Slit2促进干细胞向神经细胞分化的作用(1)
[摘要]目的探究慢病毒介导分泌型糖蛋白神经生长导向因子2(Slit2)过表达载体转染对骨髓间充质干细胞(BMSCs)向神经细胞转化的作用。方法利用基因过表达技术，构建Slit2过表达载体;慢病毒转染BMSCs;利用流式细胞仪检测BMSCs的表面蛋白，RT`-PCR和Western`-blot方法检测空白对照组、空载体病毒组和过表达载体病毒组神经细胞表面蛋白胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)的表达。结果第2代BMSCs表面CD44、CD29、CD34和CD45的表达率分别为60.2%、58.3%、3.4%和2.6%，慢病毒转染效率为90%以上。空白对照组、空载体病毒组和过表达载体病毒组的GFAP、NSE mRNA和蛋白相对表达量比较，差异有统计学意义(F=9.089～13.893，P<0.05);其中过表达载体病毒组的GFAP、NSE mRNA和蛋白相对表达量明显高于空载体病毒组(P<0.05)，空载体病毒组和空白对照组比较差异无显著性(P>0.05)。结论Slit2过表达质粒可以提高BMSCs向神经细胞转化的效率，为神经损伤和脊髓损伤提供种子细胞。
[关键词]神经生长因子;骨髓;间充质基质细胞;神经胶质原纤维酸性蛋白质;磷酸丙酮酸水合酶
[ABSTRACT]ObjectiveTo explore the effect of lentivirus`-mediated overexpression of Slit2 on the transformation of bone marrow mesenchymal stem cells (BMSCs) into neural cells. MethodsA Slit2 overexpression vector was constructed and used to transfect BMSCs via lentivirus. Flow cytometry was used to detect surface proteins of BMSCs. RT`-PCR and Western blot were used to determine the expression of glial fibrillary acidic protein (GFAP) and neuron`-specific enolase (NSE) on the surface of neural cells in blank control group， empty lentiviral vector group， and overexpression lentiviral vector group. ResultsThe expression of CD44， CD29， CD34， and CD45 was 60.2%， 58.3%， 3.4%， and 2.6%， respectively， on the surface of the second generation BMSCs. The transfection efficiency of lentivirus was over 90%. There were significant differences in relative mRNA and protein expression of GFAP and NSE between the blank control group， the empty lentiviral vector group， and the overexpression lentiviral vector group (F=9.089-13.893，P<0.05). The overexpression lentiviral vector group had significantly higher relative mRNA and protein expression of GFAP and NSE than the blank control group and the empty lentiviral vector group (P<0.05)， while there were no significant differences between the blank control group and the empty lentiviral vector group (P>0.05). ConclusionThe Slit2 overexpression plasmid can promote the efficiency of the transformation of BMSCs into neural cells， which provide seed cells for nerve injury and spinal cord injury.
[KEY WORDS]nerve growth factor; bone marrow; mesenchymal stromal cells; glial fibrillary acidic protein; phosphopyruvate hydratase
骨髓間充质干细胞(BMSCs)具有多向分化潜能，在特定环境下，可以分化成人体内的各种细胞，比如软骨细胞、骨细胞以及神经细胞等[1`-4]。研究结果显示，BMSCs在特定诱导条件下可以转化成神经细胞，进而能促进神经损伤的修复;而且BMSCs来源于病人自体，移植后可以避免排斥反应[5];另外，BMSCs能分泌多种细胞因子，这为神经的修复提供了一个新的思路[6]。分泌型糖蛋白神经生长导向因子2(Slit2)是中枢神经系统发育过程中的一种轴突导向抑制因子，可控制神经轴突分支形成以及神经细胞迁移，能促进BMSCs向感觉神经元分化，参与损伤神经再生和功能修复[7`-9]。近年来，在交通事故中发生的神经损伤和脊髓损伤比较常见，而针对神经损伤的治疗一直是临床上的难点。本研究将Slit2过表达载体以慢病毒介导入BMSCs内，探讨慢病毒介导Slit2过表达转染促进BMSCs向神经细胞转化的作用，从而为临床神经损伤病人治疗提供新的思路。, 百拇医药(刘文忠 蔡静 李军 荣春 范磊 张海宁)
[关键词]神经生长因子;骨髓;间充质基质细胞;神经胶质原纤维酸性蛋白质;磷酸丙酮酸水合酶
[ABSTRACT]ObjectiveTo explore the effect of lentivirus`-mediated overexpression of Slit2 on the transformation of bone marrow mesenchymal stem cells (BMSCs) into neural cells. MethodsA Slit2 overexpression vector was constructed and used to transfect BMSCs via lentivirus. Flow cytometry was used to detect surface proteins of BMSCs. RT`-PCR and Western blot were used to determine the expression of glial fibrillary acidic protein (GFAP) and neuron`-specific enolase (NSE) on the surface of neural cells in blank control group， empty lentiviral vector group， and overexpression lentiviral vector group. ResultsThe expression of CD44， CD29， CD34， and CD45 was 60.2%， 58.3%， 3.4%， and 2.6%， respectively， on the surface of the second generation BMSCs. The transfection efficiency of lentivirus was over 90%. There were significant differences in relative mRNA and protein expression of GFAP and NSE between the blank control group， the empty lentiviral vector group， and the overexpression lentiviral vector group (F=9.089-13.893，P<0.05). The overexpression lentiviral vector group had significantly higher relative mRNA and protein expression of GFAP and NSE than the blank control group and the empty lentiviral vector group (P<0.05)， while there were no significant differences between the blank control group and the empty lentiviral vector group (P>0.05). ConclusionThe Slit2 overexpression plasmid can promote the efficiency of the transformation of BMSCs into neural cells， which provide seed cells for nerve injury and spinal cord injury.
[KEY WORDS]nerve growth factor; bone marrow; mesenchymal stromal cells; glial fibrillary acidic protein; phosphopyruvate hydratase
骨髓間充质干细胞(BMSCs)具有多向分化潜能，在特定环境下，可以分化成人体内的各种细胞，比如软骨细胞、骨细胞以及神经细胞等[1`-4]。研究结果显示，BMSCs在特定诱导条件下可以转化成神经细胞，进而能促进神经损伤的修复;而且BMSCs来源于病人自体，移植后可以避免排斥反应[5];另外，BMSCs能分泌多种细胞因子，这为神经的修复提供了一个新的思路[6]。分泌型糖蛋白神经生长导向因子2(Slit2)是中枢神经系统发育过程中的一种轴突导向抑制因子，可控制神经轴突分支形成以及神经细胞迁移，能促进BMSCs向感觉神经元分化，参与损伤神经再生和功能修复[7`-9]。近年来，在交通事故中发生的神经损伤和脊髓损伤比较常见，而针对神经损伤的治疗一直是临床上的难点。本研究将Slit2过表达载体以慢病毒介导入BMSCs内，探讨慢病毒介导Slit2过表达转染促进BMSCs向神经细胞转化的作用，从而为临床神经损伤病人治疗提供新的思路。, 百拇医药(刘文忠 蔡静 李军 荣春 范磊 张海宁)
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