【摘要】：研究目的： 癲癇(Epilepsy)是一種常見的神經(jīng)系統(tǒng)疾病,在研究癲癇發(fā)病機(jī)制的基礎(chǔ)上,探索抑制癲癇發(fā)作的有效途徑,具有重要的科學(xué)意義和臨床應(yīng)用價(jià)值。本論文在癲癇發(fā)生的TrkB (tropomyosin-related kinase B)受體和TrkB信號(hào)通路角度,以大鼠海馬神經(jīng)元癇樣放電模型(spontaneous recurrent epileptiform discharges, SREDs)為研究對象,通過大鼠miR-185-3p(miR-185*)激活全長型TrkB(TrkB.FL)信號(hào)通路,研究神經(jīng)元電壓門控性Ca2+通道(Voltage-gated calcium channels, VGCCs)特性和癇樣放電的抑制情況。探討TrkB.FL信號(hào)通路激活后抑制癲癇發(fā)生的機(jī)制,為臨床治療癲癇提供創(chuàng)新的調(diào)控思路。 研究方法： 1.通過無鎂液處理原代培養(yǎng)的大鼠海馬神經(jīng)元3h,構(gòu)建神經(jīng)元癇樣放電模型。 2.研究TrkB受體和TrkB信號(hào)通路在癇樣放電模型中的變化機(jī)制。 (1)通過實(shí)時(shí)熒光定量PCR和western blotting檢測全長型TrkB(TrkB.FL)和截短型TrkB(TrkB.T)受體在模型中的表達(dá)水平,并應(yīng)用鈣蛋白酶抑制劑和轉(zhuǎn)錄翻譯抑制劑研究調(diào)控TrkB受體表達(dá)變化的機(jī)制。 (2)使用BDNF處理模型,應(yīng)用western blotting檢測磷酸化TrkB.FL受體蛋白的表達(dá)水平,研究模型中TrkB受體表達(dá)變化調(diào)控TrkB.FL信號(hào)通路活性的相關(guān)機(jī)制。 3.研究miR-185*調(diào)控TrkB.FL信號(hào)通路活性后對癇樣放電的影響。 (1)利用包含miR-185*-pGLVHl/GFP表達(dá)載體的慢病毒轉(zhuǎn)染模型,實(shí)時(shí)熒光定量PCR和western blotting檢測TrkB.T受體表達(dá)和TrkB.FL信號(hào)通路活性的改變。 (2)全細(xì)胞膜片鉗檢測miR-185*轉(zhuǎn)染模型中神經(jīng)元VGCC電流和穩(wěn)態(tài)激活與失活特性的變化。 (3)全細(xì)胞膜片鉗檢測miR-185*轉(zhuǎn)染模型中神經(jīng)元放電的頻率變化情況。 4.應(yīng)用翻譯抑制劑孵育癇樣放電模型,通過全細(xì)胞膜片鉗檢測神經(jīng)元放電頻率的變化,研究TrkB.FL信號(hào)通路活性改變后對癇樣放電的影響。 研究結(jié)果： 1.大鼠海馬神經(jīng)元癇樣放電模型的鑒定結(jié)果： 與正常對照組神經(jīng)元相比,模型組中神經(jīng)元放電頻率顯著上升(P0.001, n=9), inter-event interval累計(jì)分布曲線明顯左移(P0.001,n=9)。 2.TrkB受體和TrkB信號(hào)通路在癇樣放電模型中的變化結(jié)果： (1)在癇樣放電模型中,TrkB.T受體較正常對照組表達(dá)增加(P0.001,n=6),TrkB.FL受體表達(dá)降低(P0.001,n=6)。與模型組相比,鈣蛋白酶抑制劑孵育模型后,TrkB.FL受體表達(dá)上升(P0.001,n=6)；轉(zhuǎn)錄或翻譯抑制劑孵育模型后,TrkB.T受體表達(dá)均下降(P0.001,n=6)。 (2)與正常對照孵育BDNF組相比,BDNF處理癇樣放電模型,或者BDNF處理模型再孵育鈣蛋白酶抑制劑,以上兩組磷酸化的TrkB.FL受體蛋白與總TrkB.FL受體蛋白的比值(pTrkB.FL/total TrkB.FL)均降低(P0.01,n=6),證明TrkB.FL信號(hào)通路活性被抑制。而BDNF處理癇樣放電模型再孵育翻譯抑制劑后,與BDNF處理模型組相比,pTrkB.FL/total TrkB.FL比值升高(P0.001, n=6), TrkB.FL信號(hào)通路被激活。 