大鼠骨髓间充质干细胞体外分离培养、扩增及向视网膜神经细胞诱导分化的研究

发布时间：2018-05-29 03:22

本文选题：骨髓 + 间充质干细胞　；参考：《华中科技大学》2007年硕士论文

【摘要】： 目的:研究大鼠骨髓间充质干细胞(rMSCs)的体外分离培养、扩增及其向视网膜神经细胞分化的潜能。方法:取4-6周SD大鼠双侧股骨和胫骨骨髓用培养基悬浮,接种于FN包被的六孔板中,纯化并扩增rMSCs,显微镜下观察细胞生长状态及形态学变化,并用流式细胞仪检测其表型;同时取生后1-3天SD乳鼠视网膜神经上皮层培养,定期取上清夜用作配制分化液。通过两步法不同的诱导剂诱导第8-10代rMSCs分化,显微镜下观察分化细胞的形态学变化,免疫荧光细胞化学检测分化过程中神经干细胞以及视网膜神经细胞部分特异性标记蛋白的表达。结果:rMSCs贴壁生长,表现为成纤维细胞样,呈梭形、多角形等细胞形态,达到融合时呈长梭形紧密排列,形态均一,成旋涡样生长,20代以后细胞生长逐渐衰退、出现分化倾向、形态异质;表型分析结果显示rMSCs表达CD44,CD90不表达CD34,CD45;第一步诱导分化后,细胞形态改变并逐渐交织成网状,最后形成球形,不断增大,呈现神经干细胞样生长特性,并高表达其标志nestin,第二步诱导后细胞呈双极、多极或锥形,表达NeuN、GFAP,部分表达视网膜神经节细胞标志Thy1.1。结论:rMSCs可以在体外成功分离培养、扩增纯化,具有向神经干细胞分化的潜能,并可进一步分化出视网膜神经样细胞,可用作临床药物实验研究,并有望成为移植治疗视网膜神经细胞病变的工具细胞。
[Abstract]:Aim: to study the potential of rat bone marrow mesenchymal stem cells (MSCs) to differentiate into retinal nerve cells in vitro. Methods: bone marrow of bilateral femur and tibia of 4-6 weeks Sprague-Dawley rats were suspended in culture medium and inoculated into FN-coated six-hole plate. RMSCs were purified and amplified. The growth state and morphological changes of the cells were observed under microscope and the phenotypes were detected by flow cytometry. At the same time, 1-3 days after birth SD rats retinal nerve epithelium culture, and regular supernatant night for preparation of differentiation fluid. The differentiation of 8-10 passage rMSCs was induced by different inducers of two-step method. The morphological changes of differentiated cells were observed under microscope. The expression of partial specific labeled proteins in neural stem cells and retinal nerve cells during differentiation was detected by immunofluorescence cytochemistry. Results the adherent cells of 10% rMSCs appeared as fibroblasts, fusiform, polygonal, and so on. When they reached fusion, they were arranged closely in long spindle shape and uniform in shape. After 20 generations of vortex like growth, the cell growth gradually declined, and the differentiation tendency appeared. Phenotypic analysis showed that the expression of CD44T CD90 was not expressed in rMSCs. After the first step of differentiation, the cell morphology changed and gradually intertwined into reticular shape. Finally, the cells became globular and gradually enlarged, showing the characteristics of neural stem cell like growth. After the second step, the cells showed bipolar, multipolar or conical expression, and some of them expressed NeuNa-GFAPs, and some of them expressed Thy1.1, a marker of retinal ganglion cells. Conclusion\%\ It is expected to be a tool cell for the transplantation of retinal neuropathy.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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