红细胞生成素对体外培养许旺细胞分泌神经营养因子和细胞周期的影响

发布时间：2018-04-24 13:15

本文选题：红细胞生成素 + 许旺细胞　；参考：《郑州大学》2009年硕士论文

【摘要】：目的观察和评价红细胞生成素(erythropoietin,EPO)对体外培养许旺细胞(Schwann cells,SCs)分泌神经营养因子和细胞周期的影响,探讨EPO促进周围神经再生的机制。方法选用6只两周龄的新西兰兔作为实验动物,经75%酒精浸泡20min消毒,无菌条件下取双侧坐骨神经和臂丛神经,在显微镜下剥干净神经外膜,将神经剪成0.5cm×0.5cm×0.5cm的小植块,用植块法+酶消化法+差速贴壁法培养纯化许旺细胞。实验设对照组(A组):20%胎牛血清DMEM液;实验组1(B组):20%胎牛血清DMEM液+0.5U/ml EPO;实验组2(C组):20%胎牛血清DMEM液+5U/ml EPO。 1.于细胞培养第24h、48h、72h分别对各组许旺细胞应用酶联免疫吸附试验(ELISA)测定培养上清液中的神经生长因子(NGF)和睫状神经营养因子(CNTF)。 2.于细胞培养第24h分别对各组许旺细胞进行流式细胞术检测细胞周期分布,比较SCs处于S期百分比(S%)和增殖指数PI值(S+G_2M)%。结果 1.许旺细胞培养第24h、48h、72h,在各时间点细胞培养液中NGF的含量,A组与B组、A组与C组之间差异有统计学意义(P＜0.05),B组与C组之间差异有统计学意义(P＜0.05);在各时间点细胞培养液中CNTF的含量,A组与B组、A组与C组之间差异有统计学意义(P＜0.05),B组与C组之间差异有统计学意义(P＜0.05)。 2.许旺细胞培养第24h,细胞处于S期百分比(S%),A组与B组、A组与C组之间差异有统计学意义(P＜0.05),B组与C组之间差异有统计学意义(P＜0.05):细胞增殖指数PI值(S+G_2M)%,A组与B组、A组与C组之间差异有统计学意义(P＜0.05),B组与C组之间差异有统计学意义(P＜0.05)。结论 1.EPO可促进体外培养许旺细胞分泌NGF和CNTF,且具有剂量依赖性; 2.EPO可影响体外培养许旺细胞的细胞周期,提高许旺细胞的增殖活性; 3.EPO可提高体外培养许旺细胞分泌神经营养因子的水平,并且能提高许旺细胞的增殖活性,这可能是其促进周围神经再生的作用机制之一。
[Abstract]:Objective to observe and evaluate the effect of erythropoietin erythropoietin (EPO) on the secretion of neurotrophic factor and cell cycle in Schwann cells of Schwann cells in vitro, and to explore the mechanism of EPO promoting peripheral nerve regeneration. Methods six two-week-old New Zealand rabbits were used as experimental animals. The sciatic nerve and brachial plexus nerve were removed under sterile condition by 75% alcohol immersion in 20min. The nerve was cut off the epineurium under the microscope, and the nerve was cut into the 0.5cm 脳 0.5cm 脳 0.5cm graft. Schwann cells were cultured and purified by enzyme digestion method. The experimental group was divided into two groups: control group (n = 20), control group (n = 20), control group (n = 20), control group (n = 1), group B (n = 1), group B (n = 20), 0.5U/ml (n = 20), and group 2 (n = 2), group C (n = 20), 5U/ml (n = 20). 1. The nerve growth factor NGF (NGF) and ciliary neurotrophic factor (CNTFN) in the supernatant of Schwann cells were determined by enzyme-linked immunosorbent assay (Elisa) at 24 h and 48 h and 72 h after cell culture respectively. 2. The cell cycle distribution of Schwann cells in each group was detected by flow cytometry at 24 hours after cell culture. The percentage of SCs in S phase was compared with the Pi value of proliferative index (Pi). Result 1. The content of NGF in the culture medium of Schwann cells was significantly different between group A and group B at 24 h and 48 h and 72 h after culture. There was significant difference between group A and group B and group C (P < 0.05) and between group B and group C (P < 0.05), and between group A and group B (P < 0.05), and between group B (P < 0.05) and group C (P < 0.05). The content of CNTF was significantly different between group A and group B (P < 0.05) and between group B (P < 0.05) and group C (P < 0.05). 2. At the 24h of Schwann cell culture, the percentage of cells in S phase was significantly different between group A and group B (P < 0.05) and group C (P < 0.05). The Pi value of cell proliferation index (Pi) was higher than that of group A and B (P < 0.05). The difference between group C and group C was statistically significant (P < 0.05) and the difference between group B and group C was statistically significant (P < 0.05). Conclusion 1.EPO can promote the secretion of NGF and CNTF by Schwann cells in vitro in a dose-dependent manner. 2.EPO can affect the cell cycle of Schwann cells cultured in vitro and increase the proliferation activity of Schwann cells. 3.EPO can increase the level of neurotrophic factor secreted by Schwann cells in vitro and increase the proliferative activity of Schwann cells which may be one of the mechanisms of promoting peripheral nerve regeneration.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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