甲钴胺体外诱导大鼠骨髓间充质干细胞向神经元样细胞分化的初步研究

发布时间：2018-03-17 15:24

本文选题：骨髓间充质干细胞　切入点：神经元样细胞　出处：《南华大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：通过体外培养骨髓间充质干细胞（BMSCs），初步探讨不同剂量的甲钴胺体外定向诱导BMSCs向神经元样细胞分化的可行性，以及观察分化后的细胞的生长和增殖情况，寻求一种较合理的甲钴胺诱导浓度，为临床上利用甲钴胺诱导后的BMSCs进行细胞移植更好更有效的治疗脊髓损伤提供前期研究依据。方法：1、采用密度梯度离心和贴壁培养法分离、培养和纯化大鼠骨髓间充质干细胞，倒置显微镜下连续观察细胞生长情况、生物活性及形态学变化，免疫荧光细胞化学检测BMSCs相对特异性表面标志物CD44，证实实验所需的细胞为BMSCs。 2、取生长状态良好的第4-5代BMSCs，制成单细胞悬液，调整细胞浓度，实验分为对照组（control组）和实验组（分25ug/ml、50ug/ml和100ug/ml三个浓度组），实验组分别加入不同浓度甲钴胺的10%FBS L-DMEM培养基，对照组加入10%FBS L-DMEM，不加任何诱导剂，各组以24h、48h、72h为观察记录的时间坐标轴，在倒置显微镜下连续观察细胞形态学变化和生长状态，MTT法检测诱导后细胞的生长和增殖情况，RT-PCR和western blotting方法鉴定分化的神经前体细胞的特异性标志物神经巢蛋白（neuroepithelial stem cell protein，Nestin）和神经细胞的特异性标志物神经元特异性烯醇化酶（neon-specific enolase，NSE）的表达。结果：1、原代细胞接种24-48小时后可见贴壁细胞，贴壁细胞单个散在分布，多数细胞呈小圆形或椭圆形。72小时后可见散在的梭形细胞，一周左右细胞形成集落，细胞呈梭形、三角形或星形。原代培养约两周，细胞增殖明显加快，集落中的细胞增殖成“漩涡状”，具有典型的成纤维细胞样形态，免疫荧光细胞化学鉴定显示BMSCs细胞表面标志物CD44表达阳性，造血干细胞表面抗原CD34表达阴性。2、BMSCs经25ug/ml、50ug/ml和100ug/ml诱导24h、48h和72h后，各实验组细胞折光性增强，部分细胞呈现神经元样变化，，具有神经元样形态：细胞向周围长出较长的突起，有立体感，折光性增强，倒置显微镜下观察以100ug/ml组细胞折光性及突触延长最为明显，并且相邻细胞间的突触出现连接，而对照组细胞形态无明显变化，仍然呈长梭形。MTT结果显示24h、48h和72h各实验组与对照组无显著性差异（P＞0.05），RT-PCR与western blotting结果显示不同剂量甲钴胺诱导48h后，Nestin和NSE在mRNA和蛋白水平表达均上调，其中100ug/ml组表达上调最明显，与control组比较，差异具有统计学意义（P＜0.05）；同样，100ug/ml甲钴胺诱导24h、48h和72h后，Nestin和NSE在mRNA和蛋白水平表达均上调，其中72h表达上调最明显，与control组比较，差异具有统计学意义（P＜0.05）。结论：1、甲钴胺可定向诱导BMSCs向神经元样细胞分化，分化后的细胞具有一定的增殖能力；甲钴胺对分化后的细胞生长增殖没有明显的抑制作用。2、体外实验表明，实验组中100ug/ml甲钴胺为较佳诱导浓度，能有效快捷的诱导BMSCs向神经元样细胞分化。
[Abstract]:Objective: to investigate the feasibility of BMSCs differentiation into neuron-like cells induced by different doses of mecobalamin in vitro, and to observe the growth and proliferation of differentiated cells by cultured bone marrow mesenchymal stem cells (BMSCs) in vitro. To seek a more reasonable concentration of mecobalamin induction for clinical use of mecobalamin induced BMSCs for cell transplantation better and more effective treatment of spinal cord injury to provide a preliminary study basis. Methods the bone marrow mesenchymal stem cells of rats were isolated and purified by density gradient centrifugation and adherent culture. The cell growth, biological activity and morphological changes were observed under inverted microscope. CD44, a relative specific surface marker of BMSCs, was detected by immunofluorescence cytochemistry, and it was confirmed that the cells needed for the experiment were BMSCs. 2. BMSCs, which had good growth condition, were prepared into single cell suspension and adjusted cell concentration. The experiment was divided into control group (control group) and experimental group (25ugr / ml 50ugrml and 100ugrml). The experimental group was supplemented with 10s L-DMEM medium with different concentrations of mecobalamin. In the control group, 10s L-DMEM was added without any inducer, and 24 h, 48 h and 72 h was taken as the observation time axis. The morphological changes and growth state of cells were continuously observed under inverted microscope. MTT assay was used to detect the cell growth and proliferation after induction. RT-PCR and western blotting were used to identify the specific marker of neural precursor cells, neuroepithelial nestin. The expression of stem cell protein nestin and neuron-specific enolase (NSE). Results the adherent cells were found 24 to 48 hours after inoculation. Most of the cells were small round or ellipse. After 72 hours, the cells formed colony, and the cells were fusiform. Triangle or star. After about two weeks of primary culture, cell proliferation was significantly accelerated, and the cells in the colony proliferated into a "whirlpool" with typical fibroblast-like morphology. Immunofluorescence cytochemical analysis showed that BMSCs cell surface marker CD44 was positive, and hematopoietic stem cell surface antigen CD34 expression was negative. After 24 h and 72 h after induction of 25ugmml / ml 50ugrml and 100ugrml respectively, the refractive index of the cells in each experimental group was enhanced, and some cells showed neuronal changes. It has neuron-like morphology: the cells have long protuberance, stereosensory and enhanced refraction. Under the inverted microscope, the refraction and synaptic prolongation of the cells in the 100ug-ml group are most obvious, and the synapses of the adjacent cells are connected. However, there was no significant change in cell morphology in the control group. The results showed that there was no significant difference in the expression of nestin and NSE at 24 h and 72 h between the two groups (P > 0.05). The results of RT-PCR and western blotting showed that the expression of nestin and NSE were up-regulated at the level of mRNA and protein after 48h induction with different doses of mecobalamin. The expression of nestin and NSE in 100ugr / ml group was significantly higher than that in control group (P < 0.05), and the expression of nestin and NSE were up-regulated in mRNA and protein levels at 24 h and 72 h after induction by 100ugr / ml mecobalamin, the expression of nestin and NSE was the most obvious at 72 h, which was significantly higher than that in control group. The difference was statistically significant (P < 0.05). Conclusion Mecobalamin can induce the differentiation of BMSCs into neuron-like cells, and the differentiated cells have the ability to proliferate, while mecobalamin has no obvious inhibitory effect on the growth and proliferation of differentiated cells. In the experimental group, 100ugr / ml mecobalamin was the best concentration, which could induce the differentiation of BMSCs into neuron-like cells effectively and quickly.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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