原代培养的嗅鞘细胞与异种神经支架共培养研究

发布时间：2018-06-03 06:46

本文选题：嗅鞘细胞 + 原代培养　；参考：《宁夏医科大学》2014年硕士论文

【摘要】：目的1、采用改良差速贴壁联合胰酶限时消化的方法培养大鼠乳鼠的嗅鞘细胞，并对细胞分泌的神经生长因子进行检测。2、将原代培养的嗅鞘细胞与异种神经支架在体外共同培养，观察在体外环境下支架对细胞的生长是否有影响。方法实验一：取10只SD大鼠的乳鼠（鼠龄7d），取乳鼠的嗅球，应用改良差速贴壁联合胰酶限时消化3min的方法培养细胞；对细胞进行神经生长因子的检测，细胞密度调整到密度1×106/ml，接种到24孔板内，每组密度又分为3d组、5d组、7d组、9d组，神经生长因子酶联反应检测不同时间点细胞在分泌生长因子量上的差异。实验二：萃取异种神经（青紫蓝兔胫神经）支架，对萃取后神经进行HE染色、S-100、Laminin免疫组织化学染色；在体外将嗅鞘细胞与异种神经支架进行共培养，通过扫描电镜观察细胞与支架的共存情况；神经生长因子酶联反应检测单纯嗅鞘细胞（对照组）、嗅鞘细胞与支架共培养（实验组）在3d、5d、7d、9d不同时间点上神经生长因子分泌上是否存在差异。结果实验一：应用改良差速贴壁联合胰酶限时消化3min的方法能够培养出的较高浓度的嗅鞘细胞，细胞纯度能够达到70%；统计分析数据经胰酶限时消化3min后的嗅鞘细胞分泌的神经生长因子含量随时间的延长而逐渐减少。胰酶消化后第3d神经生长因子分泌的含量最高（P=0.012P＜0.05）。实验二：萃取后的异种神经支架镜下未见雪旺氏细胞和髓鞘结构，神经基底膜、内膜、束膜和外膜均存在；嗅鞘细胞能够贴附生长在异种神经支架上；实验组与对照组在3d、5d、7d、9d四个不同时间点神经生长因子的分泌量上的差异均无统计学意义（P＞0.05）结论1、联合培养嗅鞘细胞的方法能获得更高浓度的嗅鞘细胞，细胞浓度能够达到70%，能够满足后期移植对细胞的要求。2、联合培养的嗅鞘细胞在细胞生长到大约10d（胰酶限时消化后3d）的时候分泌的神经生长因子量最多。3、萃取后的异种神经支架能够满足作为载体支架的要求。4、原代培养的嗅鞘细胞能够抓附在神经支架上生长。5、嗅鞘细胞与支架共同培养后在神经生长因子分泌量上没有受到影响，细胞能够正常分泌生长因子，异种神经支架不干扰细胞的正常生长。
[Abstract]:Objective 1. To culture olfactory ensheathing cells of neonatal rats by modified differential adherent method combined with trypsin restriction digestion, and to detect nerve growth factor (NGF) secreted by the cells. The primary cultured olfactory ensheathing cells were co-cultured with xenogeneic nerve scaffolds in vitro. To observe the effect of scaffold on cell growth in vitro. Methods: 10 SD rats (7 days old, olfactory bulb) were taken from 10 SD rats, the cells were cultured by modified differential adherent method combined with trypsin time limit digestion, and the nerve growth factor (NGF) was detected. The cell density was adjusted to 1 脳 106 / ml and inoculated into 24 hole plate. Each group was divided into 3 d group, 5 d group, 7 d group and 9 d group. Experiment 2: extraction of xenogeneic nerve (blue rabbit tibial nerve) scaffold, HE staining of nerve after extraction, S-100 laminin immunohistochemical staining, co-culture of olfactory ensheathing cells and xenogeneic nerve scaffold in vitro, The coexistence of cell and scaffold was observed by scanning electron microscope. Nerve growth factor enzyme-linked reaction was used to detect whether there were differences in the secretion of nerve growth factor (NGF) at different time points (control group, olfactory ensheathing cell and scaffold culture) at different time points (3 d ~ 5 d ~ 7 d ~ 9 d). Results in experiment 1, a high concentration of olfactory ensheathing cells could be cultured by using the modified differential adherent method combined with trypsin to digest 3min. The content of NGF secreted by olfactory ensheathing cells after digesting 3min by trypsin was gradually decreased with the increase of time. On the 3rd day after trypsin digestion, the content of nerve growth factor (NGF) was the highest (P < 0.05). Experiment 2: there were no Schwann cells and myelin sheath structure, nerve basement membrane, intima, bundle membrane and adventitia, olfactory ensheathing cells could be attached to the xenogeneic nerve scaffold under the extraction, and the olfactory ensheathing cells could grow on the xenogeneic nerve scaffold. There was no significant difference in the secretion of nerve growth factor between the experimental group and the control group on the 3rd day, 5th day, 7d and 9th day (P > 0.05). Conclusion 1. Higher concentration of olfactory ensheathing cells can be obtained by co-culture of olfactory ensheathing cells. The cell concentration can reach 70%, which can meet the cell requirement of late transplantation. The co-cultured olfactory ensheathing cells secrete nerve growth factor (NGF) at about 10 days (trypsin limit, 3 days after digestion). The xenogeneic nerve scaffold could meet the requirements of the carrier scaffold. The primary cultured olfactory ensheathing cells could grow on the nerve scaffold, and the olfactory ensheathing cells were not affected in the secretion of nerve growth factor after co-culture with the scaffold. The cells secreted growth factors normally, and the xenogeneic nerve scaffolds did not interfere with the normal growth of the cells.
【学位授予单位】：宁夏医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R745

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