骨髓间充质干细胞分化为成牙本质细胞的体内研究

发布时间：2018-05-19 13:06

本文选题：绿色荧光蛋白转基因小鼠 + 骨髓间充质干细胞　；参考：《第四军医大学》2010年硕士论文

【摘要】： 干细胞分化为成牙本质细胞是牙本质缺损修复的关键环节。骨髓间充质干细胞是一类成体干细胞,在干细胞龛的调控下能够定向分化为多种中胚层和神经外胚层来源的组织细胞,但是对于这种非牙源性的干细胞能否通过分化为成牙本质细胞的方式修复牙本质缺损的研究鲜有报道。本实验采用绿色荧光蛋白转基因小鼠的骨髓间充质干细胞的体内实验观察BMMSCs能否分化为成牙本质细胞并参与牙本质损伤的修复,为牙齿组织工程提供更具活力的种子细胞。方法:(1)取绿色荧光蛋白转基因小鼠的全骨髓,贴壁法体外培养绿色荧光蛋白转基因小鼠骨髓间充质干细胞,流式细胞术鉴定细胞表面分子标记物,体外成骨成脂诱导;(2)尾静脉注射构建绿色荧光骨髓间充质干细胞嵌合普通C57BL/6小鼠模型;(3)制备嵌合小鼠牙本质缺损修复模型,用免疫荧光检测法观察绿色荧光骨髓间充质干细胞是否参与牙本质缺损的修复,探讨骨髓间充质干细胞作为修复牙本质缺损种子细胞的可行性。结果:(1)培养的绿色荧光蛋白转基因小鼠骨髓间充质干细胞具有很强的增殖能力并可持续传代,P3代细胞流式细胞术检测干细胞表面分子阳性,绿色荧光表达稳定,具有体外分化成骨成脂能力;(2)构造的嵌合小鼠模型全骨髓再培养,发现大量具有绿色荧光标记的细胞存在,各组织脏器也有不同数量的绿色荧光细胞存在。(3)免疫组织荧光显示牙本质缺损模型嵌合小鼠牙髓腔内出现大量自发绿色荧光的细胞,牙本质缺损处的部分绿色荧光细胞表达牙本质特异性蛋白DSP和DMP-1。结论:本实验利用示踪能力强的绿色荧光蛋白转基因小鼠骨髓间充质干细胞构建荧光嵌合小鼠模型,验证骨髓间充质干细胞体内向成牙本质细胞方向分化的能力,发现骨髓间充质干细胞在体内微环境的诱导下可以向成牙本质细胞方向分化,并参与牙本质缺损的修复,可以作为牙齿组织工程的干细胞源。
[Abstract]:Differentiation of stem cells into odontoblast cells is a key link in dentin defect repair. Bone marrow mesenchymal stem cells (BMSCs) are a group of adult stem cells, which can differentiate into many mesodermal and neuroectodermal cells under the regulation of stem cell niche. However, there are few reports on whether the non-odontogenic stem cells can repair dentin defects by differentiation into odontoblast. In this study, bone marrow mesenchymal stem cells (BMSCs) of GFP transgenic mice were used to observe whether BMMSCs could differentiate into dentin cells and participate in the repair of dentin injury, and provide more active seed cells for tooth tissue engineering. Methods the whole bone marrow of transgenic mice with green fluorescent protein (GFP) was harvested. Bone marrow mesenchymal stem cells (BMSCs) of GFP transgenic mice were cultured in vitro by adherent method. Flow cytometry was used to identify the molecular markers on the surface of the cells. A green fluorescent bone marrow mesenchymal stem cell chimeric model of C57BL/6 mice was established by intravenous injection of green fluorescent bone marrow mesenchymal stem cells (C57BL/6) in vitro. Whether green fluorescent bone marrow mesenchymal stem cells were involved in the repair of dentin defect was observed by immunofluorescence assay, and the feasibility of using bone marrow mesenchymal stem cells as seed cells to repair dentin defects was discussed. Results the bone marrow mesenchymal stem cells (BMSCs) of GFP transgenic mice cultured with 1 / 1 were able to proliferate strongly. Flow cytometry was used to detect the positive surface molecules of stem cells and the expression of green fluorescence was stable. The whole bone marrow of the chimeric mouse model with the ability of adipogenic differentiation and osteogenesis in vitro was recultured and a large number of cells with green fluorescent labeling were found. There were also different numbers of green fluorescent cells in different tissues. The immunofluorescence showed that a large number of spontaneous green fluorescent cells appeared in dental pulp cavity of dental pulp of chimeric mouse model of dentin defect. Part of green fluorescent cells in dentine defect expressed dentin specific proteins DSP and DMP-1. Conclusion: in this study, a fluorescent chimeric mouse model of bone marrow mesenchymal stem cells (BMSCs) of green fluorescent protein transgenic mice with strong tracer ability was established to verify the ability of MSCs to differentiate into odontoblast cells in vivo. It was found that bone marrow mesenchymal stem cells could differentiate into odontoblast under the induction of microenvironment in vivo and participate in the repair of dentin defect, which could be used as stem cell source for tooth tissue engineering.
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R329;R78

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