海馬BDNF-p11信號通路在氯胺酮抗抑郁中的作用
發(fā)布時間：2018-08-07 06:55
【摘要】：目的：N-甲基-D-天冬氨酸(N-methyl-D-aspartate, NMDA)受體阻滯劑氯胺酮可以產(chǎn)生快速、有效、較持久的抗抑郁作用。不同分子機制、神經(jīng)環(huán)路、信號通路和相關(guān)腦區(qū)在氯胺酮抗抑郁作用中具有一定的作用,但其具體作用機制尚不清楚。新近研究表明,小分子蛋白p11能夠調(diào)節(jié)許多離子通道和五羥色胺(5-hydroxytryptamine,5-HT)受體在細胞膜上的表達,且促進突觸再生,在抑郁癥的神經(jīng)病理學機制中具有重要作用。在抑郁癥患者外周血液和抑郁模型動物相關(guān)腦區(qū)中,p11mRNA和蛋白表達均減少,而且p11的表達水平可作為判斷抑郁癥患者預(yù)后的指標。離體和在體實驗均證實腦源性神經(jīng)營養(yǎng)因子(Brain-derived neurotrophic factor, BDNF)能夠誘導并促進海馬p11表達,產(chǎn)生抗抑郁作用；而BDNF在氯胺酮抗抑郁作用中亦具有關(guān)鍵作用。因此,我們推測海馬BDNF-p11信號通路參與氯胺酮的抗抑郁作用。本研究擬觀察海馬BDNF-p11信號通路在氯胺酮抗抑郁中的變化及其作用,并探討相關(guān)機制。方法：采用慢性不可預(yù)知溫和應(yīng)激(Chronic unpredicted mild stress, CUMS)建立大鼠慢性抑郁模型。腹腔注射氯胺酮10 mg/kg或氯胺酮10 mg/kg聯(lián)合ANA-12(特異性酪氨酸激酶B (Tyrosine kinases, TrkB)受體阻滯劑)0.5 mg/kg后0.5 h和72 h,行曠場實驗(Open field test, OFT)、強迫游泳實驗(Forced swimming test,FST)和蔗糖偏好實驗(Sucrose preference test, SPT)以檢測大鼠抑郁樣行為。構(gòu)建并在離體大鼠海馬神經(jīng)元培養(yǎng)中篩選p11慢病毒載體,將高效能p11慢病毒顆粒在體注射至大鼠海馬中以沉默海馬p11的表達,10天后行OFT.FST及SPT。行為學檢測結(jié)束后,取大鼠海馬組織,采用Western blotting檢測大鼠海馬中BDNF和p11蛋白表達。結(jié)果：與對照組相比,CUMS大鼠在FST中不動時間增加,在SPT中糖水偏好百分比降低,表現(xiàn)出抑郁樣行為。注射氯胺酮后0.5 h和72 h,CUMS大鼠在FST中不動時間減少,在SPT中糖水偏好百分比增加,且與對照組差異無統(tǒng)計學意義,說明氯胺酮產(chǎn)生了快速和持續(xù)抗抑郁作用。聯(lián)合注射氯胺酮和ANA-12后0.5 h和72 h,與單獨注射氯胺酮相比,CUMS大鼠在FST中不動時間增加,在SPT中蔗糖偏好百分比減少；與注射生理鹽水相比,CUMS大鼠在FST及SPT中差異無統(tǒng)計學意義,說明ANA-12可阻斷氯胺酮的抗抑郁作用。各種處理措施對大鼠在OFT中運動總距離的影響差異均無統(tǒng)計學意義(P0.05)。與對照組相比,CUMS大鼠海馬中BDNF和p11表達顯著減少(P0.05)。氯胺酮注射后0.5 h和72 h,CUMS大鼠海馬中BDNF顯著增加,且與對照組差異無統(tǒng)計學意義。p11僅在氯胺酮注射后72 h,而不是0.5 h,顯著增加(P0.05)。聯(lián)合注射氯胺酮和ANA-12后72 h,與單獨注射氯胺酮72 h相比,CUMS大鼠海馬中p11表達顯著減少(P0.05)；與注射生理鹽水相比,CUMS大鼠海馬P11表達差異無統(tǒng)計學意義(P0.05)。與對照組相比,在體海馬注射慢病毒空載體對大鼠在FST中不動時間和SPT中蔗糖偏好百分比影響差異無統(tǒng)計學意義(P0.05)。與在體海馬注射慢病毒空載體相比,在體海馬注射p11慢病毒后13天,p11表達顯著減少,大鼠表現(xiàn)出不動時間增加和蔗糖偏好百分比降低等抑郁樣行為。與注射生理鹽水相比,注射氯胺酮對在體海馬注射p11慢病毒大鼠在FST中不動時間和SPT中蔗糖偏好百分比影響差異無統(tǒng)計學意義(P0.05),說明將大鼠海馬p11沉默后,氯胺酮抗抑郁作用消失。結(jié)論：在CUMS大鼠抑郁模型中,海馬BDNF-p11信號通路在氯胺酮抗抑郁作用的維持中起關(guān)鍵作用。
[Abstract]:Objective: N- methyl -D- aspartic acid (N-methyl-D-aspartate, NMDA) receptor blocker, ketamine, can produce rapid, effective and lasting antidepressant effects. Different molecular mechanisms, neural circuits, signal pathways and related brain regions have a certain role in the antidepressant effect of ketamine, but the specific mechanism of its action is not yet clear. It shows that small molecular protein P11 can regulate the expression of many ion channels and five serotonin (5-hydroxytryptamine, 5-HT) receptors on the cell membrane and promote synapse regeneration, which plays an important role in the neuropathological mechanism of depression. In the peripheral blood and depression model animal related brain regions of the depressive patients, the expression of p11mRNA and protein are all Decrease, and the expression level of P11 can be used as an indicator to judge the prognosis of patients with depression. In vitro and in vivo, Brain-derived neurotrophic factor (BDNF) can induce and promote the expression of P11 in hippocampus and produce antidepressant effect. BDNF also plays a key role in the antidepressant effect of ketamine. Therefore, we speculate that the hippocampal BDNF-p11 signaling pathway participates in the antidepressant effect of ketamine. This study intends to observe the changes and effects of the hippocampal BDNF-p11 signaling pathway in ketamine antidepressants and to explore the mechanisms. Methods: chronic unpredictable mild stress (Chronic unpredicted