本文選題：右美托咪定 + 鈣超載��； 參考：《青島大學(xué)》2017年博士論文

【摘要】：腦缺血是腦部組織血流量不足的一種疾病,這種情況通�？梢詫�(dǎo)致腦組織的代謝率以及能量的降低,和隨之引起來的局部區(qū)域的腦梗死。當(dāng)腦缺血這種情況發(fā)生時,由于來自于血液的氧以及營養(yǎng)物的供給減少,可能使腦部的神經(jīng)元組織產(chǎn)生饑餓感。饑餓可以誘導(dǎo)細(xì)胞產(chǎn)生凋亡,它有兩個轉(zhuǎn)移方向,一是往好的方向轉(zhuǎn)化:即恢復(fù)生存,二是往壞的方向轉(zhuǎn)化:即進(jìn)入死亡。從病理生理學(xué)上來講,再灌注損傷通常發(fā)生在缺血期后血液供應(yīng)重新返回腦組織時。與缺血性損傷相比,局部缺血/再灌注(I/R)可能是引起線粒體呼吸鏈反應(yīng)的破壞以及腦部組織腦損傷區(qū)域中炎性介質(zhì)的過量產(chǎn)生以及包漿中的Ca2+離子大量沉積從而對神經(jīng)元造成的更多的損害;在臨床外科上,有包括急性缺血性中風(fēng)以及急性創(chuàng)傷性腦損傷等很多種腦部疾病,都可以引起缺血以及再灌注損傷。再灌注損傷在臨床上被認(rèn)為是繼發(fā)性腦損傷的重要因素。線粒體在調(diào)節(jié)細(xì)胞生命活動過程中起著重要的作用,它在細(xì)胞中以較高的速度不停的運動著,通過自我分裂或者彼此間相互融合,實現(xiàn)新個體的產(chǎn)生,并可以在細(xì)胞中形成一種緊密的聯(lián)系。線粒體通常被人們稱作“細(xì)胞動力工廠”,其分裂和融合是否平衡,影響著功能和結(jié)構(gòu)的變化。經(jīng)過以前的研究表明,線粒體可以減少細(xì)胞色素C、以及調(diào)亡蛋白AFI因子等的表達(dá),從而在細(xì)胞的程序性死亡中起著重要的調(diào)控作用。我們通過對大鼠短暫性腦I/R損傷模型的分析表明,線粒體分裂的多少與細(xì)胞凋亡的程度有著密切的關(guān)聯(lián),抑制線粒體的分裂能夠減輕腦皮質(zhì)部分的I/R損傷。另外,有研究證實:線粒體的分裂過程中,鈣信號通路可以對線粒體分裂相關(guān)蛋白Drpl的磷酸化與去磷酸化產(chǎn)生影響,進(jìn)而對線粒體的分裂起到調(diào)控作用;因此如果減少線粒體攝取鈣離子的量,就可以降低線粒體的分裂能力,減少分裂線粒體的分裂次數(shù);在我們以前的實驗中也曾發(fā)現(xiàn),降低MCU活性,對腦梗死面積及自由氧產(chǎn)生有一定的抑制作用,減輕線粒體腫脹損傷。這點在大鼠短暫性I/R損傷模型中得到了很好的體現(xiàn)。右美托咪定是一種通過選擇性激動發(fā)揮其作用的鎮(zhèn)靜劑以及鎮(zhèn)痛劑,主要作用于α2腎上腺素受體。以前的研究已經(jīng)證明過,右美托咪定可防止缺血再灌注損傷誘導(dǎo)的對心肌,腎,腸,肝,肺以及腦的損傷。同時Kuhmonen等人發(fā)現(xiàn)右美托咪定能夠?qū)Υ笫笾心X動脈閉塞引起的腦梗死有很大的益處。右美托咪定還可以減少炎性介質(zhì)誘導(dǎo)的Ca2+釋放以及鈣超載減少炎癥的繼續(xù)進(jìn)展。最新研究表明右美托咪定可以抑制氧糖剝奪或者Ca2+誘導(dǎo)線粒體腫脹而保持線粒體形態(tài)與功能的穩(wěn)定,抑制海馬神經(jīng)元鈣超載,減少鈣調(diào)神經(jīng)磷酸酶的激活,進(jìn)而抑制線粒體分裂,其機制可能是通過抑制鈣調(diào)神經(jīng)磷酸酶的活性,減少其對Drp1-ser637的磷酸化,進(jìn)而抑制Drp1的轉(zhuǎn)位,減少與線粒體外膜蛋白Fis1的結(jié)合;另外,它還可以抑制Drp1-ser637與Fis1的表達(dá),減少它們在線粒體外膜的共定位結(jié)合,從而抑制線粒體分裂的啟動。凋亡是I/R誘導(dǎo)的神經(jīng)元損傷中的主要途徑。線粒體在涉及細(xì)胞凋亡調(diào)節(jié)的內(nèi)在途徑中起重要作用。I/R誘發(fā)引起來炎癥因子可以引起線粒體的結(jié)構(gòu)和功能異常,導(dǎo)致Cyt C從線粒體釋放到細(xì)胞質(zhì)中和隨后級聯(lián)激活半胱天冬酶-9,-3和-6,進(jìn)而引起相關(guān)的凋亡級聯(lián)反應(yīng),Caspase-3的激活是線粒體凋亡必須經(jīng)歷的過程,它通過與其他家族成員的級聯(lián)反應(yīng),可以加速細(xì)胞的凋亡。因此我們就假設(shè)右美托咪定可以通過抑制鈣超載來減少線粒體分裂、保持線粒體形態(tài)與功能的穩(wěn)定,進(jìn)而通過抑制線粒體-細(xì)胞色素C-半胱氨酸蛋白酶凋亡途徑,來減少神經(jīng)元細(xì)胞的凋亡,從而發(fā)揮腦保護(hù)作用。目的:探討右美托咪定在大鼠海馬神經(jīng)元缺氧復(fù)氧損傷中的作用,以及右美托咪定對線粒體分裂的的影響。方法:新生24h之內(nèi)的SD大鼠,斷頭分離大腦海馬區(qū)神經(jīng)組織,收集獲得神經(jīng)元細(xì)胞進(jìn)行原代海馬神經(jīng)元培養(yǎng),培養(yǎng)至第8天,氧糖剝奪法建立海馬神經(jīng)元缺氧復(fù)氧模型,按照隨機數(shù)字表法將其隨機分為6組::空白對照組(C組),細(xì)胞未給予任何處理;賦形劑組(V組),賦形劑二甲基亞砜(DMSO),終濃度為0.01%]加入細(xì)胞培養(yǎng)基;缺氧復(fù)氧組(H/R組);右美托咪定組:D1、D2、D3組,在細(xì)胞缺氧復(fù)氧期間分別加入右美托咪定0.1、1、10μmol/L。分組處理之后,用細(xì)胞增殖與毒性檢測試劑盒檢測各組細(xì)胞活性(確定右美托咪定合適的濃度范圍)、用透射電鏡觀察線粒體的超微結(jié)構(gòu)、用激光共聚焦顯微鏡觀察各組神經(jīng)元細(xì)胞質(zhì)Ca2+熒光強度、用ELISA法檢測細(xì)胞鈣調(diào)神經(jīng)磷酸酶活性、用Western-blot檢測Drp1、Fis1、Cyt C、capase3蛋白的表達(dá)。用流式細(xì)胞學(xué)檢測神經(jīng)元細(xì)胞的凋亡率。結(jié)果:與對照組比較,H/R組的細(xì)胞數(shù)目以及活性顯著減少,與H/R組比較,右美托咪定組(D1、D2、D3)細(xì)胞數(shù)量相對增加,并且D2組的細(xì)胞活性比D1和D3相對要高。