成神经分化的骨髓间充质干细胞向干细胞因子的趋化性迁移研究

发布时间：2018-05-04 18:06

本文选题：骨髓间充质干细胞(BMSCs) + 细胞分化　；参考：《苏州大学》2010年硕士论文

【摘要】： 目的恶性胶质瘤等原发脑瘤对人类危害极大,骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)能够向胶质瘤发生趋化性迁移,并且具有易于获得、多向分化等优点,因而成为治疗胶质瘤最具希望的治疗手段。但是,关于不同分化状态细胞迁移的研究还较少。本实验旨在探讨成神经分化的BMSCs向干细胞因子(stem cell factor,SCF)的迁移行为。方法(1)采用Percoll密度梯度分离法分离BMSCs,并在体外扩增培养,通过免疫细胞化学染色的方法检测表面抗原,以及成脂、成骨诱导分化鉴定。(2)采用抗氧化剂丁羟基茴香醚(BHA)及bFGF联合诱导BMSCs向神经样细胞分化,首先用浓度为10 ng/ml的bFGF预诱导24 h,之后加入浓度为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,并且通过免疫荧光染色方法检测神经细胞特异性标志物nestin、β-III-tubulin和NSE的表达情况。(3)用Dunn chamber研究成神经分化BMSCs向SCF的定向迁移行为,通过德国Leica AF6000活细胞工作站拍摄迁移图像,应用NIH Image J分析图像,计算细胞的迁移速率、迁移效率以及迁移轨迹。并研究了PI3K抑制剂LY294002处理后BMSCs迁移行为的变化。结果(1)采用Percoll细胞分离液密度梯度法,成功分离培养出大鼠骨髓间充质干细胞,免疫荧光染色显示CD29、CD90、CD106呈阳性,CD34呈阴性,并且能够分化成脂肪细胞和成骨细胞。(2)用bFGF/BHA诱导BMSCs向神经样细胞分化,预诱导24 h,细胞形态变化较小,仍为长梭形;诱导5 h后细胞胞体出现回缩、有突触伸出,形态改变较大,初步展现出神经细胞的形态特征;维持培养18h,细胞分叉稍有增多;维持48 h后,细胞分叉数目增多,并且产生二级分叉,类似于成熟神经元的形态。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细胞标志物nestin、β-III-tubulin以及NSE。(3)Dunn chamber迁移实验显示,SCF诱导实验组细胞的迁移速率和迁移效率明显高于对照组,表明SCF对BMSCs具有趋化作用。此外,分化不同状态的BMSCs向SCF的趋化迁移程度也不同。另外,PI3K信号通路抑制剂LY294002能够降低BMSCs的迁移速率和效率。结论BMSCs可以分化为神经样细胞,其对SCF的趋向性迁移与分化状态密切相关,并且PI3K信号通路参与该迁移过程。
[Abstract]:Objective malignant glioma and other primary brain tumors are very harmful to human beings. Bone marrow mesenchymal stem cells (bone marrow mesenchymal stem cells, BMSCs) can migrate to glioma, and have the advantages of easy to obtain, multidifferentiation and so on. Therefore, it has become the most promising treatment for glioma. There are few studies on cell migration. The aim of this study was to investigate the migration of BMSCs stem cell factor (SCF) into neural differentiation.
Methods (1) BMSCs was isolated by Percoll density gradient method, and was amplified and cultured in vitro. The surface antigen was detected by immunocytochemical staining, and the differentiation of osteogenesis and osteogenesis induced differentiation. (2) the combination of antioxidant butyl anisole (BHA) and bFGF was used to induce BMSCs to differentiate into neural like cells. First, B with a concentration of 10 ng/ml was used. FGF preinduced 24 h, then Ding Qiangji anisole (BHA) and 2% two methyl sulfoxide (DMSO) were added to 5 h, and the morphological changes of BMSCs were induced in the differentiation process during the induction of differentiation with H-DMEM containing N2, and the expression of neuron specific markers was detected by immunofluorescence staining. (3) Dunn chamber was used to study the directional migration of neurogenic BMSCs to SCF. The migration images were taken by the Leica AF6000 live cell workstation in Germany, and the NIH Image J analysis images were used to calculate the migration rate, migration efficiency and migration path of the cells. The change of BMSCs migration behavior after LY294002 treatment of PI3K inhibitors was also studied.
Results (1) the rat bone marrow mesenchymal stem cells were successfully isolated and cultured by the density gradient method of Percoll cell separation solution. Immunofluorescence staining showed that CD29, CD90, CD106 were positive, CD34 was negative, and could differentiate into adipocytes and osteoblasts. (2) bFGF/BHA induced BMSCs to differentiate into neurolike cells, preinduced 24 h, and cell morphological changes Small, still long spindle shape. After 5 h induction, cell body retracted, synapses protruded, morphologic changes were larger, and the morphological characteristics of nerve cells were shown initially; maintaining 18h, cell bifurcation slightly increased; after maintaining 48 h, the number of cell bifurcations increased and produced two grade bifurcation, similar to the form of mature neurons. The control group cells were not clear. The immunofluorescence staining showed that the induced cell expression nestin, beta -III-tubulin and NSE. (3) Dunn chamber migration experiment showed that the migration rate and migration efficiency of SCF induced experimental group were significantly higher than those of the control group, which indicated that SCF had chemotaxis to BMSCs. Moreover, BMSCs to S in different states was differentiated to S. The degree of chemotactic migration of CF is also different. In addition, PI3K signaling pathway inhibitor LY294002 can reduce the migration rate and efficiency of BMSCs.
Conclusion BMSCs can be differentiated into neuron like cells, which is closely related to the trend migration and differentiation of SCF, and PI3K signaling pathway is involved in the migration process.

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

【共引文献】