胶质细胞源性神经营养因子基因修饰后间充质干细胞的生长与分化

背景：近年来,研究表明外源性神经营养因子或化学诱导剂可诱导大鼠骨髓间充质干细胞向神经样细胞分化,但外源性诱导剂诱导作用短暂且化学性物质不可避免的对细胞活力造成负面影响,一定程度上限制了骨髓间充质干细胞的基础研究与临床应用空间。 目的：以载胶质细胞源性神经营养因子和绿色荧光蛋白基因重组腺病毒(Ad-rGDNF-GFP)转染大鼠骨髓间充质干细胞,观察目的基因在靶细胞内的表达、对细胞的营养作用及对骨髓间充质干细胞向神经样细胞分化的影响。 方法：转染组中以Ad-rGDNF-GFP转染骨髓间充质干细胞,感染复数分别为10,50,80,100,150,200;对照组加入等量的培养液作为对照。转染后12 h,倒置荧光显微镜观察2组中绿色荧光蛋白的表达情况;转染后72 h,流式细胞仪测定转染组中转染效率;MTT法测定2组中细胞活力;转染后5,10 d,PCR检测转染组中胶质细胞源性神经营养因子mRNA的表达;转染后5 d免疫荧光鉴定2组中神经元烯醇化酶的表达情况,转染后10 d鉴定微管相关蛋白2的表达。 结果与结论：转染后12 h,细胞中可观察到绿色荧光蛋白的表达,随时间延长荧光强度逐渐加强。以感染复数=100时绿色荧光蛋白的表达强度、稳定性较好,转染后3 d,转染效率近90%;转染后转染组细胞活力增加;转染5 d后,骨髓间充质干细胞表达神经元烯醇化酶,细胞伸出神经样细胞突起;转染10 d后,胶质细胞源性神经营养因子mRNA表达显著增强,骨髓间充质干细胞表达神微管相关蛋白2,细胞突触样结构明显且相互连接成网,呈现典型神经样细胞形态。说明以Ad-rGDNF-GFP转染骨髓间充质干细胞,感染复数=100时,可获得较高的转染效率,胶质细胞源性神经营养因子表达后明显提高了骨髓间充质干细胞活力,并诱导骨髓间充质干细胞向神经样细胞定向分化。

Proliferation and differentiation of mesenchymal stem cells modified with glial cell line-derived neurotrophic factor

BACKGROUND: Exogenous neurotrophic factors or chemical induction can induce rat bone marrow mesenchymal stem cells to differentiate into neuron-like cells. However, exogenous inductors exert a short inducible action, and their chemical substances inevitably have a negative impact on cell viability to limit the application prospects of bone marrow mesenchymal stem cells to a certain extent. OBJECTIVE:To investigate the effect of glial cell line-derived neurotrophic factor, green fluorescent protein gene transfection by adenovirus vector on biological characteristics of rat bone marrow mesenchymal stem cells, to observe the expression of glial cell line-derived neurotrophic factor and green fluorescent protein and the role of nutrition on bone marrow mesenchymal stem cells, and to explore the ability to differentiate into neuron-like cells induced by glial cell line-derived neurotrophic factor. METHODS: The bone marrow mesenchymal stem cells at passage 3 were transfected by recombinant adenovirus (Multiplicity of infection=10, 50, 80, 100, 150, 200). The experiment had two groups according to target genes: bone marrow mesenchymal stem cells were transfected by Ad-GDNF-GFP in transfection group, and bone marrow mesenchymal stem cells were not transfected in control group. The expression of green fluorescent protein was detected by inverted fluorescence microscope. Transfection efficiency was calculated by flow cytometry. Cells viability and the morphological changes of cells were compared respectively by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and inverted fluorescence microscope between the two groups. On days 5 and 10 after transfection, the expression of glial cell-derived neurotrophic factor mRNA was detected by PCR. On day 5, the expression of neuron-specific enolase was determined by immunofluorescence examination. On day 10, the expression of microtubule-associated protein 2 was identified. RESULTS AND CONCLUSION: By the end of 12 hours after transfection, the green fluorescent protein expressed in cells, and the fluorescence intensity gradually increased with time. When the multiplicity of infection was 100, the fluorescence intensity was strong and stable, and the transfection rate was nealy 90% on day 3 after transfection. Cell viability in the transfection group was strengthened after transfection. On day 5 after transfection, bone marrow mesenchymal stem cells expressed neuron-specific enolase, and neuron-like protrusions gradually extended. On day 10 after transfection, bone marrow mesenchymal stem cells expressed microtubule-associated protein 2 and glial cell line-derived neurotrophic factor mRNA, and exhibited neuron-like morphology and interconnected synpases. The recombinant adenovirus, Ad-GDNF-GFP, can highly transfect bone marrow mesenchymal stem cells when the multiplicity of infection is 100, and glial cell line-derived neurotrophic factor can promote the proliferation of bone marrow mesenchymal stem cells and induce bone marrow mesenchymal stem cells to differentiate into neuron-like cells.