與對照組相比,H/R組的細(xì)胞凋亡率顯著增多,與H/R組比較,右美托咪定組(D1、D2、D3)細(xì)胞凋亡率相對降低,并且D2組的細(xì)胞凋亡率比D1和D3相對要低。與對照組相比,神經(jīng)元Ca2+熒光強度、鈣調(diào)神經(jīng)磷酸酶活性及Drp1、Fis1、Cyt C、caspase3蛋白的表達(dá)升高(P0.05);與H/R組比較,D1、D2、D3組神經(jīng)元活性增強、線粒體超微結(jié)構(gòu)破壞明減輕,神經(jīng)元Ca2+熒光強度、鈣調(diào)神經(jīng)磷酸酶活性及Drp1、Fis1、Cyt C、caspase3的表達(dá)均降低(P0.05);D2組又較D1、D3組細(xì)胞活性增強,細(xì)胞質(zhì)Ca2+熒光強度、鈣調(diào)神經(jīng)磷酸酶活性及Drp1、Fis1、Cyt C、caspase3的表達(dá)均降低(P0.05)。結(jié)論:右美托咪定0.1、1、10μmol/L可以明顯改善大鼠海馬神經(jīng)元在缺氧復(fù)氧中的損傷,其中1μmol/L是最佳的保護(hù)濃度,其機制可能是與右美托咪定抑制鈣超載介導(dǎo)的線粒體分裂及線粒體凋亡途徑有關(guān)系。
[Abstract]:Cerebral ischemia is a disease of the brain tissue with insufficient blood flow, which usually leads to the metabolic rate of brain tissue and the decrease of energy, and the consequent cerebral infarction in the local area. When the condition of cerebral ischemia occurs, the brain tissue may be caused by the reduction of oxygen from the blood and the supply of nutrients. Starvation produces a sense of hunger. Starvation can induce cell apoptosis. It has two direction of metastasis, one is to turn in a good direction, that is to restore survival, and the two is to turn into the bad direction: into death. From pathophysiology, reperfusion injury usually occurs after the ischemia period, and the blood supply returns to the brain tissue. Local ischemia / reperfusion (I/R) may be the damage to the mitochondrial respiratory chain reaction and the excessive production of inflammatory mediators in the brain tissue of the brain tissue and the deposition of Ca2+ ions in the plasma in the plasma, thus causing more damage to the neurons. In clinical surgery, acute ischemic stroke and acute traumatic brain damage are included. Many kinds of brain diseases, such as injuries, can cause ischemia and reperfusion injury. Reperfusion injury is considered to be an important factor in secondary brain damage in clinical. Mitochondria play an important role in regulating cell life activities. It is moving at a high speed in cells, by self splitting or by each other. Interfusion, the production of new individuals, and the formation of a close association in cells. Mitochondria are often called "cell power plants", and their division and fusion are balanced, affecting the changes in function and structure. After previous studies, mitochondria can reduce cytochrome C, and AFI factor, and so on. Expression, which plays an important role in the programmed cell death of the cell, has been analyzed by the analysis of the transient brain I/R damage model in rats. It is shown that the number of mitochondrial divisions is closely related to the degree of apoptosis, and the inhibition of mitochondrial division can reduce the I/R damage in the cortical part of the brain. The calcium signaling pathway can affect the phosphorylation and dephosphorylation of mitochondrial mitotic protein Drpl and regulate the mitosis