激活态雪旺细胞联合骨髓间充质干细胞移植修复脊髓损伤的蛋白组学分析

发布时间：2018-04-17 18:52

本文选题：脊髓损伤 + 骨髓间充质干细胞　；参考：《天津医科大学》2017年硕士论文

【摘要】：【目的】采用iTRAQ蛋白组学技术筛选脊髓损伤(Spinal Cord Injury,SCI)后激活态雪旺细胞(Activated Schwann cells,ASCs)联合骨髓间充质干细胞(Bone marrow derived mesenchymal stem cells,BMSCs)移植在损伤微环境中的蛋白表达水平,并对差异表达蛋白进行功能注释,分析其所涉及神经修复相关的重要功能,初步揭示细胞移植修复SCI在基因与分子水平上的调控机制,寻找关键调节蛋白,分析其作用方式,为进一步优化细胞移植修复脊髓损伤策略和临床转化应用提供理论依据。【方法】1.细胞分离培养:采用骨髓腔冲洗分离培养大鼠BMSCs,并应用流式细胞术检测胞膜表面分子鉴定BMSCs。应用坐骨神经预损伤激活和胶原酶消化分离培养SCs,采用S100免疫荧光染色鉴定SCs。2.实验分组:54只Wistar成年雌性大鼠随机分成6组(每组9只):A组,DMEM空白对照7天组;B组,DMEM空白对照14天组;C组,DMEM空白对照28天组;D组,BMSC联合ASC移植7天组;E组,BMSC联合ASC移植14天组;F组,BMSC联合ASC移植28天组。3.动物模型制作与细胞移植:采用成年雌性Wistar大鼠,利用NYU Impactor II型打击装置建立胸10节段脊髓挫伤模型,打击重量为10g,高度为2.5cm。在损伤后7天,使用10μl Hamilton微量注射器分别将DMEM,ASCs与BMSCs混合悬液缓慢注射入脊髓损伤区域,注射剂量为每只15μl,细胞密度为1×105/μl。分别在移植后7天,14天,28天行心脏灌注,取出损伤区域0.5cm组织。4.蛋白组学分析:应用裂解液提取法提取脊髓组织蛋白,应用2D Quant试剂盒测定每组蛋白浓度,应用Mascot软件对二级质谱图信息进行定性定量计算,采用iTRAQ/TMT质谱定量方法鉴定差异蛋白,对差异蛋白分别进行GO功能富集分析与String网络分析。【结果】BMSCs联合ASCs移植修复脊髓损伤后7天,差异表达蛋白数为105个,其中,39个表达上调,66个表达下调;移植后14天,差异表达蛋白数为185个,其中,151个表达上调,34个表达下调;移植后28天,差异表达蛋白数为81个,其中,46个表达上调,35个表达下调。差异表达蛋白的GO功能富集分析结果发现,移植后7天差异蛋白主要富集于炎症反应、免疫应答、急性期反应等免疫相关生物学过程。移植后14天组差异蛋白主要富集于神经突触、神经递质运输、金属离子代谢等生物学过程。移植后28天组,差异蛋白主要富集于胞外空间重塑、细胞外基质、层黏连蛋白、细胞粘附、髓鞘化等生物学过程与细胞组分。String网络互作分析发现表达下调的STAT1蛋白在移植7天后位于蛋白互作网络的核心位置。【结论】1.本研究初步绘制了ASCs联合BMSCs移植后脊髓损伤微环境的蛋白表达谱,发现了神经丝蛋白,钙网织蛋白,Lyn蛋白等与脊髓损伤修复相关的差异蛋白。2.ASCs联合BMSCs移植主要通过抑制炎症免疫反应、抗凋亡、重塑细胞外基质、调节损伤局部微环境等作用机制促进脊髓损伤修复,并提示STAT1蛋白在ASCs联合BMSCs移植修复脊髓损伤中可发挥免疫调理和抗凋亡的作用。3.深入探讨ASCs联合BMSCs联合移植促进修复的分子机制有助于优化移植策略的探索研究,为细胞移植修复脊髓损伤的临床治疗应用奠定基础。
[Abstract]:[Objective] using the screening of spinal cord injury group (Spinal Cord Injury iTRAQ protein, SCI) after activated Schwann cells (Activated Schwann cells, ASCs) combined with bone marrow mesenchymal stem cells (Bone marrow derived mesenchymal stem cells, BMSCs) transplantation in injury microenvironment in protein expression level, and the differences in protein expression for functional annotation, analysis the important function of the neural repair, preliminarily reveal the mechanism of regulation of cell transplantation in repair of SCI gene and molecular level, to find the key regulatory protein, analyze the effect, provide a theoretical basis for the further optimization strategy of cell transplantation in repair of spinal cord injury and clinical application. [methods] 1. cells isolation: the bone marrow cavity flushing rat BMSCs were isolated and cultured, and flow cytometry was used to detect the cell membrane surface molecular identification of BMSCs. application of sciatic nerve injury pre activation Isolation of SCs and collagenase digestion by S100, immunofluorescence staining of SCs.2. experimental groups: 54 adult female Wistar rats were randomly divided into 6 groups (n = 9): A group, DMEM control group for 7 days; B group, DMEM control group for 14 days; C group, DMEM control group for 28 days; group D, BMSC and ASC 7 days of transplantation group; group E, BMSC and ASC 14 days of transplantation group; group F, BMSC and ASC 28 days of transplantation group.3. animal model and cell transplantation: the adult female Wistar rats, striking device of thoracic spinal cord contusion