无镁诱导神经元体外惊厥样放电后细胞微环境的变化

发布时间：2018-03-05 01:14

本文选题：神经元　切入点：星形胶质细胞　出处：《重庆医科大学》2012年硕士论文　论文类型：学位论文

【摘要】：目的无镁诱导体外培养神经元（neurons，N）放电，研究放电后N和星形胶质细胞(astrocytes，AST)共培养体系中脑源性神经营养因子（brain-derived neurotrophic factor， BDNF）、神经生长因子（nerve growth factor，NGF）、谷氨酸（glutamate，GLU）、白介素-1beta（interleukin-1beta,IL-1β）动态变化及乳酸脱氢酶（lacticdehydrogenase，LDH）含量。方法以纯化的胎鼠海马N和新生鼠AST为研究对象，按照是否经过短暂的无镁标准细胞外液处理诱导反复的自发性惊厥样放电，分为N对照组、N无镁组、N+AST对照组和N+AST无镁组。 ELISA方法测定惊厥后不同时间点细胞培养上清液中的BDNF、NGF、IL-1β含量；高效液相色谱法测定不同条件下各时间点细胞上清液中GLU含量；细胞培养上清液中LDH的变化采用全自动生化分析仪测定。结果（1）N经无镁细胞外液处理，恢复正常培养72h后，仍旧存在反复的自发性“惊厥样放电”。（2）N+AST经无镁处理后48h，AST的体积增大，突起及细胞数目增多。（3）N+AST对照组在24h、48h细胞上清液BDNF较N对照组24h、48h细胞上清液BDNF升高（p㩳0.05）。N+AST经无镁处理后，在12h、24h、48h分泌的BDNF均较N+AST对照组相同时间点的BDNF升高，尤其12h、24h升高明显（p㩳0.01）；而N经同样的无镁细胞外液处理后，各个时间点BDNF含量也有升高，但与N对照组比较，差异无统计学意义。N+AST经无镁处理后在24h分泌的BDNF较N无镁组24h分泌的BDNF升高（p㩳0.05）。（4）N+AST经无镁处理后，各个时间点NGF的分泌量与共培养对照组相同时间点相比均没有发现明显变化，而且各个时间点间NGF的含量也趋于平稳。（5）N+AST经无镁标准细胞外液处理后，细胞上清液中GLU的含量从6h开始就有增加，，在48h、72h含量均较对照组同时间点GLU含量显著性增高，差异有统计学意义（p㩳0.05）。（6）N+AST经过无镁诱导反复自发的惊厥样放电后，细胞上清液中IL-1β的含量在12h、24h、48h较对照组相同时间点IL-1β的含量显著性增高，差异具有统计学意义（p㩳0.05，p㩳0.01）。（7）N+AST共培养体系经无镁标准细胞外液处理后，72h细胞上清液中乳酸脱氢酶显著高于N+AST对照组（p㩳0.01）。结论（1）N+AST在正常培养情况下分泌的BDNF由N和AST共同参与，N可能起主要分泌作用。短暂无镁处理的N+AST，经诱导放电后BDNF的分泌显著增多，并且可能主要来自惊厥微环境活化的AST。（2）无镁诱导N放电对共培养体系中NGF的分泌影响较小。（3）无镁诱导N放电后与AST共培养微环境中GLU和IL-1β的分泌均显著性增多，并且IL-1β的变化比GLU出现较早。（4）体外惊厥微环境对N具有损伤作用，导致LDH漏出增多。
[Abstract]:Objective to induce neuronal neuronsn (NN) discharges in cultured neurons without magnesium. To study the dynamic changes of brain-derived neurotrophic factor (BDNF), brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), nerve growth factor (NGF), glutamate glutamate (GLU), interleukin-1 beta (IL-1 尾) and the content of lactate dehydrogenase (LDH) in the co-cultured system of N and astrocytes. Methods the purified fetal rat hippocampal N and newborn rat AST were used to induce recurrent spontaneous convulsion-like discharges according to whether they were treated with transient magnesium free standard extracellular fluid. They were divided into N control group, N mg free group, N AST control group and N AST without magnesium group. ELISA method was used to determine the content of BDNF NGF FN IL-1 尾 in the supernatant of cell culture at different time points after convulsion, and high performance liquid chromatography (HPLC) was used to determine the content of GLU in cell supernatant at different time points after convulsion. The changes of LDH in supernatant of cell culture were measured by automatic biochemical analyzer. Results after treated with Mg-free extracellular solution for 72 h, there was still repeated spontaneous "convulsive discharges". The volume of AST was increased 48 h after treatment without magnesium. The cell supernatant BDNF in the control group was significantly higher than that in the control group at 24 h or 48 h. The BDNF in the supernatant of the cell supernatant was significantly higher than that in the control group at 24 h or 48 h. After mg free treatment, the BDNF secreted by 0.05U 路N AST at 12h ~ 24h was significantly higher than that of N AST control group at the same time, especially at 12h / 24h. After treated with the same magnesium free extracellular solution, the content of BDNF increased at different time points, but there was no significant difference compared with the control group. The BDNF secreted by N AST at 24 hours after magnesium free treatment was higher than that of BDNF secreted at 24 hours in N mg free group. There was no significant change in the secretion of NGF at each time point after mg free treatment of 0.05N AST, and the content of NGF at each time point tended to be stable after treated with magnesium free standard extracellular fluid. The content of GLU in the supernatant was increased from 6 h to 72 h, and the content of GLU in the supernatant was significantly higher than that in the control group at 48 h and 72 h, and the difference was statistically significant. After repeated spontaneous convulsion-like discharges induced by mg free, the content of IL-1 尾 in cell supernatant was significantly higher than that in control group at 12h ~ 24h ~ 48h, and the difference was statistically significant. 0.05,p? The lactate dehydrogenase (LDH) in the supernatant of 0. 01 AST / 0. 01% N AST was significantly higher than that in the N AST control group after being treated with magnesium free standard extracellular fluid for 72 h. 0.01g. Conclusion the BDNF secreted by N AST in normal culture may play a major role in the secretion of N by N and AST. The secretion of BDNF after induced discharge of N ASTs treated with transient mg free treatment is significantly increased. [WT5HZ] [WT5 "BZ] [WT5" BZ] [WT5 "BZ]. And it may be that the activation of AST.2from convulsion microenvironment has little effect on the secretion of NGF in co-culture system, and that the secretion of GLU and IL-1 尾 in microenvironment of co-culture with AST is significantly increased after Mg-free N discharges, and the effect of Mg-free N discharges on the secretion of GLU and IL-1 尾 in co-cultured microenvironment is significant. The change of IL-1 尾 was earlier than that of GLU.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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