of mitochondria. Therefore, if the amount of calcium ions in mitochondria is reduced, the splitting ability of mitochondria can be reduced and the number of split mitochondria is reduced. In our previous experiments, it was also found that reducing the activity of MCU has a certain inhibitory effect on the area of cerebral infarction and the production of free oxygen and alleviated the swelling and injury of mitochondria. This is a good manifestation of the transient I/R damage model in rats. Right metoimidin is a sedative and analgesic agent that plays its role by selective excitation. A previous study has shown that dexmedetomidine can prevent damage to the myocardium, kidney, intestines, liver, lungs, and brain induced by ischemia reperfusion injury. Kuhmonen et al. Found that right metoimidin could be of great benefit to cerebral infarction caused by occlusion of the middle cerebral artery in rats. Right metomomidin can also be used. Reduction of inflammatory mediators induced Ca2+ release and calcium overload to reduce inflammation. The latest research shows that dexmedetomidine can inhibit oxygen glucose deprivation or Ca2+ induced mitochondrial swelling to maintain mitochondrial morphologic and functional stability, inhibit calcium overload in hippocampal neurons, reduce the activation of calcineurin, and then inhibit line particles. The mechanism may be to inhibit the phosphorylation of Drp1-ser637 by inhibiting the activity of calcineurin, thus inhibiting the transposition of Drp1 and reducing the binding of the mitochondrial membrane protein Fis1. In addition, it also inhibits the expression of Drp1-ser637 and Fis1, and reduces the co localization of the mitochondria in the outer membrane of the mitochondria, and thus inhibits the line particles. Apoptosis is the main pathway of I/R induced neuronal damage. Mitochondria play an important role in the internal pathway involving apoptosis regulation..I/R induces the structure and function of mitochondria caused by inflammatory factors, resulting in the release of Cyt C from mitochondria to cytoplasm and subsequent cascade activation of cysteine and asparagus. The enzyme -9, -3 and -6 cause the associated apoptosis cascade reaction, and the activation of Caspase-3 is the necessary process of mitochondrial apoptosis. It can accelerate cell apoptosis by cascade reaction with other family members. Therefore, we hypothesized that dexmedetomidin could reduce mitochondrial division by inhibiting calcium overload and maintain mitochondrial morphology. Function stability, and then by inhibiting the mitochondrial cytochrome C- cysteine protease apoptosis pathway, to reduce the apoptosis of neuron cells and to play the role of brain protection. Objective: To explore the role of dexmedetomidine in the hypoxia reoxygenation injury of rat hippocampal neurons, and