杏仁核电点燃致痫大鼠神经肽Ghrelin水平表达变化的研究

发布时间：2018-10-16 11:02

【摘要】：目的观察神经肽Ghrelin在杏仁核电点燃大鼠外周血与脑组织中的变化，探讨其原因及意义。 方法健康雄性SD大鼠64只，随机分为点燃组（n=48）、假手术组（n=8）、空白组（n=8），点燃组以最后一次刺激为零点，再分为3h、6h、12h、24h、48h、72h六个亚组，每个亚组8只。点燃组植入电极后给予电刺激，制作杏仁核点燃膜型；假手术组只植入电极不予刺激；空白组不予处理。观察大鼠行为学及脑电图变化，采用ELISA、免疫组化和实时荧光定量PCR分别检测血清中和下丘脑中辛酰化Ghrelin表达情况。采用SPSS17.0统计软件进行数据分析，计量资料用均数±标准差（x s）表示，，采用单因素方差分析，组间比较用最小显著法（LSD-t）。 结果（1）杏仁核电点燃致痫大鼠癫痫发作情况：点燃大鼠在刺激最初表现为胡须抖动、点头，随着刺激增多，出现抽搐、前肢阵挛等症状，完全点燃时表现为后肢站立并跌倒，全身抽动，湿狗样抖动，发作时间约15s到数分钟。点燃组后放阈为（441.54±52.43）μA，点燃速度为（7.12±2.13）d。实验组大鼠均可达到ⅴ级发作。 （2）杏仁核电点燃致癫痫大鼠血清中辛酰基化Ghrelin的浓度变化：空白组与假手术组之间差异无统计学意义（P0.05），各时间点辛酰基化Ghrelin水平与假手术组比较均降低（P0.05）。各时间点组间比较，3h组与6h组、6h组与12h组、24h组与48h组、48h组与72h组比较，差异有统计学意义（P0.05），12h组与24h组比较差异无统计学意义（P0.05）。 （3）杏仁核电点燃致痫大鼠下丘脑中辛酰基化Ghrelin蛋白的表达：空白组与假手术组比较，差异无统计学意义（P0.05）；与假手术组比较，癫痫组各个时间点的辛酰基化Ghrelin表达水平均降低，各相邻组间比较3h组与6h组、6h组与12h组、12h组与24h组、24h组与48h组、48h组与72h组间比较，差异有统计学意义（P0.05）。 （4）癫痫发作后大鼠下丘脑Ghrelin mRNA的表达：空白组与假手术组比较，GhrelinmRNA相对表达量差异无统计学意义（P0.05）。各时间点组与假手术组比较，GhrelinmRNA表达水平均降低，差异有统计学意义（P0.05）；各时间点组间比较，3h组与6h组、6h组与12h组、12h组与24h组、24h组与48h组间比较，差异有统计学意义平（P0.05）；48h组与72h组比较，差异无统计学意义（P0.05）。 结论癫痫发作后，血清与下丘脑中神经肽Ghrelin水平均降低且变化趋势是一致的，通过检测血清中辛酰基化Ghrelin水平，可以反映下丘脑中相应神经肽的变化趋势，可能成为临床癫痫诊断和疗效评估的一种新型检测指标。
[Abstract]:Objective to observe the changes of neuropeptide Ghrelin in peripheral blood and brain tissue of rats ignited by nuclear amygdala. Methods Sixty-four healthy male SD rats were randomly divided into six subgroups: kindling group (n = 48), sham operation group (n = 8) and blank group (n = 8). In the igniting group, electrical stimulation was given after electrode implantation, and amygdala kindling membrane was made; in sham-operation group, only electrode implantation was not stimulated; blank group was not treated. The changes of ethology and electroencephalogram of rats were observed. The expression of octanoylated Ghrelin in serum and hypothalamus was detected by ELISA, immunohistochemistry and real-time fluorescence quantitative PCR. The data were analyzed by SPSS17.0 software. The measurement data were expressed by mean 卤standard deviation (x s), single factor analysis of variance (ANOVA) was used, and the least significant method (LSD-t) was used to compare the data between groups. Results (1) seizures of epileptic rats induced by nuclear amygdaloid kindling: the initial symptoms of epileptic rats induced by amygdaloid kindling were whiskers shaking, nodding, with the increase of stimuli, convulsions, clonus of forelimbs, and standing and falling of hind limbs when the rats were completely kindled. The whole body twitches, wet dog-like shake, attack time about 15 s to a few minutes. In the kindling group, the posterior release threshold was (441.54 卤52.43) 渭 A, and the ignition speed was (7.12 卤2.13) days. (2) the concentration of octanoylated Ghrelin in serum of epileptic rats induced by amygdaloid kindling: there was no significant difference between blank group and sham operation group (P0.05). Compared with sham operation group, the level of Ghrelin decreased (P0.05). The comparison was made between 3 h group and 6 h group, 6 h group and 12 h group, 24 h group and 48 h group, 48 h group and 72 h group. The difference was statistically significant (P0.05). There was no significant difference between 12h group and 24h group (P0.05). (3). The expression of octanoylated Ghrelin protein in hypothalamus of epileptic rats induced by nuclear amygdaloid kindling had no significant difference (P0.05). Compared with sham operation group, the expression of octanyl Ghrelin in epileptic group was decreased at all time points. The expression of octoyl Ghrelin was compared between 3 h group and 6 h group, 6 h group and 12 h group, 12 h group and 24 h group, 24 h group and 48 h group, 48 h group and 72 h group. The difference was statistically significant (P0.05). (4) the expression of Ghrelin mRNA in hypothalamus of rats after seizure: compared with sham operation group, there was no significant difference in the relative expression of GhrelinmRNA between blank group and sham operation group (P0.05). Compared with sham operation group, the expression of GhrelinmRNA in each time point group was significantly lower than that in sham operation group (P0.05), and the difference was statistically significant between 3h group and 6h group, 6h group and 12h group, 12h group and 24h group, 24h group and 48h group (P0.05). There was no significant difference between 48 h group and 72 h group (P0.05). Conclusion the levels of neuropeptide Ghrelin in serum and hypothalamus are decreased and the trend is the same after seizure. The level of octanoylated Ghrelin in serum can reflect the change trend of neuropeptide in hypothalamus. It may be a new detection index for the diagnosis and evaluation of clinical epilepsy.
【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】：R742.1

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