenchymal stem cells into neural-like cells

Objective: To investigate the effect of sonic hedgehog(SHH) gene transfection on differentiation of nasal mucosa-derived ectomesenchymal stem cells(EMSCs) into neural-like cells. Methods: EMSCs were isolated, cultured and purified by tissue adherent method.SHH gene recombinant adenovirus was transfected into EMSCs(SHH-EMSCs).The stem cells markers included Connexin-43, Nestin, and Vimentin in the EMSCs and SHH-EMSCs were stained by immunofluorescence. The expression of SHH was detected by Western blotting.Then these cells were induced to neural cells.The experiments were divided into four groups:SHH-EMSCs inducing group,SHH-EMSCs normal culture group,EMSCs inducing group and EMSCs normal culture group.The morphology of the cells were observed by the inverted microscope.The expression of neural markers included GAP-43, TUBB3, MBP and GFAP were detected by immunofluorescence and Western Blotting.In addition,the SHH-EMSCs suspension was injected into the spinal cord. Two weeks after transplantation, the survived SHH-EMSCs in the rat spinal cord and secreted SHH were examined by immunohistochemistry. Results: Immunofluorescence results showed that the staining positive rates of Connexin-43,Nestin and Vimentin of SHH-EMSCs were all about 90%.Western blotting data showed that SHH-EMSCs highly expressed SHH. Differentiated SHH-EMSCs showed typical bipolar, multipolar and pyramidal neural-like morphology after inducing for 7 days in experimental group. Immunofluorescence and Western-blotting data showed that differentiated SHH-EMSCs expressed higher GAP-43,β3-tubin, MBP and Smoothen compared with the other three groups; GFAP of cells were weakly expressed in the four groups, with no statistical significance. The experimental results in vivo showed that transplanted SHH-EMSCs have higher survival rate and could express TUBB3 and secrete SHH in the spinal cord. Conclusion: SHH gene modified EMSCs are easy to differentiate into neural-like cells and can be used as the excellent seed cells for repairing spinal cord injury.   ［Key words］nasal mucosa; ectomesenchymal stem cells; sonic hedgehog; gene transfection; neural lile cells; differentiation; spinal cord injury