大鼠miR-185-3p调控的TrkB.FL信号通路抑制神经元痫样放电的研究
发布时间：2018-08-11 09:21
【摘要】：研究目的： 癫痫(Epilepsy)是一种常见的神经系统疾病,在研究癫痫发病机制的基础上,探索抑制癫痫发作的有效途径,具有重要的科学意义和临床应用价值。本论文在癫痫发生的TrkB (tropomyosin-related kinase B)受体和TrkB信号通路角度,以大鼠海马神经元痫样放电模型(spontaneous recurrent epileptiform discharges, SREDs)为研究对象,通过大鼠miR-185-3p(miR-185*)激活全长型TrkB(TrkB.FL)信号通路,研究神经元电压门控性Ca2+通道(Voltage-gated calcium channels, VGCCs)特性和痫样放电的抑制情况。探讨TrkB.FL信号通路激活后抑制癫痫发生的机制,为临床治疗癫痫提供创新的调控思路。 研究方法： 1.通过无镁液处理原代培养的大鼠海马神经元3h,构建神经元痫样放电模型。 2.研究TrkB受体和TrkB信号通路在痫样放电模型中的变化机制。 (1)通过实时荧光定量PCR和western blotting检测全长型TrkB(TrkB.FL)和截短型TrkB(TrkB.T)受体在模型中的表达水平,并应用钙蛋白酶抑制剂和转录翻译抑制剂研究调控TrkB受体表达变化的机制。 (2)使用BDNF处理模型,应用western blotting检测磷酸化TrkB.FL受体蛋白的表达水平,研究模型中TrkB受体表达变化调控TrkB.FL信号通路活性的相关机制。 3.研究miR-185*调控TrkB.FL信号通路活性后对痫样放电的影响。 (1)利用包含miR-185*-pGLVHl/GFP表达载体的慢病毒转染模型,实时荧光定量PCR和western blotting检测TrkB.T受体表达和TrkB.FL信号通路活性的改变。 (2)全细胞膜片钳检测miR-185*转染模型中神经元VGCC电流和稳态激活与失活特性的变化。 (3)全细胞膜片钳检测miR-185*转染模型中神经元放电的频率变化情况。 4.应用翻译抑制剂孵育痫样放电模型,通过全细胞膜片钳检测神经元放电频率的变化,研究TrkB.FL信号通路活性改变后对痫样放电的影响。 研究结果： 1.大鼠海马神经元痫样放电模型的鉴定结果： 与正常对照组神经元相比,模型组中神经元放电频率显著上升(P0.001, n=9), inter-event interval累计分布曲线明显左移(P0.001,n=9)。 2.TrkB受体和TrkB信号通路在痫样放电模型中的变化结果： (1)在痫样放电模型中,TrkB.T受体较正常对照组表达增加(P0.001,n=6),TrkB.FL受体表达降低(P0.001,n=6)。与模型组相比,钙蛋白酶抑制剂孵育模型后,TrkB.FL受体表达上升(P0.001,n=6)；转录或翻译抑制剂孵育模型后,TrkB.T受体表达均下降(P0.001,n=6)。 (2)与正常对照孵育BDNF组相比,BDNF处理痫样放电模型,或者BDNF处理模型再孵育钙蛋白酶抑制剂,以上两组磷酸化的TrkB.FL受体蛋白与总TrkB.FL受体蛋白的比值(pTrkB.FL/total TrkB.FL)均降低(P0.01,n=6),证明TrkB.FL信号通路活性被抑制。而BDNF处理痫样放电模型再孵育翻译抑制剂后,与BDNF处理模型组相比,pTrkB.FL/total TrkB.FL比值升高(P0.001, n=6), TrkB.FL信号通路被激活。 3.利用miR-185*调控TrkB.FL信号通路活性后影响痫样放电的研究结果： (1)利用miR-185*转染痫样放电模型72h后,miR-185*在模型中持续大量的表达,并调控模型中TrkB.T受体表达减少(P0.001, n=6)。miR-185*转染痫样放电模型后再经BDNF处理,与BDNF处理模型组相比,pTrkB.FL/total TrkB.FL比值上升(P0.001,n=4),TrkB.FL信号通路同样被激活。 (2)痫样放电模型组中神经元VGCC电流和稳态激活与失活特性的相关参数与正常对照组比较后发现,VGCC最大电流密度升高(P0.001,n=10-15)；激活曲线的V1/2和斜率因子k显著降低(P0.001,n=7-10)。表示VGCC激活的阈电位减小,激活速度加快,通道相对容易被激活。模型组VGCC失活曲线的V1/2显著增加(P0.001,n=7-9),而斜率因子k却显著降低(P0.001,n=7-9)。表示VGCC失活的阈电位增加,失活速度变慢,通道相对更难失活。 而miR-185*转染痫样放电模型后,VGCC特性的相关参数与未经转染的模型组相比,VGCC最大电流密度降低(P0.001,n=10.15)；激活曲线的V1/2显著增加(P0.01,n=7-10),斜率因子k也显著升高(P0.001,n=7-10)。表示VGCC激活的阈电位升高,激活速度降低,通道相对难以激活。