TG2基因修饰的鼻粘膜间充质干细胞向神经样细胞分化的研究

Differentiation of TG2 gene modified nasal mucosa mesenchymal stem cells into neural-like cells

目的:研究重组transglutaminase 2(TG2)腺病毒转染对鼻粘膜骨髓间充质干细胞(EMSCs)向神经样细胞分化影响。方法:利用贴壁筛选法分离培养和扩增EMSCs,通过免疫荧光方法分析EMSCs的纯度,采用免疫荧光和免疫印迹方法测定转染效率。实验分组:重组TG2腺病毒转染EMSCs(ad EMSCs)组、重组GFP腺病毒转染EMSCs组(GFP-EMSCs)和空白对照组(control),诱导细胞向神经样细胞分化,倒置显微镜观察分化过程中细胞形态的变化。免疫荧光和免疫印迹方法检测诱导后EMSCs的轴突膜蛋白(GAP-43),微管相关蛋白(MAP2)和髓磷脂碱性蛋白(MBP)的表达情况。向大鼠脊髓内注射ad EMSCs,2周后取脊髓。结果:实验数据表明转染TG2腺病毒的EMSCs可稳定表达TG2。诱导7 d后,ad EMSCs组细胞呈现双极和多极的典型神经样细胞形态,并且GAP-43,MAP2和MBP的表达水平较对照组高。移植入体内2周的ad EMSCs存活良好且部分细胞表达GAP-43。结论:TG2基因修饰有利于EMSCs在体外向神经样细胞分化,并且具有良好的可移植性。

Objective： To investigate the effects of recombinant Transglutaminase2（ TG2） adenovirus transfection on the differentiation of ectomesenchymal stem cells（ EMSCs） into neural-like cells. Methods： EMSCs were isolated and expanded by adherent screening method. The purity of EMSCs was determined by immunofluorescence. The efficiency of transfection was determined by immunofluorescence and western-blotting. Experimental groups： Recombinant TG2 adenovirus transfected EMSCs（ adEMSCs）,recombinant GFP adenovirus transfected EMSCs group（ GFP-EMSCs） and control group,induced cell differentiation into neural-like cells. The cells were observed under inverted microscope Changes in cell morphology. Immunofluorescence and Western-blotting were used to detect the expression of axonal membrane protein（ GAP-43）,microtubule-associated protein 2（ MAP2） and myelin basic protein（ MBP） in EMSCs after inducing. Ad EMSCs were injected into the spinal cord of rats and spinal cord was taken after 2 weeks. Results：The data showed that TG2 was stably expressed in EMSCs,which were transfected with TG2 adenovirus. After 7 days of induction,the cells of adEMSCs group showed typical bipolar and multipolar neural-like cells morphology,and the expression levels of GAP-43,MAP2 and MBP were higher than control groups. Ad EMSCs transplanted into the body for2 weeks,which survived well,and part of cells expressed GAP-43. Conclusion： TG2 gene modification is conducive tothe differentiation of EMSCs into neuron-like cells in vitro,and have superior portability.