血小板源性生长因子对成神经分化的间充质干细胞定向迁移研究

发布时间：2018-01-10 01:12

本文关键词：血小板源性生长因子对成神经分化的间充质干细胞定向迁移研究　出处：《苏州大学》2010年硕士论文　论文类型：学位论文

更多相关文章： 骨髓间充质干细胞 分化 PDGF Rac1 迁移

【摘要】： 目的目前恶性胶质瘤是最为盛行的原发性脑瘤,传统的方法,如化疗,放疗难以从根本上对其进行治疗,但由于骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)在体内外均显示出很强的趋瘤性,所以BMSCs有望成为胶质瘤治疗的优选种子细胞。然而,关于分化的BMSCs细胞是否有同样的趋化性以及BMSCs向胶质瘤趋化性迁移机制研究的却很少,本研究旨在探讨成神经分化的BMSCs向胶质瘤分泌的趋化因子之一血小板源性生长因子(platelet-derived growth factor, PDGF)的迁移行为及其分子机制。方法本实验分四部分进行研究,其中前三部分研究成神经分化的BMSCs向PDGF的迁移,最后一部分研究的是PDGF引起的BMSCs迁移的分子机制。各个部分内容具体为:(1)采用Percoll密度梯度离心法体外分离培养大鼠BMSCs,通过免疫组织化学染色和多向分化的方法对培养的细胞进行鉴定。(2)采用加入抗氧化剂的方法诱导BMSCs向神经样细胞分化,即先用10 ng/ml的bFGF预诱导24 h,再加入200μM的丁羟基茴香醚(BHA)和2%的二甲基亚砜(DMSO)诱导5 h,最后用含有10μl/ml N2的H-DMEM维持培养48 h。观察诱导分化过程中BMSCs的形态变化,通过免疫荧光染色检测诱导细胞的神经细胞特异性标志物nestin、β-III-tubulin和NSE的表达情况。(3)运用Dunn chamber研究成神经分化不同阶段BMSCs向胶质瘤分泌的因子之一PDGF的趋化性迁移。细胞的迁移通过德国Leica AF6000活细胞工作站拍摄得到图像,应用NIH Image J软件分析图像,每个状态随机抽取30个细胞进行统计得到细胞的迁移速率、迁移效率和单个细胞的迁移轨迹。(4) Rac1对PDGF诱导的BMSCs迁移行为的影响通过实时显微摄像系统进行研究,而PI3K信号通路对该趋化性迁移行为的作用通过实时显微摄像系统和Dunn chamber两种方法进行探索。两种方法均用德国Leica AF6000活细胞工作站拍摄得到图像,应用NIH Image J分析图像,每个状态随机抽取30个细胞进行统计得到细胞的迁移速率和效率。结果(1)采用Percoll密度梯度离心法成功分离培养出大鼠BMSCs,可稳定传至20代以上。免疫组织化学鉴定结果为CD29、CD90、CD106阳性,CD34阴性;多向性分化实验证明其既可以成骨分化又可以成脂分化,证实实验所培养的细胞具备BMSCs的基本特征。(2)用bFGF/BHA诱导BMSCs向神经样细胞分化,预诱导24 h,细胞胞体出现收缩,细胞边缘变得不规整;诱导5 h,细胞胞体进一步收缩,折光性增强,伸出细长的突起,并且多为两极突起,小部分细胞出现死亡并脱落;维持48 h细胞出现更多的分叉,并出现二级分叉;对照组细胞形态上则无显著性变化。免疫荧光染色结果显示诱导的细胞表达神经前体细胞标志物nestin,未成熟神经元标志β-III-tubulin和成熟神经元标志物NSE。(3) Dunn chamber分析显示,BMSCs能够趋化PDGF定向迁移,细胞的迁移速率和迁移效率均明显高于对照组,单个细胞迁移轨迹也证实了这一结果。成神经分化不同时期的细胞对PDGF的趋化能力不同:未诱导的BMSCs、预诱导24 h和诱导5 h的分化细胞的迁移速率得到明显提高,而未诱导的BMSCs、维持18 h和维持48 h的分化细胞的迁移效率显著升高。(4)实时显微摄像系统分析显示,提高Rac1的总量或者激活其表达水平会提高PDGF诱导的BMSCs的迁移速率,反之则会显著性的降低迁移速率,但对细胞的迁移效率均无显著性的影响;同时PI3K信号通路的抑制剂LY294002的加入会降低迁移速率,Dunn chamber获得同样的结果,说明Rac1和PI3K信号通路均参与PDGF诱导的BMSCs的迁移。结论BMSCs可以趋向PDGF迁移,其对PDGF的趋向性迁移与其分化状态密切相关,在此过程中有Rac1和PI3 K信号通路的参与。
[Abstract]:The purpose of the malignant glioma is the most prevalent primary brain tumors, traditional methods, such as chemotherapy, radiotherapy is fundamentally to treatment, but because of bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) in vivo showed strong tumor tendency, so BMSCs is expected to become the preferred treatment for glioma seed cells. However, whether on the differentiation of BMSCs cells have the same chemotaxis and BMSCs to glioma migration mechanism research is seldom, this study aims to investigate the neural differentiation of BMSCs glioma cells to secrete chemotactic factor of platelet-derived growth factor (platelet-derived growth factor, PDGF) the migration behavior and its molecular mechanism.
Methods the study was divided into four parts, the first three parts research into neural differentiation of BMSCs to PDGF migration, the last part is the study of the molecular mechanism of PDGF induced BMSCs migration. Each part content is as follows: (1) using Percoll density gradient centrifugation in vitro cultured rat BMSCs were identified. In cultured cells by immunohistochemistry staining and differentiation. (2) by adding antioxidants to induce BMSCs to differentiate into neuron like cells. That is using bFGF pre 10 ng/ml followed by 24 h, adding 200 M Ding Qiangji ANISOL (BHA) two DMSO and 2% (DMSO 5) induced by H, finally with 10 l/ml N2 to maintain the H-DMEM to observe the morphological changes induced by BMSCs during the differentiation of cultured 48 h. markers of neural cell