而VGCC失活曲线的V1/2变化虽然降低,但不显著(P0.05,n=7-9),反而斜率因子k显著升高(P0.05,n=7-9),表明VGCC失活速度加快,通道相对更容易失活。 (3)miR-185*转染痫样放电模型后,与未经处理的模型相比,神经元放电频率显著降低(P0.001,n=9),其inter-event interval累计分布曲线右移(P0.001,n=9),结果显示转染模型神经元的痫样放电减弱。 4.应用翻译抑制剂孵育痫样放电模型,TrkB.FL信号通路变化后对痫样放电的调节结果： 与未经处理的模型相比,翻译抑制剂孵育模型后的神经元放电频率显著降低(P0.001,n=9), inter-event interval累计分布曲线右移(P0.001,n=9),神经元的痫样放电同样减弱。 结论： 1.大鼠海马神经元经无镁液处理3h后,成功构建痫样放电模型。 2.痫样放电模型中,钙蛋白酶调控TrkB.FL受体表达减少,转录翻译过程参与 TrkB.T受体表达的增加。TrkB.T受体的高表达抑制模型中TrkB.FL信号通路的激活。 3.miR-185*激活TrkB.FL信号通路后,抑制痫样放电的产生。 (1)miR-185*转染痫样放电模型,通过下调TrkB.T受体的表达,可以激活TrkB.FL信号通路。 (2)激活的TrkB.FL信号通路抑制miR-185*转染模型神经元的VGCC电流和通道活性。 (3)激活的TrkB.FL信号通路降低miR-185*转染模型神经元的放电频率。 4.翻译抑制剂孵育痫样放电模型,激活的TrkB.FL信号通路同样降低了神经元的放电频率,最终抑制痫样放电。
[Abstract]:Research purposes:
Epilepsy is a common nervous system disease. Based on the study of the pathogenesis of epilepsy, it is of great scientific significance and clinical value to explore effective ways to inhibit epileptic seizures. The spontaneous recurrent epileptiform discharges (SREDs) were used to study the characteristics of voltage-gated Ca2+ channels (VGCCs) and the inhibition of epileptiform discharges in neurons by activating the full-length TrkB (TrkB.FL) signaling pathway with rat microRNA185-3p (microRNA185*). The mechanism of inhibiting epilepsy after pathway activation provides innovative regulatory ideas for clinical treatment of epilepsy.
Research methods:
1. The epileptiform discharge model of primary cultured rat hippocampal neurons was established by treating them with magnesium-free solution for 3 hours.
2. to study the mechanism of TrkB receptor and TrkB signaling pathway in epileptiform discharges.
(1) The expression levels of full-length TrkB (TrkB.FL) and truncated TrkB (TrkB.T) receptors were detected by real-time fluorescence quantitative PCR and Western blotting. Calpain inhibitors and transcriptional translation inhibitors were used to study the mechanism of regulating the expression of TrkB receptors.
(2) Western blotting was used to detect the expression of phosphorylated TrkB.FL receptor protein in BDNF treatment model, and the mechanism of TrkB.FL signaling pathway activity was studied.
