Notch1信号在法舒地尔诱导MSCs向神经元样细胞分化过程中的作用

发布时间：2018-01-17 22:29

本文关键词：Notch1信号在法舒地尔诱导MSCs向神经元样细胞分化过程中的作用　出处：《郑州大学》2010年硕士论文　论文类型：学位论文

【摘要】： 背景与目的间充质干细胞具有较强的增殖、自我更新及分化的能力,可以从成熟骨髓、脂肪组织及脐带血等多种组织分离出来。它不但可以向中胚层细胞如骨骼肌细胞、心肌细胞及脂肪细胞分化,而且还可以向外胚层细胞如神经细胞、上皮细胞分化。骨髓间充质干细胞的发现,为各种神经系统变性病、中枢神经损伤及脑卒中等疾病的治疗带来新的手段,给许多神经系统疾病的患者带来新的希望,具有广泛的临床运用前景。因而对骨髓间充质干细胞生物学特性、分离培养、诱导分化、向神经细胞分化的机制及移植治疗神经系统疾病等研究已经成为神经科学研究的重要领域。目前,体外诱导骨髓间充质干细胞向神经细胞分化仍然存在分化效率低及分化机制不明确等不足。因此,骨髓间充质干细胞向神经细胞分化诱导方法和分化机制尚需进一步研究。 Notch信号通路是生物体内的一条重要细胞信号传导通路,参与胚胎神经系统的发育,调控干细胞的增殖、分化和凋亡等生物活动。研究表明骨髓间充质干细胞向神经细胞分化过程中Notch信号分子表达下调,抑制Notch信号有利于其向神经元的分化,而Hes1蛋白在这过程中的作用仍未十分明确。近年研究发现,CoCl2联合Rho激酶抑制剂可以诱导骨髓间充质干细胞分化为神经细胞,这提示我们RhoA/Rho激酶信号通路可能参与调控骨髓间充质干细胞分化为神经元样细胞过程。因此,本研究旨在探讨法舒地尔在骨髓间充质干细胞分化为神经元样细胞的作用,并深入探讨Notch信号抑制后是否有利于促进骨髓间充质干细胞向神经元样细胞分化。这对于深入研究骨髓间充质干细胞向神经元样细胞分化机制,将来临床应用骨髓间充质干细胞移植联合法舒地尔治疗相关神经系统疾病提供依据等具有重要的意义。材料和方法以Wistar大鼠为实验动物,鼠龄2-3月,体重150-200g,雌雄不限,以10代以上的MSCs传代培养,进行细胞实验。实验将培养的MSCs分为未转染组、转染组(转染Rn-Notch1-siRNA)、阳性对照组(转染Rn-MAPK-1 Control siRNA)及阴性对照组(转染Negative Control siRNA)等4组。采用盐酸法舒地尔诱导大鼠MSCs分化为神经细胞。倒置荧光显微镜下观察MSCs转染后荧光表达情况；RT-PCR检测Notch1、Hes1和MAPK1 mRNA的表达变化；免疫细胞化学法检测Notch1、神经元巢蛋白(Nestin)、神经微丝蛋白亚单位(NF-M)和胶质纤维酸性蛋白(GFAP)的表达变化；MTT方法检测细胞存活率。结果 1. siRNA转染72h,MSCs荧光表达最强,转染率可达0.914±0.04；同时,转染组MSCs的Notch1和Hes1 mRNA转录下降(P0.05)；MTT提示转染组细胞存活率也显著减少(P0.05),与对照组差异有统计学意义。 2.盐酸法舒地尔可以诱导MSCs向神经细胞分化；其中以转染组诱导效果最佳,Nestin、NF-M的表达率显著的高于其它各组,差异有统计学意义(P0.05)。结论 1.盐酸法舒地尔可以诱导骨髓间充质干细胞向神经元样细胞分化。 2.抑制Notch信号,有利于盐酸法舒地尔高效诱导骨髓间充质干细胞向神经元样细胞分化, 3.细胞内可能存在Notch信号通路与RhoA/Rho激酶信号通路协同促进骨髓间充质干细胞向神经元样细胞分化。
[Abstract]:Background and purpose
Mesenchymal stem cells have strong proliferation ability of self-renewal and differentiation, from mature bone marrow tissues, adipose tissue and cord blood separated. It can not only to mesodermal cells such as skeletal muscle cells, myocardial cells and adipocyte differentiation, but also to ectoderm cells such as nerve cells, epithelial cells differentiation of bone marrow mesenchymal stem cells, for a variety of neurodegenerative disease, bring new means of treatment of CNS injury and stroke, to many nervous system disease patients new hope, has broad clinical application prospect. The biological characteristics of bone marrow mesenchymal stem cells separation. Cultivation, differentiation, to the mechanisms of neural differentiation and transplantation for the treatment of diseases of the nervous system research has become an important field of neuroscience research. At present, bone marrow mesenchymal induction in vitro The differentiation of stem cells into neural cells still has the disadvantages of low differentiation efficiency and unclear differentiation mechanism. Therefore, the differentiation and induction mechanism of bone marrow mesenchymal stem cells into neural cells need further study.
