NGF和PTEN双基因诱导大鼠骨髓间充质干细胞向神经元样细胞分化效果观察  

NGF and PTEN dual genes induce differentiation of rat bone marrow mesenchymal stem cells to neuron-like cells

作　　者：林平 陈毅 梁淑霞 李焘 邵依娜 赵有顺 吴咏军 涂迎春 黄志丹 安涛 LIN Ping;CHEN Yi;LIANG Shuxia(Department of Orthopedics,Jinhua Hospital of Zhejiang University(Jinhua Municipal Central Hospital),Jinhua 321000,China)

机构地区：[1]浙江大学金华医院(金华市中心医院)骨科,321000

出　　处：《浙江医学》2020年第10期1000-1005,I0004,共7页Zhejiang Medical Journal

基　　金：上海市周围神经显微外科重点实验室、手功能重建卫生部重点实验室基金项目(17DZ2270500);浙江省实验动物科技计划项目(2016C37094)。

摘　　要：目的探讨神经生长因子(NGF)的过表达(NGFhigh)与10号染色体上的磷酸酶和张力蛋白同源物丢失(PTEN)的下调(PTENlow)相结合对大鼠骨髓间充质干细胞(MSC)提升周围神经再生能力的影响。方法将绿色荧光蛋白(GFP)标记的OriCellTM SD大鼠MSC(MSC/GFP)分为两组,实验组通过NGF基因稳定转染和PTEN基因的小干扰RNA干扰瞬时沉默构建双基因修饰的NGFhigh/PTENlow大鼠MSC/GFP,对照组为未经基因修饰的大鼠MSC/GFP。采用细胞增殖/毒性检测试剂盒(CCK-8)和氧-葡萄糖剥夺实验检测两组细胞的增殖和凋亡情况;实时定量聚合酶链反应(qRT-PCR)和蛋白免疫印迹法(Western blot)检测两组细胞NGF、PTEN和巢蛋白-1(Nestin-1)mRNA和蛋白表达情况。结果CCK-8检测结果显示双基因修饰的NGFhigh/PTENlow大鼠MSC/GFP具有更强的增殖能力;氧-葡萄糖剥夺模型中NGFhigh/PTENlow大鼠MSC/GFP具有更强的生存能力;qRT-PCR和Western blot结果显示,双基因修饰上调了NGF、Nestin-1的表达和下调了PTEN的表达。结论双基因修饰的NGFhigh/PTENlow大鼠MSC/GFP具有更强分化为神经元样细胞的能力,因此,双基因定向诱导大鼠MSC分化有利于神经元再生,从而提升周围神经再生能力。Objective To investigate the effects of overexpression of nerve growth factor(NGF)and phosphatase and tensin homologs deleted on chromosome 10(PTEN)dual genes on the differentiation of rat bone marrow mesenchymal stem cells(MSC)to neuron-like cells.Methods The OriCellTM SD rat MSC cells were labeled with green fluorescent protein(MSC/GFP cells)was divided into 2 groups:the rat MSC/GFP cells were transfected with NGF gene and small RNA interference of PTEN to construct NGFhigh/PTENlow MSC/GFP cells in experimental group,and the rat MSC/GFP cells were not modified in control group.CCK-8 and oxygen glucose deprivation test were used to detect the proliferation and apoptosis of cells in each group;qRT-PCR and Western blot were used to detect the expression of NGF,PTEN and Nestin-1 mRNA and protein in each group.Results The double gene modified NGFhigh/PTENlow rat MSC/GFP cells had stronger proliferation ability as showed in CCK-8 results and had stronger survival ability as showed in oxygen glucose deprivation model;qRT-PCR and Western blot results showed that the expression of Nestin-1,NGF was up-regulated and PTEN was down-regulated by the double gene modification.Conclusion The double gene modified NGFhigh/PTENlow rat MSC/GFP cells have stronger ability to differentiate into neuron-like cells,indicating that the differentiation of MSC induced by double gene orientation might be used for the regeneration of neurons and peripheral nerves.

关 键 词：神经生长因子 10号染色体上的磷酸酶和张力蛋白同源物丢失 骨髓间充质干细胞 巢蛋白-1 分化和再生 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]