低频弱磁诱导移植神经干细胞上调β-Ⅲtubulin mRNA表达修复脑损伤大鼠爬行功能的研究

发布时间：2018-01-01 09:10

本文关键词：低频弱磁诱导移植神经干细胞上调β-Ⅲtubulin mRNA表达修复脑损伤大鼠爬行功能的研究　出处：《南方医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:通过体内体外实验设计观察低频弱磁(low frequency magnetic flux)在骨髓源神经干细胞移植对脑损伤大鼠(rats with brain injury)爬行功能改善的作用。观察到相关神经元标志物以及神经递质的表达改变,为脑损伤的临床治疗提供基础研究依据。方法:建立脑损伤大鼠模型,将64只SD大鼠随机分成四组,每组16只大鼠,分别为对照组(C组)、BM-NPCs组(B组)、磁场组(L组)和磁场+BM-NPCs组(LB组)。取3周龄SD大鼠骨髓来源的骨髓间充质干细胞,体外培养骨髓源神经干细胞(Bone Marrow Stroma Cells-derived Neural Progenitor Cells,BM-NPCs),在成功造模第7d,移植体外CD-Dil标记的BM-NPCs。其中,L组和LB组分别进行低频弱磁场(50Hz、5mT)干预,60min/d,连续干预60d。在实验过程中,分别在移植1d、3d、7d、30d和60 d对各组大鼠进行水平爬楼梯评分、Wayne clark行为学评分,并于移植1d、7d和30d取材进行HE染色,观察各组脑组织病理表现;在外伤区,免疫荧光检测移植细胞的神经细胞标志物NeuN、GFAP、β-Ⅲtubulin表达。在体外实验中,使用50Hz、5mT弱磁场对BM-NPCs进行处理,时间为60min/天,连续作用15天,同时设置对照组。细胞免疫荧光技术检测Q-PCR分析BM-NPCs表达Nestin(神经干细胞标志物)、PSA-NCAM、β-Ⅲtubulin(神经元标志物)、ACHE、5-HT、GABA(神经递质)等情况。结果:BM-NPCs移植后第1d,经Wayne clark评分以及水平爬楼梯评分后,各组结果差异无统计学意义(P0.05);移植后第3d、7d、30d、60d,各组Wayne clark评分、水平爬楼梯评分结果显示,组间比较差异有统计学意义(P0.05),C组与其他三组比较有明显差异,不同时间点也存在差异,不同处理因素随时间增加而行为学评分的变化趋势不同,其中LB组运动功能评分改善更为明显。HE染色结果显示,造模后第7d,各组大鼠脑损伤灶周围组织血管受压变形、组织肿胀坏死形成。BM-NPCs移植后第7d,C组和L组脑损伤灶周围组织水肿明显减轻,有较大的囊性空洞,周围炎症细胞浸润,神经细胞减少明显;B组和LB组脑损伤灶周围组织水肿较轻,出现范围局限的囊性空洞,可见胶质细胞。移植后第30d,除对照组外,其他三组囊性空洞面积均较少,B组水肿组织消失,LB组脑损伤灶周围组织细胞排类整齐,炎症细胞基本消失。移植后第60d,与对照组相比,各实验组脑损伤组织结构大部分修复。留取标本进行免疫荧光技术检测显示,BM-NPCs移植后第30d,B组和LB组大鼠脑损伤组织周围出现Dil标记的NeuN和β-Ⅲtubulin阳性细胞;移植后第60d,Dil标记的NeuN和β-Ⅲtubulin阳性细胞与脑损伤周围组织融合生长。BM-NPCs移植后第30d和第60d,C组大鼠脑损伤组织周围可见大量GFAP阳性细胞,而L组和LB组较C组少。在体外实验过程中,体外培养的BM-NPCs形成细胞球并经弱磁场干预后,可观察到神经元样细胞出现。免疫荧光技术检测发现体外培养的BM-NPCs Nestin呈阳性表达;对照组和磁场组神经元样细胞Tuj-1和GFAP均呈阳性表达。与对照组相比,Q-PCR结果显示磁场组Nestin、PSA-NCAM、Ach、GABA、5-HT基因表达水平与体外诱导前有显著性差异(P0.01),弱磁场组β-ⅢtubulinmRNA表达水平显著高于对照组(P0.05)。结论:低频弱磁能改善脑损伤大鼠患侧爬行功能,也能促进骨髓源神经干细胞移植脑损伤大鼠模型的修复,该过程可能与低频弱磁场上调β-Ⅲtubulin mRNA表达水平促进骨髓源神经干细胞向神经细胞分化有关。
[Abstract]:Objective: To observe the in vivo and in vitro experimental design of low frequency weak magnetic field (low frequency magnetic flux) in bone marrow derived neural stem cells on rat brain injury (rats with brain injury) function to improve the crawling effect. To observe related neuronal markers and neurotransmitter expression changes, and provide basic evidence for the clinical treatment of brain injury methods: the rat model of brain injury was established, 64 SD rats were randomly divided into four groups, 16 rats in each group, including control group (C group), BM-NPCs group (group B), the magnetic field and the magnetic field group (group L) +BM-NPCs group (group LB). Take 3 weeks SD rat bone marrow derived mesenchymal stem cells, neural stem cells derived from bone marrow in vitro (Bone Marrow Stroma Cells-derived Neural Progenitor Cells, BM-NPCs), the successful models of 7D, CD-Dil labeled BM-NPCs. in vitro transplantation, L group and LB group were low frequency magnetic fields (50Hz, 5 MT) 60min/d, 60d. continuous intervention, intervention in the experimental process, respectively in 3D, 7d, transplantation of 1D, 30d and D of rats in each group were 60 level stair climbing score, Wayne score, Clark behavior, and to transplant 1D, 7d and 30d were analysed by HE staining of brain tissue was observed with pathological manifestation; in the area of trauma, neural cell marker NeuN, cell immunofluorescence detection of GFAP expression of beta III tubulin. Using 50Hz in vitro, 5mT weak magnetic field treatment for BM-NPCs, time is 60min/ days, for 15 