胶质细胞源性神经营养因子体外诱导骨髓间充质干细胞向神经细胞分化的研究

发布时间：2018-01-06 21:23

本文关键词：胶质细胞源性神经营养因子体外诱导骨髓间充质干细胞向神经细胞分化的研究　出处：《扬州大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的：探讨体外分离、培养大鼠骨髓间充质干细胞(MSCs)的方法,证实提纯的骨髓间充质干细胞与胶质细胞源性神经营养因子(GDNF)共培养,可定向诱导分化为神经细胞。方法：1.选用健康SD大鼠,采用全骨髓贴壁法体外培养、纯化MSCs。2.采用流式细胞术进行细胞表型鉴定,测定CD34、CD44、CD45、CD90的阳性细胞百分率。3.分别采用对照组、GDNF组对MSCs进行诱导分化研究。4.采用倒置相差显微镜连续观察细胞生长情况及形态变化。采用免疫组化法检测神经前体细胞的特异性标志神经巢蛋白(Nestin),神经细胞的特异性标志神经元特异性烯醇化酶(neon-specific enolase, NSE)。结果：原代提取获得的骨髓间充质干细胞呈梭形,类似于成纤维细胞,培养第4代的MSCs形态均一性较好,经流式细胞仪检测纯度较高,四种细胞表型的阳性率分别是：CD34:3.98%, CD44:98.74%, CD45:1.03%, D90:98.08%。MSCs经GDNF诱导后,细胞胞体逐渐向胞核收缩,变形细胞逐渐增多,出现双极、多极和锥形典型的神经细胞形态,细胞边缘出现突起,且与邻近细胞突起逐渐相互连接。GDNF组分别于诱导后6h出现nestin表达阳性,24h后诱导达顶峰。12h检测到神经细胞的特异性标志NSE表达阳性,且表达强度随时间逐渐增加,48h后诱导达顶峰。72h观察到诱导细胞逐渐凋亡脱落。阴性对照组未出现神经细胞的形态变化及特异性标志的阳性表达。结论：大鼠骨髓间充质干细胞可通过全骨髓贴壁法在体外进行原代提取分离及传代培养,所获得细胞生长稳定、增殖迅速,经GDNF诱导后具有向神经元样细胞分化的潜能。
[Abstract]:Objective: To explore the method of isolation and culture of rat bone marrow mesenchymal stem cells (MSCs) in vitro. It is confirmed that purified bone marrow mesenchymal stem cells and glial cell line derived neurotrophic factor (GDNF) co cultured, can be induced to differentiate into neural cells.
Methods: 1. healthy SD rats by whole bone marrow adherent culture, purification of MSCs.2. by phenotype by flow cytometry, the determination of CD34, CD44, CD45, CD90 and.3. respectively by the percentage of positive cells in control group, GDNF group of MSCs induced differentiation of.4. by inverted phase contrast microscope observation the cell growth and morphological changes. The immune group specific marker nestin was detected in neural precursor cells (Nestin), a specific marker of neuron specific enolase in nerve cells (neon-specific enolase, NSE).
Results: the primary extraction of bone marrow mesenchymal stem cells were spindle shaped, similar to fibroblast cells, cultured MSCs morphological homogeneity of the fourth generation of good, high purity was detected by flow cytometry, the positive rate of four kinds of cell phenotypes were CD34:3.98%, CD44:98.74%, CD45: 1.03%, D90:98.08%.MSCs induced by GDNF the cell body, gradually to nucleus shrinkage, deformation of cells gradually increased, appeared bipolar, multipolar and typical conical nerve cell morphology, cell edge projection, and the adjacent cell processes gradually connected with each other in group.GDNF were induced after 6h nestin positive expression reached the peak detected by.12h nerve cells induced by 24h after specific marker NSE expression, and the expression increased gradually with time, reached a peak at 48h after induction of.72h induced cell apoptosis observed gradually fall off. The negative control group did not show morphological changes of nerve cells. Positive expression of specific markers.
Conclusion: rat bone marrow mesenchymal stem cells can be extracted, separated and cultured in vitro by whole bone marrow adherent method. The cells obtained are stable and proliferated rapidly. After GDNF induction, they have the potential to differentiate into neuron like cells.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329.2

【参考文献】