3. to study the effect of miR-185* on the epileptiform discharges after regulating the activity of TrkB.FL signaling pathway.
(1) The expression of TrkB.T receptor and the activity of TrkB.FL signaling pathway were detected by real-time fluorescence quantitative PCR and Western blotting using a lentiviral transfection model containing the expression vector of Mi-185*-pGLVHl/GFP.
(2) Whole-cell patch clamp technique was used to detect the changes of VGCC current and steady-state activation and inactivation of neurons in the model of microarray-185 * transfection.
(3) whole cell patch clamp test was used to detect the frequency of neuronal firing in miR-185* transfection model.
4. Using the model of epileptiform discharges incubated by translation inhibitors, the changes of neuronal discharge frequencies were detected by whole-cell patch clamp to study the effects of changes in TrkB.FL signaling pathway activity on epileptiform discharges.
Research findings:
1. the epileptic discharge model of rat hippocampal neurons was identified.
Compared with the normal control group, the discharge frequency of neurons in the model group increased significantly (P 0.001, n = 9), and the cumulative distribution curve of inter-event interval shifted significantly to the left (P 0.001, n = 9).
The changes of 2.TrkB receptor and TrkB signaling pathway in epileptiform discharges model:
(1) In the epileptiform discharge model, the expression of TrkB.T receptor increased (P 0.001, n = 6) and the expression of TrkB.FL receptor decreased (P 0.001, n = 6) compared with the normal control group.
(2) The ratio of phosphorylated TrkB.FL receptor protein to total TrkB.FL receptor protein (pTrkB.FL/total TrkB.FL) in BDNF-treated epileptiform discharges model or BDNF-treated model was lower than that in BDNF-treated group (P 0.01, n=6), suggesting that the activity of TrkB.FL signaling pathway was inhibited. The ratio of pTrkB.FL/total TrkB.FL increased (P 0.001, n=6) and the TrkB.FL signaling pathway was activated in the BDNF treated group after incubation of the TrkB.FL inhibitor.
3. using miR-185* to regulate the activity of TrkB.FL signaling pathway, the results of epileptiform discharges were studied.
(1) After 72 hours of transfection of epileptiform discharge model with microRNAs-185 * the expression of microRNAs-185 * in the model continued to increase, and the expression of TrkB.T receptor decreased (P 0.001, n = 6). After transfection of epileptiform discharge model with microRNAs-185 * and BDNF treatment, the ratio of pTrkB.FL/total TrkB.FL increased (P 0.001, n = 4), and the TrkB.FL signaling pathway was the same as that of BDNF treatment group. Activated.
(2) Compared with the normal control group, the parameters related to VGCC current and steady-state activation and inactivation of neurons in the epileptiform discharge model group showed that the maximum current density of VGCC increased (P 0.001, n = 10-15), and the V1/2 and slope factor K of activation curve decreased significantly (P 0.001, n = 7-10). V1/2 of the VGCC inactivation curve increased significantly (P 0.001, n=7-9) while the slope factor K decreased significantly (P 0.001, n=7-9). This indicated that the threshold potential of VGCC inactivation increased, the inactivation rate slowed down, and the channel was relatively more difficult to inactivate.
Compared with the untransfected group, the maximum current density of VGCC decreased (P 0.001, n = 10.15), the V1/2 of the activation curve increased significantly (P 0.01, n = 7-10), and the slope factor K increased significantly (P 0.001, n = 7-10). V1/2 of VGCC inactivation curve decreased, but not significantly (P 0.05, n = 7-9), but the slope factor K increased significantly (P 0.05, n = 7-9), indicating that VGCC inactivation speed increased, and the channel was relatively easier to inactivate.
(3) Compared with untreated model, the discharge frequency of neurons was significantly decreased (P 0.001, n = 9) and the cumulative distribution curve of inter-event interval shifted to the right (P 0.001, n = 9) after transfection of microRNA185* into the epileptiform discharge model. The results showed that the epileptiform discharge of neurons in the transfected model was decreased.
4. Using the model of epileptiform discharges incubated by translation inhibitors, the regulation of TrkB.FL signaling pathway on epileptiform discharges was studied.
