甲基泼尼松龙对体外培养许旺细胞分泌神经营养因子的研究

发布时间：2018-01-04 16:10

本文关键词：甲基泼尼松龙对体外培养许旺细胞分泌神经营养因子的研究　出处：《郑州大学》2009年硕士论文　论文类型：学位论文

【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 mm~3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5×10~4/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℃、CO_2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。
[Abstract]:Objective:
To study the effect of methylprednisolone on the secretion of neurotrophic factors from Schwann cells in vitro.
Method:
Two week old New Zealand rabbits were sacrificed after 6, with 75% alcohol for 20min, under sterile conditions, take the sciatic nerve and brachial plexus, microscope stripping epineurium after the nerve was cut into 1 mm~3 size, spacing of 1cm grown in culture flask paved gelatin; 7d after subculture, continue to apply trypsin digestion after 3D culture, combined with differential centrifugation and purified S-100 protein immunohistochemical staining of Schwann cell purity. The identification of Schwann cells with 5 x 10~4/ml single cell suspension, each hole 1ml in 4 well culture plate.
According to the different groups of each hole by adding methyl prednisolone concentration in culture medium: A hole for the control group, containing 20% calf serum anti double high glucose DMEM medium 2ml medium containing 0.1mg/L 2ml; DMEM B methyllprednisolone hole; the medium 2ml containing 1.0mg/L methylprednisolone prednisolone DMEM C hole in the 2ml. of the medium; the culture plates were placed in the temperature of 37 DEG DMEM containing 5.0mg/L methylprednisolone in D hole, the volume fraction of CO_2 in the training of 5% incubation, cell growth was observed every day; every 2D by counting the number of cells. Then the estimation of each 2D aspirate culture liquid are stored in the centrifuge tube and 4 DEG C refrigerator; draw clean, PBS repeatedly flushing the culture hole, then add the appropriate medium.
The two times before and after the calculation of the number of cells were subtracted, that increase the value of the number of cells, one way ANOVA was used for statistical comparison. The 20 culture liquid mixing, is divided into three parts, NGF, CNTF and GDNF respectively by ELISA method, quantitative determination, the two values were measured before and after subtraction. The added value of neurotrophic factor secretion, one way ANOVA was used for statistical comparison. The experimental data using SPSS13.0 statistical software, P < 0.05, the difference was statistically significant.
Result:
(1) the growth rate of Schwann cells in group B, C and D was faster than that in group A, and the secretion of three neurotrophic factors was significantly higher than that in group A (P < 0.05). There was no significant difference between B, C and D group (P > 0.05).
The test results of NGF were better than that of CNTF and GDNF (P < 0.01), and there was no significant difference in the secretion of CNTF and GDNF (P > 0.05).
Conclusion:
Methylprednisolone can directly act on Schwann cells to promote the proliferation of Schwann cells in vitro and the secretion of neurotrophic factors.
The dosage of methylprednisolone has no significant difference in promoting the proliferation of Schwann cells in vitro and the secretion of neurotrophic factors in vitro.
Methylprednisolone can promote the regeneration and repair of injured peripheral nerves by promoting the proliferation of Schwann cells and the secretion of neurotrophic factors in vitro.

【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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