specific detection of nestin cells induced by immunofluorescence staining, -III-tubulin beta and NSE table Respectively. (3) using Dunn chamber differentiation into neurons in different stages of BMSCs secretion to glioma factor PDGF chemotactic migration. Cell migration through the German Leica AF6000 live cell station captured images, image analysis using NIH Image J software, 30 cells each state random migration statistics the rate of cell migration, the migration trajectory of efficiency and single cells. (4) the effect of Rac1 on the migration behavior of BMSCs induced by PDGF were investigated by real-time imaging system, and the PI3K signaling pathway to explore the chemotactic migration to function by real-time microscopic imaging system and Dunn chamber two. Two methods cell live by German Leica AF6000 workstation captured images, using NIH Image J image analysis, 30 cells were randomly selected from each state statistics obtained moving speed move cells Rate and efficiency.
Results (1) cultured rat BMSCs isolated by Percoll density gradient centrifugation, stably passaged more than 20 passages. Immunohistochemical identification results for CD29, CD90, CD106 positive, CD34 negative; differentiation experiment shows that it can not only make the osteogenic differentiation and adipogenic differentiation, confirmed the basic characteristics of experimental training the cells with BMSCs. (2) bFGF/BHA induced BMSCs differentiated into neuron like cells induced by pre 24 h, the cell body contraction, cell edge becomes irregular; induced by 5 h, the cell body further contraction, strong refraction, slender processes, and two processes, die and fall off the emergence of a small number of cells; 48 h cells maintain more divergent, and the emergence of the two split; control group cells had no significant changes. Immunofluorescence staining showed that the neural precursor cell marker nestin expression induced by the cell, not Mature neuronal markers -III-tubulin and beta neuronal marker NSE. (3) Dunn chamber analysis showed that BMSCs can chemoattract PDGF directional migration, migration rate and migration efficiency of cells were significantly higher than the control group, single cell migration path also confirmed this result. Cells in different periods after differentiation of PDGF chemotactic God different: not induced by BMSCs, the pre induction 24 h and induced the migration rate of differentiated cells of 5 h has been significantly improved, but not induced by BMSCs, to maintain 18 h and maintain the migration efficiency of 48 h cells increased significantly. (4) real-time image system analysis, improve the total amount of Rac1 or activation its expression level will increase the migration rate of PDGF induced by BMSCs, otherwise it will reduce the migration rate significantly, but had no significant effect on cell migration efficiency; at the same time, the PI3K signaling pathway inhibitor LY294002 The addition of Dunn chamber has the same result, indicating that both Rac1 and PI3K signaling pathways are involved in the migration of PDGF induced BMSCs.
Conclusion BMSCs tends to migrate to PDGF, and its tendency to migrate to PDGF is closely related to its differentiation state. In this process, there are Rac1 and PI3 K signaling pathways involved.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329

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