Notch signal pathway is an important signal transduction pathway in living organisms, involved in embryonic development of the nervous system, the regulation of stem cell proliferation, differentiation and apoptosis. The results show that the biological activity of bone marrow mesenchymal stem cells to form Notch signaling molecules during neural differentiation were reduced, inhibition of the Notch signal is conducive to neuron the role of Hes1 protein and differentiation, not in this process is very clear. Recent studies have found that CoCl2 and Rho kinase inhibitors can induce differentiation of bone marrow mesenchymal stem cells into neural cells. This suggests that RhoA/Rho kinase signaling pathway may be involved in the regulation of bone marrow mesenchymal stem cells to differentiate into neuron like cells.
Therefore, the purpose of this study was to investigate the effects of fasudil on differentiation of bone marrow mesenchymal stem cells into neuron like cells, and explore whether inhibition of Notch signaling is conducive to the promotion of bone marrow mesenchymal stem cells to differentiate into neuron like cells. In the study of bone marrow mesenchymal stem cells into neuron like cell differentiation mechanism, the future clinical application of bone marrow mesenchymal stem cell transplantation and fasudil plays an important role in the treatment of diseases of the nervous system to provide related evidence.
Materials and methods
The Wistar rats were used as experimental animal, rats aged 2-3 months, weight 150-200g, male or female, with more than 10 generations of MSCs cells were subcultured. The experiment cultured MSCs were divided into non transfection group, transfection group (transfected with Rn-Notch1-siRNA), positive control group (transfected with Rn-MAPK-1 Control siRNA) and negative control group (Negative Control siRNA 4 transfection) group. Rat MSCs induced differentiation into neural cells fasudil hydrochloride. Inverted fluorescence microscope MSCs after transfection, the expression of fluorescence detection; RT-PCR Notch1, the expression of Hes1 and MAPK1 mRNA detection; Notch1 immunocytochemistry, nestin neurons (Nestin), neurofilament protein unit (NF-M) and glial fibrillary acidic protein (GFAP) expression; MTT method to detect cell survival rate.
Result
1. siRNA transfected 72h, MSCs showed the strongest fluorescence expression, and the transfection rate reached 0.914 + 0.04. Meanwhile, the transcription of Notch1 and Hes1 mRNA decreased (P0.05) in the transfection group, and MTT indicated that the cell survival rate of transfection group was also significantly reduced (P0.05), and the difference between the transfection group and the control group was statistically significant (MSCs).
2. fasudil hydrochloride can induce MSCs to differentiate into neural cells; the transfection group was the best, Nestin, the expression rate of NF-M was significantly higher than other groups, the difference was statistically significant (P0.05).
conclusion
1. fasudil hydrochloride can induce bone marrow mesenchymal stem cells to differentiate into neuron like cells.
2. inhibition of the Notch signal, is conducive to the efficiency of fasudil hydrochloride induced bone marrow mesenchymal stem cells to differentiate into neuron like cells,
3. cells may have Notch signaling pathway and RhoA/Rho kinase signaling pathway to promote bone marrow
The mesenchymal stem cells differentiate into neuron like cells.

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

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