3.利用miR-185*調(diào)控TrkB.FL信號(hào)通路活性后影響癇樣放電的研究結(jié)果： (1)利用miR-185*轉(zhuǎn)染癇樣放電模型72h后,miR-185*在模型中持續(xù)大量的表達(dá),并調(diào)控模型中TrkB.T受體表達(dá)減少(P0.001, n=6)。miR-185*轉(zhuǎn)染癇樣放電模型后再經(jīng)BDNF處理,與BDNF處理模型組相比,pTrkB.FL/total TrkB.FL比值上升(P0.001,n=4),TrkB.FL信號(hào)通路同樣被激活。 (2)癇樣放電模型組中神經(jīng)元VGCC電流和穩(wěn)態(tài)激活與失活特性的相關(guān)參數(shù)與正常對照組比較后發(fā)現(xiàn),VGCC最大電流密度升高(P0.001,n=10-15)；激活曲線的V1/2和斜率因子k顯著降低(P0.001,n=7-10)。表示VGCC激活的閾電位減小,激活速度加快,通道相對容易被激活。模型組VGCC失活曲線的V1/2顯著增加(P0.001,n=7-9),而斜率因子k卻顯著降低(P0.001,n=7-9)。表示VGCC失活的閾電位增加,失活速度變慢,通道相對更難失活。 而miR-185*轉(zhuǎn)染癇樣放電模型后,VGCC特性的相關(guān)參數(shù)與未經(jīng)轉(zhuǎn)染的模型組相比,VGCC最大電流密度降低(P0.001,n=10.15)；激活曲線的V1/2顯著增加(P0.01,n=7-10),斜率因子k也顯著升高(P0.001,n=7-10)。表示VGCC激活的閾電位升高,激活速度降低,通道相對難以激活。而VGCC失活曲線的V1/2變化雖然降低,但不顯著(P0.05,n=7-9),反而斜率因子k顯著升高(P0.05,n=7-9),表明VGCC失活速度加快,通道相對更容易失活。 (3)miR-185*轉(zhuǎn)染癇樣放電模型后,與未經(jīng)處理的模型相比,神經(jīng)元放電頻率顯著降低(P0.001,n=9),其inter-event interval累計(jì)分布曲線右移(P0.001,n=9),結(jié)果顯示轉(zhuǎn)染模型神經(jīng)元的癇樣放電減弱。 4.應(yīng)用翻譯抑制劑孵育癇樣放電模型,TrkB.FL信號(hào)通路變化后對癇樣放電的調(diào)節(jié)結(jié)果： 與未經(jīng)處理的模型相比,翻譯抑制劑孵育模型后的神經(jīng)元放電頻率顯著降低(P0.001,n=9), inter-event interval累計(jì)分布曲線右移(P0.001,n=9),神經(jīng)元的癇樣放電同樣減弱。 結(jié)論： 1.大鼠海馬神經(jīng)元經(jīng)無鎂液處理3h后,成功構(gòu)建癇樣放電模型。 2.癇樣放電模型中,鈣蛋白酶調(diào)控TrkB.FL受體表達(dá)減少,轉(zhuǎn)錄翻譯過程參與 TrkB.T受體表達(dá)的增加。TrkB.T受體的高表達(dá)抑制模型中TrkB.FL信號(hào)通路的激活。 3.miR-185*激活TrkB.FL信號(hào)通路后,抑制癇樣放電的產(chǎn)生。 (1)miR-185*轉(zhuǎn)染癇樣放電模型,通過下調(diào)TrkB.T受體的表達(dá),可以激活TrkB.FL信號(hào)通路。 (2)激活的TrkB.FL信號(hào)通路抑制miR-185*轉(zhuǎn)染模型神經(jīng)元的VGCC電流和通道活性。 (3)激活的TrkB.FL信號(hào)通路降低miR-185*轉(zhuǎn)染模型神經(jīng)元的放電頻率。 4.翻譯抑制劑孵育癇樣放電模型,激活的TrkB.FL信號(hào)通路同樣降低了神經(jīng)元的放電頻率,最終抑制癇樣放電。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R742.1

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