MiRNAs调控BDNF-TrkB通路抑制神经元痫样放电的机制研究

发布时间：2018-03-22 19:22

本文选题：癫痫　切入点：酪氨酸激酶受体B　出处：《天津医科大学》2016年博士论文　论文类型：学位论文

【摘要】：研究目的:Micro RNA(mi RNA)是一类内源性小分子非编码RNA,作为转录后调控因子,参与癫痫发病机制的多个蛋白编码基因的表达。脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)-酪氨酸激酶受体B(tropomyosin-related kinase receptor type B,Trk B)信号通路是癫痫发生的关键通路,选择性中枢神经系统内敲除Trk B基因能完全阻止点燃诱发的癫痫发作。有关研究发现mi R-204-5p(mi R-204)能通过调控Trk B受体影响肿瘤的发生和转移,深入研究mi R-204是否能通过Trk B信号通路调控癫痫的发生,有望为临床癫痫治疗提供新的思路。根据上述研究背景提出本论文的研究目的如下:1.以原代培养的大鼠海马神经元的痫样放电模型为研究对象,通过Trk B受体及其下游信号通路,研究mi R-204抑制神经元痫样放电和电压门控性Ca2+通道(voltage-gated calcium channel,VGCC)电流的机制。2.在mi R-204抑制神经元痫样放电的研究基础上,选取前期研究的在癫痫神经元模型中异常表达并且与BDNF-Trk B通路相关的mi R-132-3p(mi R-132)为靶点,研究过表达mi R-132后神经元痫样放电和VGCC电流的改变,为探索其他类型mi Rs在癫痫发病机制中的作用提供创新思路与方法。研究方法:1.原代培养的海马神经元经无镁细胞外液处理3h,建立神经元痫样放电模型。2.Mi R-204调控Trk B信号通路抑制神经元痫样放电的机制研究。(1)通过实时荧光定量PCR(real-time quantitative polymerase chain reaction,q RT-PCR)检测神经元痫样放电模型中mi R-204的表达改变。(2)应用包含mi R-204-p GLVH1/GFP的慢病毒载体转染原代培养的海马神经元以过表达mi R-204,通过q RT-PCR检测过表达mi R-204后Trk B m RNA的表达改变。(3)Trk B下游通路的代表性信号分子包括Akt,PLCγ1,ERK1/2和CREB。通过western blotting以这些信号分子的磷酸化蛋白水平和总蛋白水平的比值反映信号分子的激活程度,研究神经元痫样放电模型模型中mi R-204调控Trk B下游信号分子活性的相关机制。(4)应用全细胞膜片钳电流钳技术检测神经元痫样放电模型的放电特性,神经元痫样放电模型过表达mi R-204后检测痫样放电频率的变化。(5)应用全细胞膜片钳电压钳技术检测神经元痫样放电模型VGCC电流和稳态激活与失活特性的变化,神经元痫样放电模型过表达mi R-204后VGCC特性的改变。3.Mi R-132调控BDNF影响神经元痫样放电的机制研究。为研究其他mi Rs在癫痫发病机制中的作用是否一致,选取前期研究发现在神经元痫样放电模型中表达上调的mi R-132为靶点,研究mi R-132对神经元痫样放电和VGCC电流的调控,并进一步探讨其调控机制。(1)通过q RT-PCR检测神经元痫样放电模型中BDNF m RNA的表达,慢病毒转染过表达mi R-132后观察BDNF m RNA的表达改变,研究mi R-132调控BDNF表达的机制。(2)应用全细胞膜片钳电流钳技术检测神经元痫样放电模型分别过表达mi R-132和孵育外源性BDNF后痫样放电频率的变化。(3)应用全细胞膜片钳电压钳技术检测神经元痫样放电模型分别过表达mi R-132和孵育外源性BDNF后VGCC电流特性的改变。研究结果:1.Mi R-204调控Trk B信号通路抑制神经元痫样放电。(1)神经元痫样放电模型中mi R-204的表达下调:与正常对照组神经元相比,癫痫模型组中mi R-204的表达水平明显下降(P0.001,n=6)。(2)Mi R-204负调控Trk B受体m RNA的表达:Trk B m RNA的表达水平在癫痫模型组和对照组无明显差异(P0.05,n=6)。癫痫模型组过表达mi R-204后Trk B m RNA表达明显降低(P0.001,n=6)。(3)Mi R-204抑制Trk B受体下游信号通路的活性:神经元痫样放电模型中Akt信号通路活性无明显改变,PLCγ1(P0.001,n=6)、ERK1/2(P0.001,n=6)以及CREB(P0.001,n=6)信号活性均明显增强。在神经元痫样放电模型中过表达mi R-204后PLCγ1信号活性无明显改变(P0.05,n=6),而ERK1/2信号活性(P0.01,n=6)以及CREB信号活性(P0.001,n=6)均明显降低。证明mi R-204主要调控Trk B受体下游的ERK1/2-CREB信号通路。(4)Mi R-204抑制神经元的痫样放电:全细胞电流钳检测正常对照神经元表现为偶发的动作电位发放;而癫痫模型组表现为持续高频的动作电位发放,放电频率比正常对照组明显增高(P0.001,n=14)。癫痫模型组过表达mi R-204后神经元的痫样放电频率明显降低(P0.001,n=14)。(5)Mi R-204抑制神经元痫样放电模型中神经元VGCC电流和活性:神经元痫样放电模型VGCC的最大电流密度增加(P0.001,n=10),稳态激活曲线的半数激活电压降低(P0.01,n=10),稳态失活曲线的半数失活电压升高(P0.01,n=10),表明神经元痫样放电模型VGCC电流增强,通道更易激活难以失活。神经元痫样放电模型过表达mi R-204后,最大电流密度降低(P0.001,n=10),稳态激活曲线的半数激活电压增高(P0.05,n=10),稳态失活曲线的半数失活电压降低(P0.01,n=10),说明mi R-204抑制神经元痫样放电模型中神经元的VGCC电流和活性。2.神经元痫样放电模型中mi R-132抑制BDNF加重痫样放电。(1)在神经元痫样放电模型中mi R-132抑制BDNF m RNA的表达:癫痫模型中BDNF m RNA的表达水平明显上调(P0.001,n=6)。对照神经元过表达mi R-132后BDNF m RNA的表达水平明显增高(P0.001,n=6),而神经元痫样放电模型过表达mi R-132后BDNF m RNA的表达水平明显下降(P0.001,n=6)。(2)BDNF抑制神经元痫样放电,而mi R-132逆转这一作用加重神经元痫样放电:癫痫模型组过表达mi R-132后放电频率明显增高(P0.01,n=14)。癫痫模型组孵育BDNF后放电频率明显降低(P0.001,n=14),但在此基础上过表达mi R-132后放电频率又增高到癫痫模型组水平(Mg2+-free+BDNF+mi R-132:Mg2+-free=7.11±2.90:6.54±2.64,P0.5,n=14)。说明BDNF对神经元痫样放电有抑制作用,而在mi R-132过表达时这种抑制作用消失,mi R-132能明显加重神经元痫样放电。(3)BDNF抑制痫样放电神经元VGCC电流和活性,而mi R-132能逆转这一作用加重VGCC电流和活性:癫痫模型组过表达mi R-132后VGCC最大电流密度增加(P0.01,n=10)。癫痫模型组孵育BDNF后VGCC最大电流密度降低(P0.05,n=10),稳态激活曲线的半数激活电压增高(P0.001,n=10),稳态失活曲线的半数失活电压降低(P0.01,n=10),但在此基础上过表达mi R-132后逆转了VGCC特性的改变。说明BDNF对痫样放电神经元增强的VGCC有抑制作用,而mi R-132则相反加强痫样放电神经元VGCC的电流和活性。结论:1.Mi R-204调控Trk B信号通路抑制神经元痫样放电。(1)神经元痫样放电模型中mi R-204的表达下调。(2)Mi R-204抑制Trk B m RNA的表达。(3)在神经元痫样放电模型中Trk B下游信号通路PLCγ1和ERK1/2-CREB激活,而其中ERK1/2-CREB信号通路的激活能被mi R-204抑制。(4)Mi R-204抑制神经元痫样放电模型的痫样放电和VGCC电流。2.Mi R-132抑制BDNF加重神经元痫样放电。(1)在神经元痫样放电模型中mi R-132抑制BDNF m RNA的表达。(2)BDNF抑制神经元痫样放电和VGCC电流活性,而mi R-132能逆转这一作用加重神经元痫样放电和加强VGCC电流活性。
