腺病毒介导的胶质细胞源性神经营养因子基因通过Wnt信号通路诱导骨髓间充质干细胞向神经样细胞分化

发布时间：2018-01-27 06:49

  本文关键词： 骨髓间充质干细胞 胶质细胞源性神经营养因子 神经细胞 分化 Wnt信号通路　出处：《扬州大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的观察腺病毒介导的胶质细胞源性神经营养因子(GDNF)基因对大鼠骨髓间充质干细胞(BMSCs)向神经样细胞分化的影响,并进一步研究Wnt信号通路在骨髓间充质干细胞向神经样细胞分化中的作用。方法分离、培养BMSCs,流式细胞仪鉴定其细胞表型分子CD11b、CD34、CD29、CD90。将第3代培养的BMSCs分为3组:转染组、DKK-1组、对照组。转染组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-GDNF-GFP)转染BMSCs,DKK-1组以AD-GDNF-GFP转染BMSCs同时加入DKK-1以阻断Wnt信号通路,对照组仅以含10%胎牛血清DMEM培养基培养。于转染后第1、5、7、14天荧光显微镜下观察BMSCs对绿色荧光蛋白GFP的表达情况并以Western blot技术检测转染组BMSCs对GDNF蛋白的表达情况。MTT法测定转染后BMSCs活力。于转染后以免疫荧光技术检测BMSCs对神经特异性蛋白NSE、MAP-2、β-Tublin Ⅲ的表达情况。RT-PCR检测BMSCs对神经营养因子bFGF、EGF、BDNF、NT-3基因的表达情况。为探讨Wnt信号通路在腺病毒介导的胶质细胞源性神经营养因子基因诱导BMSCs神经分化过程中的作用,转染后以免疫荧光、蛋白质印迹技术检测β-catenin、NSE的表达情况,并应用RT-PCR法进一步检测Wnt信号通路相关分子Wnt1a、Wnt3a、Wnt7a基因表达水平。结果第3代培养的骨髓间充质干细胞呈鱼群样、漩涡状贴璧生长,高表达CD29、CD90,阳性率分别为99.8%、95.6%;低表达CD11b、CD34,且阳性率分别为1.4%,1.5%。载GDNF基因重组腺病毒转染BMSCs24小时后,在荧光显微镜下即可观察到绿色荧光蛋白GFP的表达,随着时间的延长荧光蛋白GFP的荧光强度逐渐增强,转染7天后绿色荧光蛋白GFP的荧光强度最高,转染后14天荧光强度明显减弱。分别于转染后第1天、3天、7天、14天以WesternBlot技术均可检测到GDNF蛋白的表达。转染后5天,免疫荧光技术检测结果显示转染组BMSCs表达NSE,转染后10天MAP-2、β-Tublin Ⅲ呈阳性表达,且多数细胞呈典型的神经细胞形态,胞体明显皱缩并向周围形成突起。而对照组均未见NSE、MAP-2、β-Tublin Ⅲ表达。转染5天后免疫荧光技术检测发现转染组β-catenin、NSE呈阳性表达,而DKK-1组β-catenin荧光强度明显低于对照组,对照组未见β-catenin表达,且DKK-1组、对照组无NSE表达。RT-PCR检测结果表明,转染组Wnt信号通路相关分子Wnt1a、Wnt3a、Wnt7a基因表达量明显上调,与DKK-1组及对照组比较存在明显差异,且有统计学意义(P0.05)。结论(1)腺病毒介导的胶质细胞源性神经营养因子基因可以促进骨髓间充质干细胞表达神经元标志蛋白,能诱导其向神经样细胞分化;(2)腺病毒介导的胶质细胞源性神经营养因子基因可以通过激活Wnt信号通路诱导骨髓间充质干细胞向神经样细胞分化。
[Abstract]:Objective to investigate the effect of glial cell derived neurotrophic factor (GDNF) gene mediated by adenovirus on the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into neuroblast-like cells (NSCs). To further study the role of Wnt signaling pathway in the differentiation of bone marrow mesenchymal stem cells into neural like cells. Methods BMSCs were isolated and cultured. Flow cytometry was used to identify the phenotypic molecule CD11b. CD34, CD29, CD90. The BMSCs cultured in the third generation were divided into three groups: transfection group and DKK-1 group. In control group, BMSCs was transfected with recombinant adenovirus containing glial cell derived neurotrophic factor (GDNF) and green fluorescent protein gene (GFP). In DKK-1 group, BMSCs was transfected with AD-GDNF-GFP and DKK-1 was added to block Wnt signaling pathway. The control group was only cultured on DMEM medium containing 10% fetal bovine serum. The expression of green fluorescent protein GFP by BMSCs was observed under 14 days fluorescence microscope and Western was used to detect the expression of green fluorescent protein GFP. Blot technique was used to detect the expression of GDNF protein in BMSCs of transfection group. The activity of BMSCs after transfection was detected by MTT method. The specificity of BMSCs to nerve was detected by immunofluorescence technique after transfection. Protein NSE. Expression of MAP-2, 尾 -Tublin 鈪，

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