model in 10 segments by NYU Impactor II, hit the weight is 10g, the height is 2.5cm. in 7 days after injury, using 10 L Hamilton micro syringe respectively DMEM, ASCs and BMSCs mixed suspension was slowly injected into the area of spinal cord injury, the dose per injection is only 15 l, the cell density of 1 * 105/ L. respectively in 7 days after transplantation, 14 days. 28 days after cardiac perfusion, remove the damage zone The domain 0.5cm.4. proteomics analysis: extraction of spinal cord tissue proteins by lysis method, determination of protein concentration in each group using 2D Quant kit, Mascot software is applied to the qualitative and quantitative calculation of the two grade mass spectrum information, the quantitative method for iTRAQ/TMT mass spectrometric identification of differential proteins, the differential proteins were analyzed with String GO enrichment analysis network. [results] BMSCs combined with ASCs transplantation for repair of spinal cord injury after 7 days, the number of differentially expressed protein is 105, among them, 39 upregulated and 66 downregulated; 14 days after transplantation, the differentially expressed protein number 185, among them, 151 upregulated and 34 downregulated; 28 days after transplantation, the differentially expressed proteins in number 81, among them, 46 upregulated and 35 downregulated. Enrichment analysis results show that the protein expression difference of GO function, the 7 day after transplantation proteins mainly enriched in inflammation, immune response A biological reaction in acute stage of immune process. 14 days after transplantation group proteins are mainly enriched in synapses, neurotransmitter transport, metal ion metabolism and other biological processes. 28 days after the transplantation group, the difference of protein enriched in the extracellular space remodeling, extracellular matrix, laminin, cell adhesion, myelin sheath such as biological processes and cellular components of.String network interaction analysis showed that down-regulation of the expression of STAT1 protein in 7 days after transplantation in the protein interaction network core position. [Conclusion] this study draws 1. ASCs combined with BMSCs transplantation after spinal cord injury microenvironment protein expression profile, found neurofilament protein, calreticulin fabric of protein, Lyn protein and repair of spinal cord injury related proteins.2.ASCs combined with BMSCs transplantation mainly by inhibiting the inflammatory immune response, anti apoptosis, extracellular matrix remodeling, regulating the local micro environment damage The mechanism to promote the repair of spinal cord injury, and suggest that STAT1 protein may play immune regulation and anti apoptosis effect of.3. ASCs combined with BMSCs transplantation of promoting the repair mechanism will help to explore the optimization strategy research on ASCs transplantation combined with BMSCs transplantation for repair of spinal cord injury, lay the foundation for clinical application of cell transplantation for repair of spinal cord injury.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.2

【参考文献】