[Abstract]:Objective: Micro RNA (MI RNA) is a class of endogenous small molecule non encoding RNA, as a factor regulating transcription, expression of multiple genes encoding proteins involved in the pathogenesis of epilepsy. Brain derived neurotrophic factor (brain-derived neurotrophic, factor, BDNF) - tyrosine kinase receptor B (tropomyosin-related kinase receptor type B. Trk B) signaling pathway is a key of epilepsy, selective central nervous system Trk B gene knockout can completely prevent the kindling induced seizures. The study found that MI R-204-5p (MI R-204) can be affected by the regulation of Trk B receptor in tumor progression and metastasis, in-depth study of MI R-204 is Trk through B signaling pathway the regulation of the occurrence of epilepsy, is expected to provide new ideas for clinical treatment of epilepsy. According to the above research objective of this dissertation was put forward as follows: 1. rat hippocampal neurons in primary culture The epileptiform discharge model element as the research object, through the Trk B receptor and its downstream signaling pathways, inhibition of neuronal epileptiform discharges and voltage-gated Ca2+ channels of MI R-204 (voltage-gated calcium channel, VGCC) based on the mechanism of.2. current in MI R-204 inhibits neuronal epileptiform discharge on the selection of the previous studies in epileptic neuron the model and associated with the abnormal expression of BDNF-Trk B mi R-132-3p (MI R-132 pathway) as the target, studied the changes of neurons epileptiform discharge and VGCC current expression of MI R-132, provides a new idea and method for exploring the role of other types of MI Rs in the pathogenesis of epilepsy. Methods: hippocampal neurons after 3H liquid magnesium free cell culture 1. primary, establish mechanism of epileptiform discharges of neuron model.2.Mi R-204 control Trk B signal pathway and inhibition of neuronal epileptiform discharges (1) by real-time fluorescence. Light PCR (real-time quantitative polymerase chain quantitative reaction, Q RT-PCR) to change the expression of epileptiform discharges of neuron model in MI R-204. (2) lentiviral vector containing mi R-204-p GLVH1/GFP transfection in primary cultured hippocampal neurons by overexpression of MI R-204, by Q RT-PCR detection expression of MI R-204 changed after Trk B m RNA. (3) the representative signal molecule Trk B downstream pathways including Akt, PLC ERK1/2 and CREB. gamma 1, the ratio of the total protein level and phosphorylated protein levels of these molecules reflect the degree of activation of signal molecules by Western blotting, the related mechanism of epileptiform discharges of neuron model in MI R-204 the regulation of Trk B downstream signal molecular activity. (4) the discharge characteristics using the whole cell patch clamp current clamp technique to detect neuronal epileptiform discharge model, epileptiform discharges of neuron model over expression of MI R-204 After the change detection of epileptiform discharges. (5) change and inactivation activation using the whole cell patch clamp voltage clamp technique to detect neuronal epileptiform discharge model of VGCC current and steady state, the change of.3.Mi R-132 regulatory mechanism of