单唾液酸四己糖神经节苷脂在骨髓间充质干细胞向神经元样细胞分化中的作用

发布时间：2018-07-21 19:05

【摘要】： 目的:近年来研究表明骨髓间充质干细胞(Bone marrow stromal cells ,BMSCs)可以体外诱导分化为神经细胞,因此BMSCs有望成为神经细胞移植的种子细胞来治疗神经系统疾病,但BMSCs定向诱导分化率低,且分化后的细胞存活时间短而不能有效增殖以满足移植的需要。如何能够找到稳定高效的诱导方法及提高诱导后神经细胞的存活率是需要解决的一个关键问题。单唾液酸四己糖神经节苷脂(monosialotetrahexosyl ganglioside ,GM1)在神经细胞的发育、分化、修复和信号转导中有相当重要的作用。研究表明, GM1有诱导BMSCs分化为神经元样细胞的作用,本文就不同浓度GM1对BMSCs的诱导分化作用作一探讨。方法:以全骨髓培养法分离成人BMSCs,进行原代和传代培养。用流式细胞仪检测BMSCs表面CD44, CD45, CD90,CD105的表达以鉴定纯度。以含有10μg/ml、60μg/ml、90μg/ml GM1的DMEM无血清培养基诱导第五代的BMSCs,阴性对照组不加任何诱导剂。至诱导后7d观察细胞形态变化并计数,诱导后6h以免疫细胞化学法测定各组细胞神经细胞特异性表面标志神经元特异性烯醇化酶(NSE)、神经元特异性微管相关蛋白(MAP-2),以鉴定是否诱导为神经元样细胞并计算分化百分率。各组间分化百分率进行比较和统计学处理。并用MTT法测定诱导后0.5h、6h、24h、3d细胞生长状况。结果:细胞接种2天后给予PBS冲洗,全量换液可见贴壁细胞呈集落状生长。8～10天后细胞可基本铺满瓶底,为均一梭状细胞。以1:3比例传代,传代后1天左右细胞可以恢复活力并贴壁生长,5～6天后可铺满瓶底。传代至4～5代时细胞形态比较均一,混杂细胞较少。流式细胞术检测细胞表面标志CD44(+),CD90(+), CD105(+),CD45(-),基本证实BMSCs的纯度。BMSCs经预诱导后,细胞体积增大,形态变长。加入诱导液30～40 min后,细胞开始发生形态变化,胞体向内收缩,呈球形或圆形,并向周围长出突起,有立体感,折光性增强;诱导4 h后,细胞形态发生明显改变,胞体进一步收缩形成突起,胞体立体感增强,周围出现光晕,可见到很多双极和多极细胞;6 h后大多数细胞形成锥形、三角形等不规则形状,突起增多变长,细胞间突起相互连接,交错成网,呈现典型的神经细胞样形态。到诱导后3天,神经元样细胞增多不明显。5天后有少量细胞悬浮死亡,突起延长多交织成网状;7天时细胞形态与5天时基本相同,但悬浮死亡细胞较多。阴性对照组细胞仍呈均一长梭状细胞,但细胞生长速度明显减慢。细胞诱导6 h免疫细胞化学检测不同浓度GM1组和阴性对照组细胞均有MAP-2,NSE表达,但未检测到GFAP的表达。10μg/ml、60μg/ml、90μg/ml GM1组NSE、MAP-2细胞阳性率均较阴性对照组明显高(P0.05)。组间比较:60μg/ml、90μg/ml组NSE、MAP-2细胞阳性率均较10μg/ml组高(P0.05),60μg/ml、90μg/ml组细胞阳性率差异不显著(P0.05)。MTT结果显示经GM1诱导后的神经元样细胞比阴性对照组生长状态好。结论 1、不同浓度GM1均具有诱导BMSCs向神经细胞分化的作用,诱导6 h后BMSCs向神经细胞分化的分化率最高。 2、中、高浓度GM1诱导BMSCs分化为神经元样细胞的百分率较高,诱导后的神经元样细胞生长状态较好。
[Abstract]:Objective: in recent years, studies have shown that Bone marrow stromal cells (BMSCs) can be induced to differentiate into nerve cells in vitro, so BMSCs is expected to be the seed cell of the nerve cell transplantation to treat nervous system diseases, but the differentiation rate of BMSCs is low, and the survival time of the differentiated cells is short and can not be effectively increased. Monosialotetrahexosyl Ganglioside (GM1), a single sialic acid four hexose Ganglioside (GM1), is of considerable importance in the development, differentiation, repair and signal transduction of nerve cells. The research shows that GM1 has the function of inducing BMSCs to differentiate into neuron like cells. In this paper, the induction and differentiation of BMSCs with different concentrations of GM1 are discussed.
