本文關鍵詞： 骨髓間充質干細胞 膠質細胞源性神經營養(yǎng)因子 神經細胞 分化 Wnt信號通路　出處：《揚州大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的觀察腺病毒介導的膠質細胞源性神經營養(yǎng)因子(GDNF)基因對大鼠骨髓間充質干細胞(BMSCs)向神經樣細胞分化的影響,并進一步研究Wnt信號通路在骨髓間充質干細胞向神經樣細胞分化中的作用。方法分離、培養(yǎng)BMSCs,流式細胞儀鑒定其細胞表型分子CD11b、CD34、CD29、CD90。將第3代培養(yǎng)的BMSCs分為3組:轉染組、DKK-1組、對照組。轉染組以載膠質細胞源性神經營養(yǎng)因子和綠色熒光蛋白基因重組腺病毒(AD-GDNF-GFP)轉染BMSCs,DKK-1組以AD-GDNF-GFP轉染BMSCs同時加入DKK-1以阻斷Wnt信號通路,對照組僅以含10%胎牛血清DMEM培養(yǎng)基培養(yǎng)。于轉染后第1、5、7、14天熒光顯微鏡下觀察BMSCs對綠色熒光蛋白GFP的表達情況并以Western blot技術檢測轉染組BMSCs對GDNF蛋白的表達情況。MTT法測定轉染后BMSCs活力。于轉染后以免疫熒光技術檢測BMSCs對神經特異性蛋白NSE、MAP-2、β-Tublin Ⅲ的表達情況。RT-PCR檢測BMSCs對神經營養(yǎng)因子bFGF、EGF、BDNF、NT-3基因的表達情況。為探討Wnt信號通路在腺病毒介導的膠質細胞源性神經營養(yǎng)因子基因誘導BMSCs神經分化過程中的作用,轉染后以免疫熒光、蛋白質印跡技術檢測β-catenin、NSE的表達情況,并應用RT-PCR法進一步檢測Wnt信號通路相關分子Wnt1a、Wnt3a、Wnt7a基因表達水平。結果第3代培養(yǎng)的骨髓間充質干細胞呈魚群樣、漩渦狀貼璧生長,高表達CD29、CD90,陽性率分別為99.8%、95.6%;低表達CD11b、CD34,且陽性率分別為1.4%,1.5%。載GDNF基因重組腺病毒轉染BMSCs24小時后,在熒光顯微鏡下即可觀察到綠色熒光蛋白GFP的表達,隨著時間的延長熒光蛋白GFP的熒光強度逐漸增強,轉染7天后綠色熒光蛋白GFP的熒光強度最高,轉染后14天熒光強度明顯減弱。分別于轉染后第1天、3天、7天、14天以WesternBlot技術均可檢測到GDNF蛋白的表達。轉染后5天,免疫熒光技術檢測結果顯示轉染組BMSCs表達NSE,轉染后10天MAP-2、β-Tublin Ⅲ呈陽性表達,且多數(shù)細胞呈典型的神經細胞形態(tài),胞體明顯皺縮并向周圍形成突起。而對照組均未見NSE、MAP-2、β-Tublin Ⅲ表達。轉染5天后免疫熒光技術檢測發(fā)現(xiàn)轉染組β-catenin、NSE呈陽性表達,而DKK-1組β-catenin熒光強度明顯低于對照組,對照組未見β-catenin表達,且DKK-1組、對照組無NSE表達。RT-PCR檢測結果表明,轉染組Wnt信號通路相關分子Wnt1a、Wnt3a、Wnt7a基因表達量明顯上調,與DKK-1組及對照組比較存在明顯差異,且有統(tǒng)計學意義(P0.05)。結論(1)腺病毒介導的膠質細胞源性神經營養(yǎng)因子基因可以促進骨髓間充質干細胞表達神經元標志蛋白,能誘導其向神經樣細胞分化;(2)腺病毒介導的膠質細胞源性神經營養(yǎng)因子基因可以通過激活Wnt信號通路誘導骨髓間充質干細胞向神經樣細胞分化。
[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，

本文編號：1467871