黄芪诱导人脐带wj-mscs分化过程中nse蛋白的表达.doc

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黄芪诱导人脐带wj-mscs分化过程中nse蛋白的表达,该论文为《昆明理工大学》硕士论文,内容就脐带间充质干细胞的生物学特性、分化潜能和机制做了详细的概括和总结。并通过实验证明了黄芪在诱导干细胞分化为神经细胞过程中的作用,并摸索出最佳诱导条件。论文对一些实验方法,如western blot 等有比较详细的记录。摘 要中枢神经系统疾病一直以来都是临床治疗上难点。随着现代生物技...
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该论文为《昆明理工大学》硕士论文,内容就脐带间充质干细胞的生物学特性、分化潜能和机制做了详细的概括和总结。并通过实验证明了黄芪在诱导干细胞分化为神经细胞过程中的作用,并摸索出最佳诱导条件。论文对一些实验方法,如Western Blot 等有比较详细的记录。
 摘  要
中枢神经系统疾病一直以来都是临床治疗上难点。随着现代生物技术的发展,利用干细胞移植技术来治疗神经系统疾病成为可能的治疗策略。人脐带Wharton’s jelly源性间充质干细胞(Wharton’s jelly mesenchymal stem cells,WJ-MSCs)是来源于分娩废弃脐带的一种成体干细胞,具有自我更新和多向分化潜能,且有独特的免疫调节特性。与胚胎干细胞和骨髓间充质干细胞相比,WJ-MSCs具有来源广泛,在体外易于分离培养和扩增,无伦理道德问题的争议等优势,被认为是组织工程中理想的种子细胞源。已有研究表明,WJ-MSCs具有分化为三个胚层来源细胞的潜能。近年来,国内外学者通过不同的诱导方式,在体内、外观察到WJ-MSCs分化为神经样细胞。这无疑为神经系统疾病的治疗带来了希望。
黄芪是一味扶正固本、补中益气之要药,在传统中药配方中被广泛使用。据报道,黄芪对神经组织具有一定的保护作用,能诱导WJ-MSCs分化为神经样细胞。本课题组已建立培养脐带间充质干细胞的技术平台,并观察基本的生物学特性。基于我们是立足于服务临床的研究部门,需要实实在在的建立我们自身的实验数据。于此,本实验采用组织块贴壁培养法获取WJ-MSCs,用蛋白质免疫印迹(Western blot)检测NSE蛋白在不同浓度黄芪诱导WJ-MSCs分化过程中的表达以及时效关系。旨在探讨黄芪诱导的适宜条件,以期为提高黄芪诱导效率奠定理论基础。
WJ-MSCs的培养及蛋白提取:采用组织块贴壁法分离培养原代WJ-MSCs,通过胰酶消化来纯化、扩增细胞。我们选用P3~P5的细胞进行实验。在借鉴文献的基础上,设黄芪浓度为12.5mg/ml、25mg/ml、37.5mg/ml、50mg/ml、62.5mg/ml以及对照组,诱导24h后检测NSE蛋白的表达。随后,以50 g/L黄芪分别诱导WJ-MSCs6h、12h、24h、36h、48h后,检测NSE蛋白的表达。
依照上述实验时间点,收集细胞,提取蛋白,用BCA法进行蛋白定量,分装,-20℃冻存备用。
采用Western blot免疫印迹方法检测NSE蛋白在黄芪诱导WJ-MSCs分化过程中的表达:两组细胞蛋白中神经元标志物神经元特异性烯醇化酶(NSE)的表达变化情况。
实验结果示,用组织块贴壁培养法结合胰酶消化法获取的WJ-MSCs为成纤维样细胞,细胞增殖能力强,折光性好。经不同浓度的黄芪诱导24h后,在12.5g/L诱导组中,NSE蛋白条带亮度最高。提示在此浓度条件下,细胞中NSE蛋白表达量可能最高。50 g/L黄芪诱导不同时间后,在6hNSE蛋白条带亮度最强,12h次之。
结论:NSE蛋白在? g/L黄芪诱导WJ-MSCs分化6h的表达量最大。


 关键词:脐带;间充质干细胞;黄芪;NSE;免疫印迹

 

 

 

 

 

 

