神经干细胞体外培养体系的建立

发布时间：2018-06-18 20:50

本文选题：神经干细胞 + 饲养层　；参考：《广西医科大学》2007年硕士论文

【摘要】： 目的:探讨替代细胞因子培养神经干细胞的适合技术和条件,建立神经干细胞体外培养体系,降低实验成本,为建立细胞资源库打下基础。方法: 1.分离海马制成单细胞悬液。用含2%B27的DMEM/F12无血清培养液(bFGF+EGF各20ng/ml)培养细胞。采用Nestin的免疫细胞化学方法鉴定原代和传代细胞中的NSCs。免疫细胞化学方法检测诱导分化细胞的β-Tublin、GFAP。采用Brdu标记原代神经干细胞,传代后检测Brdu。采用Brdu标记神经干细胞,并进行同质小鼠大脑海马和侧脑室内移植,检测细胞存活情况。 2.选择胚胎成纤维细胞、Sertoli细胞、骨髓基质细胞,建立细胞培养体系,使用10μg/ml的丝裂霉素C制备饲养层。 3.实验分为五组,倒置显微镜下观察计数细胞,并绘制生长曲线。结果: 1.添加细胞因子培养可以形成大量的悬浮方式生长的细胞球。细胞球经过多次传代可以形成次代细胞球。原代、次代细胞经免疫细胞化学染色检测Nestin,结果为阳性。免疫细胞化学染色分别检测分化细胞的β-Tublin、GFAP,结果为阳性。采用Brdu标记神经干细胞,免疫细胞化学检测Brdu,原代、次代NSC均为阳性。体内移植7d后在海马和侧脑室可检测到Brdu标记阳性的细胞。 2.分离培养小鼠胚胎成纤维细胞、Sertoli细胞及骨髓基质细胞,并用丝裂霉素C处理,成功制备饲养层。 3.三种饲养层都能使NSC增殖,在胚胎成纤维细胞、骨髓基质细胞饲养层上的生长状况优于Sertoli细胞饲养层。细胞因子组培养效果优于饲养层组,对照组细胞分化明显。结论: 1.新生昆明小鼠大脑海马能分离出NSC。 2.NSC大脑内移植能继续存活。 3.NSC细胞在三种饲养层上均能稳定的增殖并保持未分化状态,在胚胎成纤维细胞、骨髓基质细胞饲养层上的生长状况优于Sertoli细胞饲养层。 4.细胞因子组培养NSC效果优于饲养层组,但细胞因子的价格昂贵,大规模培养NSC成本过高。饲养层组可较好地模拟体内细胞生长微环境,且培养费用低廉、增殖稳定等方面较细胞因子组有优势,适宜在普通实验室推广。
[Abstract]:Objective: to explore the suitable techniques and conditions for alternative cytokine culture of neural stem cells, to establish the culture system of neural stem cells in vitro, to reduce the experimental cost, and to lay a foundation for the establishment of cell resource bank. Methods: 1. The hippocampus was isolated and made into single cell suspension. The cells were cultured in the serum-free medium of DMEM / F _ (12) containing 27% bFGF EGF (20 ng / ml). Nestin immunocytochemistry was used to identify NSCsin primary and passage cells. Immunocytochemical method was used to detect 尾 -Tublinus GFAPs. Primary neural stem cells (NSCs) were labeled with Brdu. Neural stem cells (NSCs) were labeled with Brdu and transplanted into hippocampus and lateral ventricle of homogenous mice. Sertoli cells and bone marrow stromal cells were selected from embryonic fibroblasts to establish a cell culture system. The feeder layer was prepared with mitomycin C of 10 渭 g/ml. The experiment was divided into five groups. The count cells were observed under inverted microscope and the growth curve was drawn. Results: 1. The addition of cytokines can form a large number of suspension growth cells. The cell ball can form the secondary cell ball after multiple passages. Nestin was detected by immunocytochemical staining in primary and secondary cells. The 尾-Tublinus GFAPs of differentiated cells were detected by immunocytochemical staining, and the results were positive. Brdu labeled neural stem cells were used to detect Brdu. primary and secondary NSC were all positive by immunocytochemistry. Brdu labeled positive cells were detected in hippocampus and lateral ventricle 7 days after transplantation in vivo. 2. 2. Sertoli cells and bone marrow stromal cells were isolated from mouse embryonic fibroblasts and treated with mitomycin C. All the three feeding layers could make NSC proliferate. The growth of NSC in the feeder layer of embryonic fibroblasts and bone marrow stromal cells was better than that of Sertoli cell feeder layer. The culture effect of cytokine group was better than that of feeder layer group, and the cell differentiation of control group was obvious. Conclusion: 1. NSC2. 2. NSC can survive. 3. NSC cells can proliferate stably and remain undifferentiated in the three feeder layers, and NSC cells can be transplanted into embryonic fibroblasts. The growth status of bone marrow stromal cell feeder layer was better than that of Sertoli cell feeder layer. 4. The effect of cultured NSC in cytokine group was better than that in feeder layer group, but the price of cytokines was high, and the cost of large scale culture of NSC was too high. The feeding layer group can simulate the microenvironment of cell growth in vivo, and the culture cost is low, the proliferation is stable and so on, which is suitable to be popularized in the common laboratory.
【学位授予单位】：广西医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R329

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