基于iTRAQ技术的骨髓间充质干细胞与雪旺细胞共培养后的蛋白质组学分析

发布时间：2018-06-25 05:55

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】：研究目的:脊髓损伤(spinal cord injuries,SCI)是中枢神经系统创伤性疾病,具有高发病率,高致残率,目前尚无有效治愈的方法。本课题组前期研究发现自体激活雪旺细胞(SCs)不仅能分泌神经营养因子,抑制神经元凋亡,还可以诱导骨髓间充质干细胞(BMSCs)向神经元方向分化,促进轴突再生和功能恢复,但具体的激活及诱导机制尚不清楚。本课题应用iTRAQ(Isobaric Tag for Relative and Absolute Quantitation)技术深入研究BMSCs与SCs共培养后的蛋白质组学变化,建立其差异蛋白谱,寻找参与细胞移植干预修复的潜在细胞信号转导通路,确立细胞信号通路中关键调节位点的改变和调控方式,进一步明确BMSCs与SCs共移植的修复SCI机理。研究内容与方法:本课题应用采用胰酶消化组织块和机械分离法培养雪旺细胞,同时应用胰酶快速消化法和双30分钟差速贴壁法纯化SCs。用改进的全骨髓贴壁法,分离培养大鼠BMSCs。应用S100免疫荧光染色鉴定SCs,应用流式细胞仪和三系分化鉴定BMSCs。本实验共分为3组,单纯BMSCs组(SC0),BMSCs与SCs共培养3天组(SC3),BMSCs与SCs共培养7天组(SC7)。本实验采用半定量换液的方式进行共培养。应用裂解液提取法提取各组蛋白,应用2D Quant试剂盒测定每组蛋白浓度,采用iTRAQ/TMT质谱定量方法鉴定差异蛋白。应用Mascot软件对二级谱图信息进行定性定量计算。对差异蛋白分别进行GO、KEGG、Interpro功能的注释与功能分析,对差异蛋白进行显著性富集分析,应用STRING数据库对差异蛋白进行网络互作关系分析。研究结果:本实验以表达上调或下调1.3倍以上作为差异表达蛋白。SC3d组与SC0d组相比,上调蛋白29个,下调蛋白45个;SC7d组与SC0d组相比,上调蛋白43个,下调蛋白32个;SC7d组与SC3d组相比,上调蛋白83个,下调蛋白24个。对差异表达蛋白进行GO注释和亚细胞定位分析显示:差异表达蛋白共涉及13种生物过程,9种细胞组份,9种分子功能;主要位于细胞核、细胞质、胞外蛋白,质膜等亚细胞结构中。对鉴定的差异表达蛋白进行蛋白功能(GO/level2)、功能结构域、KEGG Pathway富集分析显示,发现SC3d vs.SC0d比较组的差异表达蛋白显著富集的功能term相对较少,SC7d vs.SC0d组差异蛋白主要富集在脂类代谢、糖类代谢,生物降解等生物过程,显著富集在溶酶体、细胞骨架和细胞外基质等细胞组份中,显著富集在水解酶活性,糖胺聚糖结合和糖类结合等分子功能中,3个比较组差异蛋白显著富集的代谢通路为鞘糖脂生物合成代谢,神经鞘脂类代谢,溶酶体代谢途径,显著富集的功能结构域为糖苷键键水解酶催化结构域,钙调蛋白同源结构域,糖基水解酶家族13。基于对各组差异蛋白GO(三个本体)、KEGG和Interpro共五项功能富集分析的结果cluster分析显示,总共54个生物过程term得以聚类,38个细胞组份功能term得以聚类,38个分子功能term得以聚类。KEEG聚类分析主要集中在SC7 vs.SC3比较组,主要为神经节鞘磷脂生物合成代谢、溶酶体代谢途径、鞘磷脂代谢途径等,功能结构域聚类分析同样主要集中在SC7 vs.SC3比较组,主要为硫酸酯酶、糖苷水解酶催化结构域、糖苷水解酶超家族等结构域。结论:本研究应用定量蛋白组学技术,绘制了BMSCs与SCs共培养后的差异蛋白表达谱,为揭示BMSCs与SCs共移植到脊髓损伤部位发挥作用的深层机制奠定了一定的基础,对于这些问题的研究将有助于深化和扩大干细胞临床治疗应用。
[Abstract]:Objective: spinal cord injuries (SCI) is a traumatic disease of the central nervous system, with high incidence and high disability rate. There is no effective cure at present. In our previous study, it was found that autoactivated Schwann cells (SCs) can not only secrete a god management factor, inhibit neuronal apoptosis, but also induce bone marrow mesenchymal cells. Stem cells (BMSCs) differentiate into neuron direction, promote axonal regeneration and function recovery, but the specific activation and induction mechanism is not clear. This subject uses iTRAQ (Isobaric Tag for Relative and Absolute Quantitation) technology to study the changes in egg white substance after BMSCs and SCs co culture, and establish its differential protein spectrum and seek participation. Cell transplantation intervention to repair the potential cell signal transduction pathway, establish the change and regulation of key regulatory sites in cell signaling pathway, and further clarify the mechanism of BMSCs and SCs transplantation for the repair of SCI. Enzyme rapid digestion method and double 30 minute differential adherence method were used to purify SCs. with improved full bone marrow adherence method. The isolated and cultured rat BMSCs. was identified by S100 immunofluorescence staining, and SCs was identified by S100 immunofluorescence staining. The use of flow cytometry and three line differentiation identification were divided into 3 groups, pure BMSCs group (SC0), BMSCs and SCs co culture 3 days group (SC3), BMSCs and SCs co culture for 7 days Group (SC7). In this experiment, a