ABSTRACT
                                   
      Diseases of the central nervous system has been a clinical difficult. With the development of modern biotechnology, the stem cell transplantation to treat nervous system diseases become possible treatment strategies. Umbilical cord mesenchymal stem cells derived from umbilical cord delivery abandoned. Is a kind of adult stem cells. Embryonic stem cells and bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells in vitro has extensive sources, easy isolation and culture and proliferation, no ethical issues and other advantages, is considered to be the ideal source of tissue engineering seed cells. In addition, umbilical cord mesenchymal stem cells also have the differentiation potential, in appropriate culture conditions in vitro, can be induced to differentiate into various tissue cells. In recent years, scholars at home and abroad through different ways, in vitro to complete the umbilical cord mesenchymal stem cells differentiate into neuronlike cells. In this process, the application of traditional Chinese medicine China attention, has been reported to use traditional Chinese medicine Astragalus mongholicus induced differentiation of umbilical cord mesenchymal stem cells into neuron like cells. This is the clinical treatment of diseases of the nervous system brings hope. But at the same time, the problem of Astragalus membranaceus induced differentiation is not clear, this for clinical cell transplantation in the treatment of some limitations.
      Based on this, the technique and Western blot method for primary cell culture, the umbilical cord mesenchymal stem cells differentiation in Astragalus, Astragalus induced concentration and suitable for induction time problems are investigated and discussed. In order to lay a theoretical foundation for improving the efficiency of Astragalus membranaceus induced differentiation. The experiment is divided into two parts.
     1.Isolation and culture of primary human umbilical cord mesenchymal stem cells by tissue adherent method, through the passage to purified cells. P3-P5 as a substitute for good growth state of cells as experimental products, in accordance with the Astragalus concentrations were 12.5mg/ml, 25mg/ml, 37.5mg/ml, 50mg/ml, 62.5mg/ml and blank control group 6 group, the induction time was 24h, another group according to the induction time were 6h, 12h, 24h, 36h, 48h and blank control group 6 group, Astragalus membranaceus induced concentration was 50mg/ml. To observe the morphological changes of cells, induced group discovered in adherent cell shedding, but no obvious changes in cell morphology. Protein extraction, quantification by BCA protein concentration determination of cell protein, the protein -20 ℃ storage backup.
     2.The two groups were detected in neurons cell protein markers of neuron specific enolase by Western blot blotting method (NSE) expression changes. The experimental results showed the expression of NSE protein in display, the control group and the experimental group in the experimental group, difference in the concentration of Astragalus, induced by 24h, the concentration of 12.5mg/ml in the group of Astragalus, electrophoresis band maximum brightness, the concentration of 25mg/ml group. Show that this concentration condition, is the highest expression of NSE protein in cells. In the induction time difference group, control group and experimental group has band appeared in the experimental group, the induction time for the brightness of 6h bands was the strongest, followed by 12h, showed that the induction time in the 6-12h, the highest expression of NSE proteins in the cell rate. We believe in Astragalus membranaceus induced mesenchymal stem cells differentiate into neuron like cells in the process of Astragalus, the suitable concentration of 12.5-25mg/ml, the best time induced by 6-12h.
       Conclusion: the tissue culture method can conveniently obtain umbilical cord mesenchymal stem cells, and the cell growth, proliferation speed; in Astragalus membranaceus induced differentiation of mesenchymal stem cells, cell morphology did not change, Western blot, the experimental group and the control group. NSE protein expression, the band comparing the brightness of Astragalus, concentration of 12.5mg/ml and induced 6h two group with the highest brightness, it also shows that under these conditions, the NSE expression in cells of the maximum. Therefore, we believe that in Astragalus membranaceus induced differentiation of mesenchymal stem cells, the optimal concentration range of 12.5-25mg/ml, the best induction when the long range 6-12h.

Keywords: Umbilical cord mesenchymal stem cells;Astragalus membranaceus;
             Neuron specific enolase;Western blot

目  录
摘  要 I
ABSTRACT III
目  录 VI
插图和附表清单 XI
英文缩写表 XII
第一章 绪论 1
1.1 前言 1
1.2 干细胞研究概况 1
1.2.1 胚胎干细胞 1
1.2.2 脐带间充质干细胞 2
  1.2.2.1 间充质干细胞生物学特性 2
  1.2.2.2 间充质干细胞的鉴别 3
  1.2.2.3 间充质干细胞分化潜能 3
  1.2.2.4 间充质干细胞的免疫原性 4
1.3 UCMSC的诱导分化及其机制 4
1.3.1 UCMSC的诱导分化方法 4
1.3.2 UCMSC的诱导分化机制 5
  1.3.2.1 外源性调控 5
  1.3.2.2内源性调控 6
1.4 中药诱导MSC分化神经样细胞 8
1.4.1 MSC向神经样细胞分化的特点 8
  1.4.1.1 MSC诱导分化为神经样细胞 8
  1.4.1.2 MSC促进其他细胞分化为神经样细胞 9
1.4.2 中药诱导MSC分化为神经样细胞 9
  1.4.2.1中药复方制剂诱导MSC分化为神经样细胞 9
  1.4.2.2单味中药诱导MSC分化为神经样细胞 10
  1.4.2.3中药有效成分诱导MSC分化为神经样细胞 11
1.5 黄芪药理作用及诱导MSC分化为神经样细胞 13
1.5.1 黄芪药理作用 13
  1.5.1.1对免疫系统的作用 13
  1.5.1.2抗肿瘤作用 14
  1.5.1.3神经保护作用 14
1.5.2 黄芪诱导MSC分化为神经样细胞 14
第二章 黄芪诱导MSC及蛋白提取 16
2.1 引言 16
2.2 材料与方法 17
2.2.1 实验材料 17
2.2.2 实验药品与试剂 17
2.2.3 实验仪器 18
2.2.4 脐带MSCs的原代培养 19
2.2.5 脐带MSCs的传代 20
2.2.6 脐带MSCs生长曲线 20
2.2.7 脐带MSCs的冻存与复苏 21
2.2.8 黄芪诱导脐带MSC 21
2.2.9 细胞蛋白提取及定量 24
2.3 实验结果 26
2.3.1 脐带MSCs分离、纯化及形态学观察 26
2.3.2 细胞生长曲线分析 29
2.3.3 黄芪诱导脐带MSC 29
2.3.4 细胞蛋白定量标准曲线 29
2.3 实验结果 30
2.5 本章小结 32
第三章 黄芪诱导MSC中NSE的表达差异 33
3.1 引言 33
3.2 实验材料与仪器 33
3.2.1 实验试剂 33
3.2.2 实验仪器 34
3.2.3 实验准备 34
3.3 实验方法 35
    3.3.1 SDS-PAGE电泳 35
3.3.2 转膜 37
3.3.3 免疫反应 37
3.3.4 化学发光显影 37
3.5 讨论 39
3.6 本章小结 40
致  谢 41
参 考 文 献 42