mild stress, CUMS) is used to establish chronic depression models in rats. Type. Intraperitoneal injection of ketamine 10 mg/kg or ketamine 10 mg/kg combined with ANA-12 (specific tyrosine kinase B (Tyrosine kinases, TrkB) receptor blocker) 0.5 h and 72 h after 0.5 mg/kg (Open field), forced swimming test and sucrose preference test The P11 lentivirus vector was screened in the cultured rat hippocampal neurons in vitro, and the high efficient P11 lentivirus particles were injected into the hippocampus of the rat to silence the expression of P11 in the hippocampus. After 10 days of OFT.FST and SPT. behavior detection, the hippocampus tissue was taken and B in the hippocampus of rats was detected by Western blotting. DNF and P11 protein expression. Results: compared with the control group, the duration of CUMS rats increased in FST, and the percentage of sugar water preference decreased in SPT, showing depressive behavior. After injection of ketamine, 0.5 h and 72 h, CUMS rats were less active in FST, and the ratio of sugar to water in SPT increased, and there was no significant difference from the control group. The combination of ketamine and ANA-12 0.5 h and 72 h after injection of ketamine and ANA-12, compared with the single injection of ketamine, increased the duration of CUMS rats in FST and decreased the percentage of sucrose preference in SPT; compared with the injection of saline, the difference in FST and SPT in CUMS rats was not statistically significant, indicating ANA-12 can be found. The antidepressant effect of ketamine was blocked. There was no significant difference in the effect of various treatment measures on the total distance of exercise in OFT (P0.05). Compared with the control group, the expression of BDNF and P11 in the hippocampus of CUMS rats decreased significantly (P0.05). The BDNF of 0.5 h and 72 h in the rats after injection of ketamine, and the BDNF of the hippocampus in the rats of CUMS significantly increased, and there was no difference between the control group and the control group. .p11 was only 72 h after ketamine injection, but not 0.5 h, significantly increased (P0.05). Compared with ketamine and ANA-12 72 h after injection of ketamine and ANA-12, the expression of P11 in hippocampus of CUMS rats decreased significantly (P0.05). Compared with the injection of saline, there was no significant difference in the expression of hippocampal P11 expression in CUMS rats. There was no significant difference in the effect of lentivirus no-load in the body hippocampus on the duration of FST and the percentage of sucrose preference in SPT (P0.05). Compared with the lentivirus empty vector injected in the hippocampus, the expression of P11 was significantly reduced at 13 days after the injection of P11 lentivirus in the hippocampus, and the rats showed increased time and sucrose preference. Compared with the injection of physiological saline, there was no significant difference in the effect of injection of ketamine on the duration of P11 lentivirus injection in FST rats in the hippocampus and the percentage of sucrose preference in SPT in the hippocampus of the rats (P0.05), indicating that the antidepressant effect of chloramine ketamine disappeared after the hippocampal P11 was silent in the rat. Conclusion: depression in CUMS rats In the model, the hippocampal BDNF-p11 signaling pathway plays a key role in the maintenance of ketamine antidepressant effect.