days, the control group at the same time. The detection of Q-PCR by immunofluorescence analysis of the expression of BM-NPCs Nestin (neural stem cell marker), PSA-NCAM, beta III tubulin (neuron marker), ACHE, 5-HT, GABA (neurotransmitter) etc. RESULTS: after transplantation, BM-NPCs 1D, Wayne Clark score and the level of the stair climbing score, no significant difference between the results of each group (P0.05); After transplantation, 3D, 7d, 30d, 60d, Wayne were Clark score, the level of stair climbing scores showed that there were significant differences between the groups (P0.05), C group compared with the other three groups have obvious differences, there are differences in different time, different factors and behavioral trends with different scores time increases, which group LB movement function score improved more significantly.HE staining showed that after modeling 7d, vascular compression tissue surrounding the brain injury rats focal deformation, formation of.BM-NPCs and 7d after transplantation of tissue swelling and necrosis, C group and L group brain injury perihematomal edema was reduced, cystic large voids, inflammatory cell infiltration, nerve cells decreased significantly; B group and LB group of brain injury foci surrounding tissue edema, cystic confined cavity, visible glial cells. After transplantation, 30d, except the control group, the other three groups of cystic cavity surface Products are less, edema in group B disappeared, LB group of brain injury foci surrounding tissue cells row class neatly, inflammatory cells disappeared. After transplantation, 60d, compared with the control group, the experimental group of brain tissue damage repair. Display structure of most specimens for immunofluorescence detection of BM-NPCs after transplantation, 30d, Dil mark NeuN and beta III tubulin positive cells appeared around the B group and LB group rat brain tissue injury after transplantation; 60d, Dil labeled NeuN and beta III tubulin positive cells surrounding tissue and brain injury fusion growth after transplantation of.BM-NPCs 30d and 60d C around the rats brain injury tissue a large number of GFAP positive cells, L group and LB group than in C group. In vitro experiment, cultured BM-NPCs cells and the formation of the ball by the weak magnetic field intervention can be observed in neuron like cells. Immunofluorescence assay showed that BM-NPCs cultured in vitro Ne The expression of stin was positive; the control group and the magnetic field group neuron like cells were positive for Tuj-1 and GFAP. Compared with the control group, Q-PCR results show that the magnetic field group of Nestin, PSA-NCAM, Ach, GABA, 5-HT gene expression level in vitro and there was significant difference before induction (P0.01), weak magnetic field group beta III tubulinmRNA expression significantly higher than the control group (P0.05). Conclusion: low frequency weak magnetic energy to improve the brain damage ipsilateral crawling function in rats, also can promote the repair of bone marrow derived neural stem cell transplantation for brain injury model in rats, which may be associated with the low frequency weak magnetic field by beta III tubulin mRNA expression of bone marrow derived neural stem cells into nerve cell differentiation.

【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R651.15

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