BDNF neurons epileptiform discharges of neurons epileptiform discharge model VGCC characteristic expression of MI after R-204. In order to investigate the role of other mi Rs in the pathogenesis of epilepsy is consistent, from previous study found that upregulation of MI R-132 expression as a target in neuronal epileptiform discharge model, MI R-132 control on epileptiform discharges of neuron and VGCC currents, and further explore its mechanism. (1) the expression of Q RT-PCR detection epileptiform discharges of neuron model with BDNF m RNA, lentivirus overexpression of MI R-132 BDNF m to observe the expression change of RNA, to study the mechanism of MI regulation of R-132 BDNF expression. (2) using the whole cell patch Detection of epileptiform discharges of neuron model clamp current clamp technique respectively the expression changes of discharge frequency of epileptiform mi R-132 and incubated with exogenous BDNF. (3) used to detect neuronal epileptiform discharge model of whole cell patch clamp technique respectively the change of expression of VGCC R-132 and current characteristics of MI incubated with exogenous BDNF. Results: 1.Mi R-204 regulation of Trk B signaling pathway inhibits neuronal epileptiform discharge. (1) expression of epileptiform discharges of neuron model in MI R-204: compared with the normal control group of neurons, the expression level of MI R-204 in epileptic model group decreased significantly (P0.001, n=6). (2) the expression of Mi R-204 Trk B negative regulation M receptor RNA: there was no significant difference in the expression level of Trk B m RNA in epilepsy model group and the control group (P0.05, n=6). The epilepsy model group expression of MI R-204 Trk B m RNA expression decreased significantly (P0.001, n=6). (3) Mi R-204 Trk inhibits B receptor downstream Pathway: Akt pathway activity of epileptiform discharges of neuron in the model did not change significantly, PLC gamma 1 (P0.001, n=6), ERK1/2 (P0.001, n=6) and CREB (P0.001, n=6) activity was significantly enhanced. The epileptiform discharges of neuron model of overexpression of MI in R-204 after PLC gamma 1 signal activity no significant change (P0.05, n=6), and ERK1/2 (P0.01, n=6) activity and CREB activity (P0.001, n=6) were significantly decreased. The results showed that ERK1/2-CREB signaling pathway downstream of MI R-204 regulatory Trk B receptor. Mi (4) R-204 inhibits epileptiform discharges of neurons: whole cell current clamp detection control neurons showed spike and occasional; epilepsy model group showed sustained spike frequency, the discharge frequency was significantly higher than the normal control group (P0.001, n=14). The epilepsy model group expression of MI R-204 after the epileptiform discharges of neurons decreased significantly (P0.0 01, n=14). (5) Mi R-204 inhibits neuronal VGCC currents and epileptiform discharges of neuron activity in the model: the maximum current density of epileptiform discharges of neuron model of VGCC (P0.001, n=10) increase, half of the steady-state activation curve of the activation voltage decreased (P0.01, n=10), half of the steady-state inactivation curve of inactivation voltage rise (P0.01, n=10), showed epileptiform discharges of neuron model VGCC current enhancement, more difficult to activate channel inactivation. Epileptiform discharges of neuron model over expression of MI R-204, the maximum current density decreased (P0.001, n=10), half of the steady-state