高频脉冲电磁场对骨髓间充质干细胞增殖影响的蛋白质组学分析

发布时间：2018-04-03 16:57

本文选题：高频脉冲电磁场　切入点：骨髓间充质干细胞　出处：《广西医科大学》2012年硕士论文

【摘要】：骨髓间充质干细胞(MSCs)因具有多向分化潜能、免疫耐受、取材方便、自我更新等特点而成为组织工程重要的种子细胞及近年来研究的一个热点。本课题的前期研究表明高频脉冲电磁场促进髓间充质干细胞的凋亡,但其机理尚不清楚,蛋白质组学是指研究和鉴定一个细胞、器官、组织在一特定时间所表达的总蛋白质的过程,蛋白质组学研究的兴起,为我们从蛋白质水平研究高频脉冲电磁场促进髓间充质干细胞的凋亡的机理奠定了基础。目的：1.利用双向电泳技术(2-DE)创建体外正常培养的和高频脉冲电磁场干预培养的骨髓间充质干细胞蛋白表达图谱。2.利用图像分析软件对蛋白表达图谱进行对比分析查找差异蛋白点,经质谱鉴定后找出高频脉冲电磁场促进骨髓间充质干细胞的凋亡有关的功能蛋白,探讨功能蛋白的作用机制。方法：1.用全骨髓贴壁法进行体外分离,培养,传代,纯化骨髓间充质干细胞,取P3代作为研究的靶细胞。2.以1×106/cm2密度接种至底面积100cm2培养瓶,分为A、B两组进行培养,A组为高频脉冲电磁场干预组,B组为对照组。3.分别提取A、B两组细胞总蛋白,纯化蛋白样本,Bradford法进行蛋白定量,进行双向电泳,获取A、B两组双向电泳图谱并进行对比分析,查找差异蛋白点。切取蛋白差异点、酶解,进行质谱鉴定,根据质谱鉴定结果进行数据库检索查找功能蛋白。结果：1.获得分辨率和重复性较好的正常培养的和高频脉冲电磁场干预培养的骨髓间充质干细胞蛋白表达图谱。2.用图像分析软件对蛋白表达图谱进行对比分析出差异蛋白点19个,其中17个蛋白点在A组表达量上升,2个表达量下降,经质谱成功鉴定11个,其中EF-hand domain-containing protein D2(又称Swiprosin-1),有促进细胞凋亡作用。结论：利用双向电泳技术(2-DE)创建了正常培养的和高频脉冲电磁场干预培养的骨髓间充质干细胞比较理想的蛋白表达图谱；EF-hand domain-containing protein D2可能是高频脉冲电磁场促进BMSCs凋亡的一个功能蛋白。
[Abstract]:Bone marrow mesenchymal stem cell (MSCs) has become an important seed cell in tissue engineering and has been a hot research topic in recent years because of its multidirectional differentiation potential, immune tolerance, convenient material collection, self-renewal and so on.Previous studies in this study have shown that high frequency pulsed electromagnetic fields promote apoptosis of marrow mesenchymal stem cells, but its mechanism is not clear. Proteomics refers to the study and identification of a cell or organ.The process of tissue expression of total protein at a specific time and the rise of proteomics provide a basis for the study of the mechanism of high frequency pulsed electromagnetic field (HFPEM) promoting the apoptosis of mesenchymal stem cells (MMSCs) from the protein level.Purpose 1.Two-dimensional electrophoretic technique (2-DE) was used to create protein expression map of bone marrow mesenchymal stem cells (BMSCs) in vitro and in high frequency pulsed electromagnetic field (HFPEM) intervention.The protein expression patterns were analyzed by image analysis software to find the differential protein spots. The functional proteins related to the apoptosis of bone marrow mesenchymal stem cells (BMSCs) were identified by high frequency pulsed electromagnetic field (HPEM) and the mechanism of functional proteins was discussed.Method 1: 1.Bone marrow mesenchymal stem cells (BMSCs) were isolated, cultured, subcultured and purified by whole-bone marrow adherent method. P3 generation was used as target cells.1 脳 106/cm2 density inoculation to the bottom area of 100cm2 culture bottle was divided into two groups: group A: high frequency pulsed electromagnetic field intervention group, group B: control group. 3.The total proteins were extracted from two groups of Agna B cells, and the protein samples were purified by Bradford method for protein quantification. The two dimensional electrophoresis patterns of two groups of Agna B were obtained and compared and analyzed to find the differential protein spots.The difference point of protein was digested, hydrolyzed by enzyme, identified by mass spectrometry, and the functional protein was searched in database according to the result of mass spectrometry.The result is 1: 1.The protein expression patterns of bone marrow mesenchymal stem cells in normal culture and in high frequency pulsed electromagnetic field were obtained with good resolution and reproducibility.19 differentially expressed protein spots were analyzed by image analysis software. Among them, 17 protein spots increased in group A and 2 decreased. 11 spots were successfully identified by mass spectrometry.Among them, EF-hand domain-containing protein D 2 (also known as Swiprosin-1) can promote apoptosis.Conclusion: the ideal protein expression profile of bone marrow mesenchymal stem cells (BMSCs) in normal culture and high frequency pulsed electromagnetic field intervention has been created by using two-dimensional electrophoresis technique. EF-hand domain-containing protein D2 may be the high frequency pulsed electromagnetic field to promote BMSCs regulation.A functional protein that kills.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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