Compared with the untreated model, the firing frequency of the neurons incubated with the translation inhibitor decreased significantly (P 0.001, n=9), the cumulative distribution curve of inter-event interval shifted to the right (P 0.001, n=9), and the epileptiform discharge of the neurons also decreased.
Conclusion:
1. rat hippocampal neurons were treated with magnesium free solution for 3h, and epileptiform discharges were successfully constructed.
2. In the epileptiform discharge model, the expression of TrkB.FL receptor is decreased and transcriptional translation is involved.
Increased expression of TrkB.T receptor. Activation of TrkB.FL signaling pathway in the TrkB.T receptor overexpression inhibition model.
3.miR-185* activates the TrkB.FL signaling pathway and suppresses epileptiform discharges.
(1) Mi-185* transfected epileptiform discharge model can activate TrkB.FL signaling pathway by down-regulating the expression of TrkB.T receptor.
(2) Activated TrkB.FL signaling pathway inhibits the VGCC current and channel activity in the model neurons transfected with miR-185*
(3) activation of TrkB.FL signaling pathway reduces the firing frequency of neurons in miR-185* transfection model.
4. Translator inhibitors incubate epileptiform discharges. Activated TrkB.FL signaling pathway also reduces the frequency of neuronal discharges and ultimately inhibits epileptiform discharges.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1
本文编号：2176573
[Abstract]:Research purposes:
Epilepsy is a common nervous system disease. Based on the study of the pathogenesis of epilepsy, it is of great scientific significance and clinical value to explore effective ways to inhibit epileptic seizures. The spontaneous recurrent epileptiform discharges (SREDs) were used to study the characteristics of voltage-gated Ca2+ channels (VGCCs) and the inhibition of epileptiform discharges in neurons by activating the full-length TrkB (TrkB.FL) signaling pathway with rat microRNA185-3p (microRNA185*). The mechanism of inhibiting epilepsy after pathway activation provides innovative regulatory ideas for clinical treatment of epilepsy.
Research methods:
1. The epileptiform discharge model of primary cultured rat hippocampal neurons was established by treating them with magnesium-free solution for 3 hours.
2. to study the mechanism of TrkB receptor and TrkB signaling pathway in epileptiform discharges.
(1) The expression levels of full-length TrkB (TrkB.FL) and truncated TrkB (TrkB.T) receptors were detected by real-time fluorescence quantitative PCR and Western blotting. Calpain inhibitors and transcriptional translation inhibitors were used to study the mechanism of regulating the expression of TrkB receptors.
(2) Western blotting was used to detect the expression of phosphorylated TrkB.FL receptor protein in BDNF treatment model, and the mechanism of TrkB.FL signaling pathway activity was studied.
3. to study the effect of miR-185* on the epileptiform discharges after regulating the activity of TrkB.FL signaling pathway.
(1) The expression of TrkB.T receptor and the activity of TrkB.FL signaling pathway were detected by real-time fluorescence quantitative PCR and Western blotting using a lentiviral transfection model containing the expression vector of Mi-185*-pGLVHl/GFP.
(2) Whole-cell patch clamp technique was used to detect the changes of VGCC current and steady-state activation and inactivation of neurons in the model of microarray-185 * transfection.
(3) whole cell patch clamp test was used to detect the frequency of neuronal firing in miR-185* transfection model.
4. Using the model of epileptiform discharges incubated by translation inhibitors, the changes of neuronal discharge frequencies were detected by whole-cell patch clamp to study the effects of changes in TrkB.FL signaling pathway activity on epileptiform discharges.
Research findings:
1. the epileptic discharge model of rat hippocampal neurons was identified.
Compared with the normal control group, the discharge frequency of neurons in the model group increased significantly (P 0.001, n = 9), and the cumulative distribution curve of inter-event interval shifted significantly to the left (P 0.001, n = 9).
The changes of 2.TrkB receptor and TrkB signaling pathway in epileptiform discharges model:
(1) In the epileptiform discharge model, the expression of TrkB.T receptor increased (P 0.001, n = 6) and the expression of TrkB.FL receptor decreased (P 0.001, n = 6) compared with the normal control group.