【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R614
[Abstract]:Objective: N- methyl -D- aspartic acid (N-methyl-D-aspartate, NMDA) receptor blocker, ketamine, can produce rapid, effective and lasting antidepressant effects. Different molecular mechanisms, neural circuits, signal pathways and related brain regions have a certain role in the antidepressant effect of ketamine, but the specific mechanism of its action is not yet clear. It shows that small molecular protein P11 can regulate the expression of many ion channels and five serotonin (5-hydroxytryptamine, 5-HT) receptors on the cell membrane and promote synapse regeneration, which plays an important role in the neuropathological mechanism of depression. In the peripheral blood and depression model animal related brain regions of the depressive patients, the expression of p11mRNA and protein are all Decrease, and the expression level of P11 can be used as an indicator to judge the prognosis of patients with depression. In vitro and in vivo, Brain-derived neurotrophic factor (BDNF) can induce and promote the expression of P11 in hippocampus and produce antidepressant effect. BDNF also plays a key role in the antidepressant effect of ketamine. Therefore, we speculate that the hippocampal BDNF-p11 signaling pathway participates in the antidepressant effect of ketamine. This study intends to observe the changes and effects of the hippocampal BDNF-p11 signaling pathway in ketamine antidepressants and to explore the mechanisms. Methods: chronic unpredictable mild stress (Chronic unpredicted mild stress, CUMS) is used to establish chronic depression models in rats. Type. Intraperitoneal injection of ketamine 10 mg/kg or ketamine 10 mg/kg combined with ANA-12 (specific tyrosine kinase B (Tyrosine kinases, TrkB) receptor blocker) 0.5 h and 72 h after 0.5 mg/kg (Open field), forced swimming test and sucrose preference test The P11 lentivirus vector was screened in the cultured rat hippocampal neurons in vitro, and the high efficient P11 lentivirus particles were injected into the hippocampus of the rat to silence the expression of P11 in the hippocampus. After 10 days of OFT.FST and SPT. behavior detection, the hippocampus tissue was taken and B in the hippocampus of rats was detected by Western blotting. DNF and P11 protein expression. Results: compared with the control group, the duration of CUMS rats increased in FST, and the percentage of sugar water preference decreased in SPT, showing depressive behavior. After injection of ketamine, 0.5 h and 72 h, CUMS rats were less active in FST, and the ratio of sugar to water in SPT increased, and there was no significant difference from the control group. The combination of ketamine and ANA-12 0.5 h and 72 h after injection of ketamine and ANA-12, compared with the single injection of ketamine, increased the duration of CUMS rats in FST and decreased the percentage of sucrose preference in SPT; compared with the injection of saline, the difference in FST and SPT in CUMS rats was not statistically significant, indicating ANA-12 can be found. The antidepressant effect of ketamine was blocked. There was no significant difference in the effect of various treatment measures on the total distance of exercise in OFT (P0.05). Compared with the control group, the expression of BDNF and P11 in the hippocampus of CUMS rats decreased significantly (P0.05). The BDNF of 0.5 h and 72 h in the rats after injection of ketamine, and the BDNF of the hippocampus in the rats of CUMS significantly increased, and there was no difference between the control group and the control group. .p11 was only 72 h after ketamine injection, but not 0.5 h, significantly increased (P0.05). Compared with ketamine and ANA-12 72 h after injection of ketamine and ANA-12, the expression of P11 in hippocampus of CUMS rats decreased significantly (P0.05). Compared with the injection of saline, there was no significant difference in the expression of hippocampal P11 expression in CUMS rats. There was no significant difference in the effect of lentivirus no-load in the body hippocampus on the duration of FST and the percentage of sucrose preference in SPT (P0.05). Compared with the lentivirus empty vector injected in the hippocampus, the expression of P11 was significantly reduced at 13 days after the injection of P11 lentivirus in the hippocampus, and the rats showed increased time and sucrose preference. Compared with the injection of physiological saline, there was no significant difference in the effect of injection of ketamine on the duration of P11 lentivirus injection in FST rats in the hippocampus and the percentage of sucrose preference in SPT in the hippocampus of the rats (P0.05), indicating that the antidepressant effect of chloramine ketamine disappeared after the hippocampal P11 was silent in the rat. Conclusion: depression in CUMS rats In the model, the hippocampal BDNF-p11 signaling pathway plays a key role in the maintenance of ketamine antidepressant effect.
【學位授予單位】：南京大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R614
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