the effect of right metoimidin on mitochondrial division. In the SD rats of 24h, the nerve tissue in the hippocampus of the brain was separated and the neuron cells were collected and cultured for eighth days. The oxygen deprivation method was used to establish the hypoxic reoxygenation model of hippocampal neurons. According to the random number table method, the neurons were randomly divided into 6 groups: the blank control group (group C), the cells were not given any treatment; the cells were not treated with any treatment; The form agent group (group V), excipient two methyl sulfoxide (DMSO), the final concentration was 0.01%] added to cell culture medium, anoxic reoxygenation group (group H/R), right metoimidin group: D1, D2, D3 group, after cell hypoxia reoxygenation was added to right metomomidin 0.1,1,10 u mol/L. group treatment, the cell activity was detected by cell proliferation and toxicity detection kit. The ultrastructure of mitochondria was observed by transmission electron microscopy. The cytoplasmic Ca2+ fluorescence intensity of each group was observed by laser confocal microscopy. The activity of calcineurin was detected by ELISA. The expression of Drp1, Fis1, Cyt C, capase3 protein was detected by Western-blot. Flow cytometry was used to detect the expression of the protein. Compared with the control group, the number and activity of the cells in the H/R group decreased significantly. Compared with the H/R group, the number of D1, D2, D3 cells in the right metoimidin group increased relatively, and the cell activity of the D2 group was higher than that of the D1 and D3. Compared with the control group, the apoptosis rate of the H /R group was significantly increased, compared with the H/R group, right metoami was compared. The apoptosis rate of D1, D2, D3 was relatively lower, and the apoptosis rate of D2 group was lower than that of D1 and D3. Compared with the control group, the neuronal Ca2+ fluorescence intensity, calcineurin activity and Drp1, Fis1, Cyt C, and the expression of Caspase3 protein increased. The fluorescence intensity of Ca2+, the activity of calcineurin and the expression of Drp1, Fis1, Cyt C, Caspase3 were all decreased (P0.05), and the activity of the D2 group was more than D1, and the cell activity of the D3 group was enhanced, the cytoplasm Ca2+ fluorescence intensity, the activity of calcineurin and the Drp1, and the expression were all decreased. L can significantly improve the damage of rat hippocampal neurons in hypoxia and reoxygenation, in which 1 mol/L is the best protective concentration. The mechanism may be related to the pathway of mitochondria division and mitochondrial apoptosis mediated by dexmedetomidine inhibiting calcium overload.

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R614

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