骨髓间充质干细胞在成神经分化过程中的定向迁移研究

发布时间：2018-04-07 18:55

本文选题：骨髓间充质干细胞(BMSCs)　切入点：分化　出处：《苏州大学》2009年硕士论文

【摘要】： 目的恶性胶质瘤是目前最为盛行的原发脑瘤,干细胞移植极有希望治疗胶质瘤。因骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)体内体外均显示很强的胶质瘤趋化性,所以应用BMSCs治疗胶质瘤即是很好的细胞治疗手段。然而,关于细胞因子参与BMSCs迁移的报道却很少,本研究旨在探讨成神经分化的BMSCs向C6细胞条件培养基和SDF-1α(stromal cell-derived factor 1α)的迁移行为。方法本实验分三个阶段研究成神经分化的BMSCs向C6细胞条件培养基和SDF-1α的迁移。(1)采用Percoll分离法在体外培养并扩增BMSCs,通过免疫荧光染色的方法检测表面抗原对BMSCs进行鉴定。(2)采用抗氧化剂诱导方案诱导BMSCs向神经样细胞分化,即先用浓度为10 ng/ml的bFGF预诱导24 h,加入浓度为200μM的丁羟基茴香醚(BHA)和2%的二甲基亚砜(DMSO)诱导5 h,再用含有N2的H-DMEM维持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的细胞表达神经细胞特异性标志物Nestin、β-III-tubulin和NSE的情况。(3)运用Dunn chamber研究了分化细胞在具有浓度梯度的趋化因子中的定向迁移行为。分化不同状态BMSCs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分析图像,每个分化点随机抽取40个细胞进行统计得到细胞的迁移速率、迁移效率和诱导5 h的迁移轨迹。接着,我们运用Boyden chamber研究了BMSCs在成神经分化过程中的趋化性迁移(Chemotaxis)和随机迁移(Chemokinesis)。以正常培养的BMSCs作为对照,随机抽取对照组和实验组各10个视野,细胞计数,计算迁移系数,迁移系数=实验组/对照组。结果(1)采用Percoll淋巴细胞分离液及不连续密度梯度法,成功分离培养出大鼠骨髓间充质干细胞,可稳定传至20代以上。表型鉴定结果为CD29、CD71、CD90、CD106呈阳性,CD34、CD45呈阴性。(2)用bFGF/BHA诱导BMSCs向神经样细胞分化,预诱导24 h,细胞变成长梭形;诱导5 h,细胞即发生剧烈的形态改变,出现胞体回缩、突触伸展等神经细胞的形态特征;维持48 h,细胞出现更多的分叉,并出现二级叉。对照组细胞无明显的形态变化,免疫荧光染色显示诱导的细胞表达神经前体细胞标志物Nestin、β-III-tubulin和成熟神经元标志物NSE。对照组不表达NSE,诱导组与对照组比较有统计学差异(P0.05)。(3) Dunnchamber分析显示,C6细胞条件培养基组和SDF-1α组诱导细胞的迁移速率和迁移效率明显高于对照组,表明C6细胞条件培养基和SDF-1α对BMSCs具有趋化作用,单个细胞迁移轨迹实验也证实了这一结果。此外,分化不同状态的BMSCs向C6细胞条件培养基和SDF-1α的趋化程度也不同。当Boyden上室为L-DMEM悬浮的细胞,下室为C6细胞条件培养基时,分化细胞的迁移数目明显多于对照;而当上下室均为C6细胞条件培养基时,分化细胞的随机迁移与对照无显著差异。结论BMSCs的定向迁移与分化状态密切相关,预诱导24 h分化细胞的定向迁移最强。
[Abstract]:Objective malignant glioma is the most prevalent primary brain tumor. Stem cell transplantation is very promising in the treatment of glioma.Because bone marrow mesenchymal stem cells (BMSCs) of bone marrow mesenchymal stem cells (BMSCs) have strong chemotaxis in vivo and in vitro, BMSCs is a good cell therapy for glioma.However, there are few reports of cytokines involved in BMSCs migration. This study aims to investigate the migration of neurogenic BMSCs to C6 cell conditioned medium and SDF-1 伪 stromal cell-derived factor 1 伪.Methods in this experiment, neural differentiation of BMSCs into C6 cell conditioned medium and migration of SDF-1 伪 were studied in three stages. BMSCs were cultured and amplified by Percoll in vitro. Surface antigens were detected by immunofluorescence staining for BMSCs.Identification. 2) differentiation of BMSCs into neuronal cells was induced by antioxidant induction.After 24 h preinduction with 10 ng/ml bFGF, 200 渭 M butadiol anisole (BHA) and 2% dimethyl sulfoxide (DMSO) were added for 5 h, and then H-DMEM containing N2 was used for 48 h.The morphological changes of BMSCs during differentiation were observed.The specific markers Nestin, 尾 -III-tubulin and NSE were detected by immunofluorescence staining. Dunn chamber was used to study the migration behavior of differentiated cells in chemokines with concentration gradient.The migration of BMSCs in different differentiation states was captured by Leica AF6000 living cell workstation in Germany. The images were analyzed by NIH Image J. 40 cells were randomly selected from each differentiation point to obtain the migration rate of the cells.Migration efficiency and migration trajectory induced for 5 h.Then, we used Boyden chamber to study the chemotaxis of BMSCs during neuronal differentiation.BMSCs cultured in normal culture was used as control group, 10 visual fields were randomly selected from control group and experimental group, cell count, migration coefficient, migration coefficient = experimental group / control group were calculated.Results 1) Rat bone marrow mesenchymal stem cells were successfully isolated and cultured by Percoll lymphocyte isolate and discontinuous density gradient method.Phenotypic identification showed that CD29, CD71, CD90, CD106, CD34, CD45, negative, and bFGF/BHA were used to induce BMSCs to differentiate into neuron-like cells, and after 24 h preinduction, the cells became long fusiform, and 5 h after induction, the cells showed drastic morphological changes and cell somatic retraction.The morphological features of nerve cells such as synaptic extension were more branched and secondary forks appeared after 48 h.The expression of Nestin, 尾 -III-tubulin and NSE were detected by immunofluorescence staining.The results of Dunnchamber analysis showed that the migration rate and migration efficiency of C6 cells in conditioned medium group and SDF-1 伪 group were significantly higher than those in control group.The results showed that C6 cell conditioned medium and SDF-1 伪 had chemotactic effect on BMSCs, and this result was confirmed by single cell migration trajectory experiment.In addition, the chemotaxis of BMSCs to C6 cell conditioned medium and SDF-1 伪 was also different.When the upper chamber of Boyden was a L-DMEM suspended cell and the lower chamber was a conditioned medium of C6 cells, the migration number of differentiated cells was significantly higher than that of the control, but when the upper and lower compartments were conditioned medium of C6 cells, there was no significant difference between the random migration of differentiated cells and that of the control.Conclusion the directional migration of BMSCs is closely related to the differentiation state, and the directional migration of differentiation cells preinduced 24 h is the strongest.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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