【作者】 朱旭；

【导师】 吴学建；

【作者基本信息】 郑州大学 ， 骨科学， 2009， 硕士

【摘要】 目的:研究甲基泼尼松龙对体外培养许旺细胞分泌神经营养因子的促进作用。方法:两周龄新西兰大白兔6只处死后,用75%酒精浸泡20min,无菌条件下取双侧坐骨神经和臂丛神经,显微镜下仔细剥除神经外膜后,将神经剪成1 mm³大小,间距1cm种植于预先铺过明胶的培养瓶中;7d后传代,继续培养3d后应用胰酶消化,联合差速贴壁法纯化,并S-100蛋白免疫组化染色检查许旺细胞纯度。鉴定过的许旺细胞配成5×10⁴/ml单细胞悬液,每孔1ml加入4孔培养板中。根据各孔所加入培养液中甲基泼尼松龙浓度的不同进行分组:A孔为对照组,加入含20%小牛血清的双抗高糖DMEM培养液2ml;B孔中加入含0.1mg/L甲基泼尼松龙的DMEM培养液2ml;C孔中加入含1.0mg/L甲基泼尼松龙的DMEM培养液2ml;D孔中加入含5.0mg/L甲基泼尼松龙的DMEM培养液2ml。将培养板置于37℃、CO₂体积分数为5%的恒温培养箱内培养,每天观察细胞生长情况;每2d用随即计数法估算细胞数。每2d吸出培养液分别存于离心管中并4℃冰箱保存;吸取干净后,PBS反复冲洗培养孔,再加入相应培养液。将前后两次计算的细胞数量分别相减,得出细胞数量的增加值,应用单因素方差分析进行统计学比较。将获得的20管培养液混匀,三等分后,用ELISA法分别做NGF、CNTF、GDNF定量测定,将前后两次的测量值分别相减,得出神经营养因子分泌量的增加值,应用单因素方差分析进行统计学比较。实验数据采用spss13.0统计学软件处理,取P＜0.05为差异有统计学意义。结果:①B、C、D组许旺细胞生长速度明显快于A组,三种神经营养因子分泌量明显多于A组,统计学有显著差异（P＜0.05）,B、C、D组之间无显著差异（P＞0.05）;②NGF的检测结果有优于CNTF、GDNF的趋势（P＜0.01）,CNTF和GDNF的促分泌情况无明显差异（P＞0.05）。结论:甲基泼尼松龙能直接作用于许旺细胞来促进体外培养许旺细胞的增殖和神经营养因子的分泌。甲基泼尼松龙的用量在促进体外培养许旺细胞的增殖和神经营养因子的分泌方面无明显差异。甲基泼尼松龙能通过促进体外培养许旺细胞的增殖和神经营养因子的分泌来促进损伤周围神经的再生和修复。更多还原

【Abstract】 Objective:To research the effects of Methylprednisolone on Schwann cells in vitro of secreting the neurotrophic factor.Methods:Chose 6 New Zealand rabbits of 2 weeks-old, killed and soak in ethyl alocohol of 75% for 20 minutes. Procurexing the ischiadic nerve and brachial plexus of both sides under asepsis, striping the epineurium and cutting to be chips of 1 mm3, and then grow in the culture flask which spreading by gelatine with 1 cm distance. Passage after 7days, go on cultivanting 3 days, then trypsinizating and depuration by differential adhesion way. Accredit the purity of cells with S-100 protein-immunohisto-chemistry stain, and preparing to be cell suspension of 5×10⁴ /ml, load into 4-hole cultivation board with 1 ml/hole.According to the concentration of the drug, there was divided into four groups: A group, control group, only giving DMEM- culture fluid 2ml; B group, adding 0.1mg / L methylprednisolone into the culture fluid; C group, adding 1.0mg/L methylprednisolone into the culture fluid; D group, to join 5.0mg/L methylprednisolone into the culture fluid. Cultivate in the homeothermia incubator of 37℃with 20% CO₂. Observe the cell growth and estimate the cell number. Every two days, draw-off the culture fluid and keep in the refrigerator of 4℃, wash the board by PBS, then join the new culture fluid with different group.Calculate the increase value of the cell number, get the quantitative assay of NGF、CNTF、GDNF by ELISA, and Calculate the increase value. The statistics test was carried out with one-side variance analysis by SPSS 13.0, choosing statistical significance of P<0.05.Results:①B, C, D groups Schwann cells faster than the growth rate of Group A, three kinds of neurotrophic factor secreted significantly more than group A, statistically significant differences （P <0.01）, B, C, D Group Between the secretion was no significant difference between the number of （P> 0.05）;②NGF of the test results are better than that of CNTF, GDNF trend （P <0.01）, CNTF and GDNF promote the secretion of the situation there was no significant difference （P> 0.05）.Conclusion:Methylprednisolone can promote the proliferation of Schwann cells in vitro and the secretion of neurotrophic factor. Application of low-dose and high-dose has no significant differences.Methylprednisolone can promote the regenerate and repair of peripheral nerve by promoting the proliferation of Schwann cells and the secretion of neurotrophic factor.更多还原