体外诱导成人骨髓间充质干细胞向神经元样细胞分化的机制研究

发布时间：2018-06-12 17:28

本文选题：体外 + 诱导　；参考：《山西医科大学》2006年硕士论文

【摘要】： 目的:研究表明,骨髓中的间充质干细胞除具备向间充质细胞分化的能力外,在一定条件下还具有向神经细胞分化的可塑性,且容易获得和导入外源基因,因而被认为是较神经干细胞更为理想的组织工程学种子细胞和基因工程学载体细胞。但目前尚不明确其分化机制,诱导分化后的细胞功能距正常神经细胞差距较大,存活时间短,不能有效增殖,不能满足移植需要。进一步了解骨髓间充质干细胞向神经细胞分化的具体机制如哪些基因、生物大分子等参与调控了分化,其途径和信号传导通路以及它们之间的相互作用和影响如何是找到确定稳定的诱导分化方法的基础。基于此,本实验从成人骨髓间充质干细胞的分离、纯化、扩增及诱导方案的实验参数优化和向神经元样细胞方向分化中Pleiotrophin(PTN)基因表达情况进行初步探索,为进一步了解骨髓间充质干细胞的分化特性,最终实现其定向调控提供实验依据。方法: 以密度梯度离心加贴壁培养方法分离成人骨髓间充质干细胞(MSCs),以含20%胎牛血清DMEM培养基进行原代培养和传代培养,用流式细胞仪检测CD45、CD35、CD105以鉴定MSCs纯度。取第6代的MSCs接种于培养板内培养成单层细胞,设对照组和实验组进行预诱导和诱导实验,诱导后30分至3天,观察细胞形态并计数目。以免疫细胞化学法和RT-PCR法测定分化后细胞神经细胞特异性表面标志NSE、MAP-2, GFAP的表达,以鉴定是否诱导为神经元样细胞并计算分化百分率。以RT-PCR法测定第6代MSCs向神经元样细胞分化过程中,诱导前和诱导后3、6、12、24小时、3天PTNmRNA的表达情况。结果: 分离的成人MSCs于细胞接种24小时后开始黏附贴壁,折光性强,呈圆形或椭圆形。3天后可见梭状或多边形细胞,呈集落状生长,10～14天可基本铺满瓶底。传代后生长迅速,5～6天即可铺满瓶底,至第5～6代时细胞呈现比较均一的成纤维细胞样形态。MSCs经流式细胞仪检测表达骨髓间充质干细胞的标志CD44、CD105,而不表达造血干细胞的特异标记CD45,从形态和表型基本证实为较纯的骨髓间充质干细胞。诱导后骨髓间充质干细胞形态发生变化,宽大扁平的细胞体开始向胞核收缩;逐渐出现双极及多极细胞,有细长突起。12小时后变形细胞增多,细长突起相互连接、交织。持续至24小时后变形细胞增多不明显,至诱导后3天悬浮死亡细胞开始增多。诱导后12小时细胞免疫化学染色显示,大部分细胞表达神经元特异性标志NSE(64.79%±0.069)、MAP-2(60.05%±0.093),而未检测到GFAP的表达,经RT-PCR半定量检测亦有NSEmRNA的表达(0.658±0.149)。实验组诱导后细胞有PTNmRNA的表达而且在分化过程中的不同时点表达不同(P0.01)。在细
[Abstract]:Objective: the study shows that mesenchymal stem cells in bone marrow have the ability to differentiate into mesenchymal cells and have the plasticity of differentiation to neural cells under certain conditions, and easy to obtain and import foreign genes. Therefore, the mesenchymal stem cells are considered to be more ideal tissue engineering seed cells and gene engineering carriers than neural stem cells. But at present, the differentiation mechanism is not clear, the differentiated cell function is far apart from the normal nerve cells, and the survival time is short. It can not effectively proliferate and can not meet the needs of transplantation. Further understanding of the specific mechanism of bone marrow mesenchymal stem cells to neural cells, such as which genes, biological macromolecules and so on, is involved in the differentiation. The pathways and signal transduction pathways and their interactions and effects are the basis for finding a stable and induced differentiation method. Based on this, the experimental parameters of the isolation, purification, amplification and induction of adult bone marrow mesenchymal stem cells were optimized and Pleiotrophin (PTN) based on the direction differentiation of the cell like cells. Preliminary investigation of the expression status will provide an experimental basis for further understanding the differentiation characteristics of bone marrow mesenchymal stem cells and ultimately achieving their directional regulation.
Method:
Adult bone marrow mesenchymal stem cells (MSCs) were isolated by density gradient centrifugation and adherent culture method. The primary culture and passage culture were carried out with 20% fetal bovine serum DMEM medium. The purity of MSCs was identified by flow cytometry, CD45, CD35 and CD105 were detected. The sixth generation of MSCs was inoculated into the cultured monolayer cells in the culture plate, and the control group and the experimental group were set up. Pre induction and induction experiments were conducted to observe cell morphology and count the number of cells from 30 to 3 days after induction. Immunocytochemical method and RT-PCR method were used to determine the expression of specific surface markers NSE, MAP-2, and GFAP after differentiation in order to identify whether the cells were induced to be neuron like cells and calculate the percentage of differentiation. The sixth generation of MSCs was determined by RT-PCR method. During the course of cell differentiation, the expression of PTNmRNA at 3 days before induction and 3,6,12,24 hours after induction was observed.
Result:
The isolated adult MSCs began to adhere to the wall after 24 hours of cell inoculation, and the refractive index was strong. The spindle shaped or polygonal cells were found in round or elliptical.3 days. The cells were covered with colony shape and were basically covered with the bottom of the bottle at 10~14 days. After the passage, the growth of the cells was rapid, and the bottom of the bottle could be spread over 5~6 days, and the cells showed a relatively uniform fibroblast like form to the fifth ~ 6 generation. Morphology and expression of bone marrow mesenchymal stem cells (CD44, CD105) were detected by flow cytometry, but the specific marker CD45 of the hematopoietic stem cells was not expressed, and the form and phenotype were basically confirmed as bone marrow mesenchymal stem cells. After induction, the morphologic changes of bone marrow mesenchymal stem cells were changed, and the large flat cells began to shrink to the nucleus after the induction of.MSCs. Bipolar and multipolar cells gradually appeared, with elongated protuberances for.12 hours, deformable cells increased, slender protuberances were interconnected and interwoven. After 24 hours, the increase of deformable cells was not obvious, and the number of suspended cells began to increase at 3 days after induction. 12 hours after induction, cell immunocytochemical staining showed that most cells expressed neuron specific markers. The expression of GFAP was not detected in NSE (64.79% + 0.069) and MAP-2 (60.05% + 0.093), and the expression of NSEmRNA was also expressed (0.658 + 0.149) by RT-PCR semi quantitative detection. The cells in the experimental group were expressed in PTNmRNA and different at different points in the process of differentiation (P0.01).
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R329.2

【共引文献】