activation curve of the activation voltage increased (P0.05, n=10), half of the steady-state inactivation curve the inactivation of voltage drop (P0.01, n=10), VGCC current and the activity of.2. neurons epileptiform discharge model mi R-204 inhibitory neurons epileptiform discharge model in MI R-132 inhibited BDNF aggravate epileptiform discharges. (1) in neuronal epileptiform discharge model in MI R-1 The expression of M RNA: 32 inhibition of BDNF was significantly up-regulated in BDNF m RNA epilepsy model (P0.001, n=6). The control over the expression level of BDNF m RNA neurons significantly increased the expression of MI R-132 (P0.001, n=6), and the epileptiform discharges of neuron model over expression of MI R-132 expression level of BDNF RNA was m decreased (P0.001, n=6). (2) BDNF inhibits epileptiform discharges of neuron, and the role of MI R-132 reversal aggravating neuronal epileptiform discharges: epilepsy model group expression of MI R-132 after the discharge frequency increased significantly (P0.01, n=14). The epilepsy model group after BDNF incubation and discharge frequency was significantly reduced (P0.001, n=14), but on the basis of over expression of MI R-132 after discharge frequency increased to the level of epilepsy model group (Mg2+-free+BDNF+mi R-132:Mg2+-free=7.11 + 2.90:6.54 + 2.64, P0.5, n=14). The results showed that BDNF had inhibitory effects on neuronal epileptiform discharge, while overexpression of MI R-132 when this suppression Mi R-132 effect disappeared, can aggravate epileptiform discharges of neuron. (3) BDNF inhibits epileptiform discharges of neurons in VGCC and MI and the current activity, R-132 can reverse the effects of increased VGCC current and activity: the epilepsy model group expression of MI R-132 VGCC the maximum current density increased (P0.01, n =10). The epilepsy model group incubation of VGCC the maximum current density decreased after BDNF (P0.05, n=10), half of the steady-state activation curve of the activation voltage increased (P0.001, n=10), half of the steady-state inactivation curve of inactivation voltage (P0.01, n=10) decreased, but based on the expression of MI R-132 after the reversal of the VGCC characteristics of the BDNF on the change. Epileptiform discharges of neurons enhanced the inhibitory effect of VGCC, MI and R-132 on the contrary strengthen current and activity of epileptiform discharges of neurons in VGCC. Conclusion: 1.Mi R-204 regulation of Trk B signaling pathway inhibits neuronal epileptiform discharge. (1) epileptiform discharges of neuron model mi R-204 table Reach down. (2) the expression of Mi R-204 B m RNA. The inhibition of Trk (3) in epileptiform discharges of neuron model Trk B signaling pathways downstream of PLC gamma 1 and ERK1/2-CREB activation, and the activation of ERK1/2-CREB signaling pathway can be inhibited. Mi R-204 (4).2.Mi epileptiform discharge and VGCC current R-132 Mi R-204 inhibition of neuronal epileptiform discharge model of inhibition of BDNF aggravate neuronal epileptiform discharge. (1) expression in neurons of epileptic discharge in the model of MI R-132 m RNA. The inhibition of BDNF (2) BDNF inhibits epileptiform discharges of neuron and VGCC current and MI activity, R-132 can reverse the effects of increased neuron epileptiform discharges and strengthening VGCC current activity.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R742.1

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