血管内皮生长因子体外诱导大鼠骨髓基质细胞分化为神经元样细胞

发布时间：2018-11-09 13:03

【摘要】： 背景与目的已有许多文献报道骨髓基质细胞(bone marrow stromal cells,BMSCs)在一定的体外条件下能分化为神经元样细胞。然而目前用以诱导BMSCs分化为神经元样细胞的诱导剂很少被应用于临床。研究者们对血管内皮生长因子(vascular endothelial growth factor,VEGF)体外诱导BMSCs分化为神经元样细胞的影响和作用机制还了解甚少。本实验旨在探索VEGF体外对BMSCs分化为神经元样细胞的影响作用,并确定实验中VEGF的最佳的诱导浓度。方法 (1) BMSCs取自6～8周雄性和雌性Sprague-Dawley大鼠体内。细胞传代至第3代时,流式细胞分析鉴定BMSCs表面干细胞标志物CD90和造血干细胞标志物CD45。 (2) BMSCS经传代3次后经10ng/mL碱性成纤维细胞生长因子(basic fibroblast growth factor,b-FGF)预诱导24h。细胞被随机分为5,10,和20ng/mLVEGF处理组和未经VEGF处理的对照组。倒置相差显微镜(×200)下逐日观察细胞形态变化。 (3)诱导后的第3天和第10天采用免疫组织化学方法鉴定神经元标志物神经特异性烯醇化酶(neuron specific enolase,NSE),分别计数每20个随机视野下NSE阳性细胞总数。结果 (1)培养第3天可见贴壁细胞,细胞形态各异,以圆形为主,少部分细胞呈三角形、梭形,有粗短突起。原代第7～10天,较多梭形细胞形成集落生长。传代培养2～3代后细胞形态变得较均一,细长梭形细胞较多,呈漩涡样生长,部分细胞呈扁平形。 (2)流式细胞分析结果示第3代BMSCs细胞表面干细胞标志物CD90(97.5%)阳性,而造血干细胞标志物CD45阴性。 (3)诱导第3天经5,10,和20ng/mLVEGF诱导的组别均可观察到细胞胞体回缩,呈锥形或圆形,胞体折光性增强,伸出较细长双或多极突起,并分出次级突起。而对照组则无此类似现象出现。 (4)诱导第10天,经10ng/mLVEGF处理的组别NSE阳性细胞数最多(P0.05)。诱导第3天和第10天,对照组中NSE阳性细胞数均最少(P0.05)。诱导第10天各组NSE阳性细胞数目均较第3天增多(P0.05)。结论 (1)在一定条件下,大鼠骨髓基质细胞体外可向神经元样细胞分化。 (2)本实验中VEGF能促进骨髓基质细胞体外向神经元样细胞分化;与5和20ng/mL相比,10ng/mL为最合适的浓度。 (3)VEGF可能具有应用于诱导BMSCs体外向神经元定向分化和修复中枢神经损伤和神经变性疾病的潜能。
[Abstract]:Background and objective many literatures have reported that bone marrow stromal cells (bone marrow stromal cells,BMSCs) can differentiate into neuron-like cells under certain conditions in vitro. However, the inducers used to induce the differentiation of BMSCs into neuron-like cells are rarely used in clinical practice. Little is known about the effects and mechanisms of vascular endothelial growth factor (vascular endothelial growth factor,VEGF) on the differentiation of BMSCs into neuron-like cells in vitro. The aim of this study was to investigate the effect of VEGF on the differentiation of BMSCs into neuron-like cells in vitro and to determine the optimal concentration of VEGF. Methods (1) BMSCs were taken from Sprague-Dawley rats of male and female at 6 weeks. BMSCs surface stem cell marker CD90 and hematopoietic stem cell marker CD45. were identified by flow cytometry at passage 3. (2) BMSCS was preinduced by 10ng/mL basic fibroblast growth factor (basic fibroblast growth factor,b-FGF) for 24 h after three passages. Cells were randomly divided into 5 cells treated with 20ng/mLVEGF and those without VEGF. The morphologic changes of cells were observed daily under inverted phase contrast microscope (脳 200). (3) nerve specific enolase (neuron specific enolase,NSE) was identified by immunohistochemical method on the 3rd and 10th day after induction, and the total number of NSE positive cells was counted in every 20 random fields. Results (1) Adhesion cells were observed on the third day of culture. The cells were mainly round and a few were triangular fusiform and had thick short protuberances. On the 7th day of primary culture, more fusiform cells formed colony growth. After 2 ~ 3 passage culture, the morphology of the cells became more homogeneous, the slender fusiform cells were more, and the cells grew like whirlpool, and some of the cells were flattened. (2) flow cytometry showed that the surface stem cell marker CD90 (97.5%) was positive in the third passage of BMSCs cells, but the hematopoietic stem cell marker CD45 was negative. (3) on the 3rd day of induction, the cell bodies were retracted in the group induced by 20ng/mLVEGF and 5 days after induction. The cell bodies were cone-shaped or circular, the refraction of the cells was enhanced, the elongated or multipolar protuberances were extended, and secondary protrusions were also observed. In the control group, there was no similar phenomenon. (4) on the 10th day of induction, the number of NSE positive cells in the group treated with 10ng/mLVEGF was the highest (P0.05). On the 3rd and 10th day, the number of NSE positive cells in the control group was the least (P0.05). On the 10th day, the number of NSE positive cells in each group was higher than that on the third day (P0.05). Conclusion (1) under certain conditions, rat bone marrow stromal cells can differentiate into neuron-like cells in vitro. (2) VEGF could promote the differentiation of bone marrow stromal cells into neuron-like cells in vitro, and 10ng/mL was the most suitable concentration compared with 5 and 20ng/mL. (3) VEGF may have the potential to induce BMSCs to differentiate into neurons in vitro and repair central nervous injury and neurodegenerative diseases.
【学位授予单位】：汕头大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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