Methods: full bone marrow culture was used to isolate adult BMSCs and carry out primary and subculture. Flow cytometry was used to detect the expression of CD44, CD45, CD90 and CD105 on the surface of BMSCs to identify the purity. Fifth generation BMSCs was induced by the DMEM serum-free medium containing 10 mu g/ml, 60 mu g/ml and 90 u g/ml GM1. Cell morphological changes were observed and counted. After induction, 6h was induced by immunocytochemical method to determine neuron specific enolase (NSE) and neuron specific microtubule related protein (MAP-2), to identify whether the neuron like cells were induced as neuron like cells and to calculate the percentage of differentiation. The percentage of differentiation among groups was compared. The growth status of 0.5h, 6h, 24h and 3D after induction was determined by MTT.
Results: after 2 days of inoculation, the cells were given PBS irrigation, and the full volume of the cells could be seen that the adherent cells were colonialized for.8 to 10 days. The cells could be basically covered with the bottom of the bottle. The cells were homogeneous fusiform cells. The cells were passaged in the 1:3 ratio. The cells could be rejuvenated and adhered to the wall for about 1 days after the passage. After 5~6 days, the cells could be filled with the bottom of the bottle. The cell morphology was compared to the 4~5 generation. The cell surface markers CD44 (+), CD90 (+), CD105 (+) and CD45 (-) were detected by flow cytometry, which basically confirmed that the cell volume increased and the morphology became longer after preinduction of the purity of BMSCs. After adding the inducer 30~40 min, the cells began to produce morphologic changes, the cell bodies contracted inward, and they were spherical or round, and protruded around them. After 4 h, the cell morphology changes obviously, the cell body is further contracted to form the protuberance, the body stereoscopic sense is enhanced, the halo around the cell appears, and a lot of bipolar and multipolar cells can be seen. After 6 h, most of the cells form a cone, triangle and other irregular shapes, the protuberances grow and grow, the intercellular protuberances are connected to each other. In the 3 day after induction, a small number of cell suspension died and the protuberance was more interwoven into the net, and the cell morphology was basically the same at the time of 7 days, but the number of dead cells was more in suspension, but the cell growth was still a long spindle cell, but the cell growth was growing at 3 days after induction. The cell induced 6 h immunocytochemistry test showed that the cells of different concentration GM1 and negative control group had MAP-2, NSE expression, but the expression of GFAP was not.10 g/ml, 60 mu g/ml, 90 u g/ml GM1 NSE, and the positive rate of MAP-2 cells was higher than that of the negative control group. The rate of sex was higher than that of the 10 g/ml group (P0.05), 60 g/ml, and the positive rate of cell positive rate in 90 mu g/ml group was not significant (P0.05).MTT results showed that the growth state of neuron like cells induced by GM1 was better than that of the negative control group.
conclusion
1, different concentrations of GM1 all have the effect of inducing BMSCs to differentiate into neurons. After 6 h induction, BMSCs has the highest differentiation rate to neural cells.
2, medium, high concentration of GM1 induced BMSCs to differentiate into neuron like cells, and the neuron like cells grew better after induction.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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