(2) The ratio of phosphorylated TrkB.FL receptor protein to total TrkB.FL receptor protein (pTrkB.FL/total TrkB.FL) in BDNF-treated epileptiform discharges model or BDNF-treated model was lower than that in BDNF-treated group (P 0.01, n=6), suggesting that the activity of TrkB.FL signaling pathway was inhibited. The ratio of pTrkB.FL/total TrkB.FL increased (P 0.001, n=6) and the TrkB.FL signaling pathway was activated in the BDNF treated group after incubation of the TrkB.FL inhibitor.
3. using miR-185* to regulate the activity of TrkB.FL signaling pathway, the results of epileptiform discharges were studied.
(1) After 72 hours of transfection of epileptiform discharge model with microRNAs-185 * the expression of microRNAs-185 * in the model continued to increase, and the expression of TrkB.T receptor decreased (P 0.001, n = 6). After transfection of epileptiform discharge model with microRNAs-185 * and BDNF treatment, the ratio of pTrkB.FL/total TrkB.FL increased (P 0.001, n = 4), and the TrkB.FL signaling pathway was the same as that of BDNF treatment group. Activated.
(2) Compared with the normal control group, the parameters related to VGCC current and steady-state activation and inactivation of neurons in the epileptiform discharge model group showed that the maximum current density of VGCC increased (P 0.001, n = 10-15), and the V1/2 and slope factor K of activation curve decreased significantly (P 0.001, n = 7-10). V1/2 of the VGCC inactivation curve increased significantly (P 0.001, n=7-9) while the slope factor K decreased significantly (P 0.001, n=7-9). This indicated that the threshold potential of VGCC inactivation increased, the inactivation rate slowed down, and the channel was relatively more difficult to inactivate.
Compared with the untransfected group, the maximum current density of VGCC decreased (P 0.001, n = 10.15), the V1/2 of the activation curve increased significantly (P 0.01, n = 7-10), and the slope factor K increased significantly (P 0.001, n = 7-10). V1/2 of VGCC inactivation curve decreased, but not significantly (P 0.05, n = 7-9), but the slope factor K increased significantly (P 0.05, n = 7-9), indicating that VGCC inactivation speed increased, and the channel was relatively easier to inactivate.
(3) Compared with untreated model, the discharge frequency of neurons was significantly decreased (P 0.001, n = 9) and the cumulative distribution curve of inter-event interval shifted to the right (P 0.001, n = 9) after transfection of microRNA185* into the epileptiform discharge model. The results showed that the epileptiform discharge of neurons in the transfected model was decreased.
4. Using the model of epileptiform discharges incubated by translation inhibitors, the regulation of TrkB.FL signaling pathway on epileptiform discharges was studied.
Compared with the untreated model, the firing frequency of the neurons incubated with the translation inhibitor decreased significantly (P 0.001, n=9), the cumulative distribution curve of inter-event interval shifted to the right (P 0.001, n=9), and the epileptiform discharge of the neurons also decreased.
Conclusion:
1. rat hippocampal neurons were treated with magnesium free solution for 3h, and epileptiform discharges were successfully constructed.
2. In the epileptiform discharge model, the expression of TrkB.FL receptor is decreased and transcriptional translation is involved.
Increased expression of TrkB.T receptor. Activation of TrkB.FL signaling pathway in the TrkB.T receptor overexpression inhibition model.
3.miR-185* activates the TrkB.FL signaling pathway and suppresses epileptiform discharges.
(1) Mi-185* transfected epileptiform discharge model can activate TrkB.FL signaling pathway by down-regulating the expression of TrkB.T receptor.
(2) Activated TrkB.FL signaling pathway inhibits the VGCC current and channel activity in the model neurons transfected with miR-185*
(3) activation of TrkB.FL signaling pathway reduces the firing frequency of neurons in miR-185* transfection model.
4. Translator inhibitors incubate epileptiform discharges. Activated TrkB.FL signaling pathway also reduces the frequency of neuronal discharges and ultimately inhibits epileptiform discharges.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1
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