MiR-132在癫痫发生过程中的作用及机制

发布时间：2018-03-02 14:47

本文选题：miR-132　切入点：antagomir　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：第一部分p-CREB/miR-132在颞叶癫痫患者及动物模型中的表达目的：探讨p-CREB和miR-132在难治性癫痫患者致痫灶的表达以及p-CREB和miR-132在氯化锂-匹罗卡品癫痫模型中海马组织中各时间点的表达规律。方法： 1.选择15例难治性癫痫患者颞叶手术切除标本和性别、年龄无差异的10例非癫痫患者颞叶组织标本。 2.选择成年SD大鼠诱导氯化锂-匹罗卡品模型，随机分为2组：正常对照组，癫痫模型组，根据癫痫进程分为7个亚组（6h，24h，3d，7d，14d，30d，60d）。 3.用westernblot技术和免疫组化技术检测p-CREB的表达水平，实时荧光定量PCR检测miR-132的表达水平。结果： 1.p-CREB在颞叶癫痫患者中及大鼠癫痫模型中各时间点表达均明显升高。 2.miR-132表达在颞叶癫痫患者组中表达降低（p＜0.05），在动物模型中24h、7d时间点miR-132表达显著升高（p＜0.05），其余时间点较正常组无显著性差异。结论：p-CREB/miR-132的表达癫痫起病的急性期和潜伏期表达升高，，呈平行关系，这条信号通路早期可能参与了癫痫的发生和发展过程。第二部分抑制miR-132的表达对大鼠癫痫发作的作用及机制目的：通过抑制miR-132的表达，研究其在癫痫发病机制中的相关作用。方法： 1.选择成年SD大鼠，分为两组Ant-132干预组和Scr阴性对照组，造模前预先立体定位脑室分别注射Ant-132和Scr，浓度分别为0.5nmol、1.0nmol、1.5nmol。2天后进行诱导氯化锂-匹罗卡品颞叶癫痫模型。分析其对癫痫模型造模过程的影响。 2.选择1.0nmol的Ant-132和Scr作为干预和控制浓度，分别获取24h、2w、1m组织标本。 3.行为学观测干预miR-132后，对大鼠癫痫发作的影响。NPY检测对苔藓纤维出芽的影响。Golgi染色检测在活体组织干预miR-132后对神经元树突形态的影响。结果： 1.在匹罗卡品造模的过程中，抑制miR-132表达可以延长诱导大鼠癫痫发作的时间，并与miR-132浓度呈正相关（p＜0.05）。 2.抑制miR-132可以有效降低慢性期癫痫的自发性发作次数（p＜0.05）。 3.抑制miR-132后，苔藓纤维出芽显著减少，逆转性降低CA3、CA1区树突出芽，降低该区树突的长度。（p＜0.05）结论：miR-132在癫痫的发生发展过程中起了重要作用。MiR-132为癫痫发病过程中重要的致病因子。抑制miR-132可能通过重塑MFs-CA3环路，减少神经环路的产生从而达到有效减缓癫痫发生的作用。
[Abstract]:The expression of p-CREBR / miR-132 in patients with temporal lobe epilepsy and animal models. Aim: to investigate the expression of p-CREB and miR-132 in epileptogenic foci in patients with intractable epilepsy and the expression of p-CREB and miR-132 in hippocampus of lithium-pilocarpine epileptic model. Methods:. 1. The temporal lobe tissue specimens of 15 patients with intractable epilepsy and 10 patients with no difference in age were selected. 2. Adult SD rats were selected to induce lithium-pilocarpine model and were randomly divided into two groups: normal control group and epileptic model group. 3. The expression of p-CREB was detected by westernblot and immunohistochemistry, and the expression of miR-132 was detected by real-time fluorescence quantitative PCR. Results:. 1. The expression of p-CREB was significantly increased in temporal lobe epilepsy patients and rat epileptic models at all time points. 2. The expression of miR-132 in the patients with temporal lobe epilepsy decreased significantly (P < 0.05), and the expression of miR-132 increased significantly at 24 h and 7 d in the animal model, but there was no significant difference between the other time points and the normal group. Conclusion the expression of the expression of the fraction p-CREB-miR-132 is increased in the acute phase and latent period of epileptic onset, which may be involved in the occurrence and development of epilepsy in the early stage. The second part: inhibitory effect of miR-132 expression on epileptic seizures in rats and its mechanism. Objective: to study the role of miR-132 in the pathogenesis of epilepsy by inhibiting its expression. Methods:. 1. Adult SD rats were divided into two groups: Ant-132 intervention group and Scr negative control group. The temporal lobe epilepsy model of lithium-pilocarpine was induced by intracerebroventricular injection of Ant-132 and scratch at a concentration of 0.5 nmol ~ 1.0 nmol ~ (-1) nmol ~ (2) 2 days before modeling, and the effects on the process of epileptic model were analyzed. 2.1.0 nmol of Ant-132 and Scr were selected as the intervention and control concentration, and the tissue samples of 24 h and 2 weeks were obtained, respectively. 3.Behavioral observation after intervention in miR-132, the effect of NPY on the sprouting of mossy fibers in rats. Golgi staining was used to detect the effect of miR-132 in vivo on the morphology of neuronal dendrites. Results:. 1. In the course of pilocarpine modeling, inhibiting the expression of miR-132 could prolong the time of epileptic seizure induced by pilocarpine in rats, and had a positive correlation with the concentration of miR-132 (p < 0.05). 2. Inhibition of miR-132 can effectively reduce the number of spontaneous seizures in chronic epilepsy (p < 0.05). 3. After inhibiting miR-132, the sprouting of moss fiber decreased significantly, the reverse effect decreased, and the length of dendrites in CA3 / CA1 region was decreased, and the length of dendrites in this area was decreased (P < 0.05). Conclusion: miR-132 plays an important role in the occurrence and development of epilepsy. MiR-132 is an important pathogenic factor in the pathogenesis of epilepsy. The inhibition of miR-132 may reduce the generation of neural loop by remodeling the MFs-CA3 loop and thus reduce the occurrence of epilepsy effectively.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

【参考文献】