semi quantitative solution was used to co culture. The protein was extracted by the lysate extraction method, the concentration of each protein was determined by 2D Quant kit and the differential protein was identified by iTRAQ/TMT mass spectrometry. Mascot software was used to make a qualitative and quantitative calculation of the two level spectrum information. The difference protein was G respectively. O, KEGG, Interpro function annotation and functional analysis, the difference protein was significantly enriched and analyzed. STRING database was used to analyze the network interaction of differential proteins. The results were as follows: in this experiment, the expression of protein.SC3d in the.SC3d group was up to 1.3 times up or down. Compared with the SC0d group, the protein was up regulated and down regulated by 45 proteins. Group SC7d was up to 43 and 32 down-regulation of protein compared with group SC0d, and 83 up and 24 down regulated proteins in group SC7d and SC3d group. The GO annotation and subcellular localization analysis of differentially expressed proteins showed that the differentially expressed proteins involved 13 biological processes, 9 cell components and 9 molecular functions, mainly located in the nucleus and cytoplasm. The protein function (GO/level2), functional domain and KEGG Pathway enrichment analysis showed that the differentially expressed proteins in the SC3d vs.SC0d group showed significant enrichment of the function term relatively less, and the SC7d vs.SC0d group difference protein was mainly enriched in lipid metabolism and saccharide generation. The biological processes such as biodegradation, such as biodegradation, were significantly enriched in the lysosome, cytoskeleton and extracellular matrix, and were significantly enriched in the function of hydrolase, glycosaminoglycan binding and saccharide binding. The metabolic pathways of the 3 comparative groups were the glycolipid biosynthesis metabolism, neurinosin metabolism, and dissolution. Enzyme metabolism pathway, the significant functional domain is glycoside bond key hydrolase domain, calmodulin homologous domain, sugar based hydrolase family 13. based on the analysis of five functions of different proteins GO (three bodies), KEGG and Interpro, cluster analysis showed that term was clustered in a total of 54 biological processes, 38 Cell component function term was clustered, and 38 molecular functional term clustering analysis was mainly concentrated in SC7 vs.SC3 comparison group, mainly ganglion sphingomyelin biosynthesis metabolism, lysosome metabolic pathway and sphingomyelin metabolic pathway, and functional domain clustering analysis was mainly concentrated in SC7 vs.SC3 comparison group, mainly sulfuric acid. Esterase, glycoside hydrolase catalyzed domain and glycoside hydrolase superfamily. Conclusion: This study used quantitative proteomics technology to draw the differential protein expression profiles of BMSCs and SCs co culture, which laid a certain foundation for revealing the deep mechanism of BMSCs and SCs transplantation to the spinal cord injury. The research will help to deepen and expand the clinical application of stem cells.
【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】