發(fā)布時間：2018-06-09 07:07

  本文選題：腦出血 + 磺酰脲類受體1�。� 參考：《第三軍醫(yī)大學》2017年碩士論文

【摘要】：腦水腫是腦出血(intracerebral hemorrhage,ICH)后繼發(fā)腦損傷的關鍵環(huán)節(jié),而血腦屏障(blood-brain barrier,BBB)的破壞是ICH后腦水腫重要的始動因素。研究保護血腦屏障完整性的有效措施對減輕臨床腦出血后的繼發(fā)腦損傷具有重要意義。格列本脲(Glibenclamide,GLC)為磺酰脲類藥物,被臨床用于口服降糖已有數(shù)十年;磺酰脲類藥物都是通過阻斷磺酰脲類受體1(sulfonylurea receptor 1,Sur1)發(fā)揮藥理作用。磺酰脲類受體1參與構(gòu)成并調(diào)節(jié)Sur1-Kir6.2和Sur1-Trpm4兩種離子通道。Sur1-Kir6.2通道和Sur1-Trpm4通道有相反的生理學作用,Sur1-Kir6.2通道開放細胞超激化,Sur1-Trpm4通道開放時細胞去激化,細胞超激化和去激化參與不同的生理過程;Sur1-Trpm4通道去極化具有減少病理性鈣離子通過電壓門控通道內(nèi)流的作用,但如果失去正常調(diào)節(jié),過度開放陽離子會通過該通道內(nèi)流引起細胞毒性水腫和細胞壞死;Sur1-Kir6.2通道開放細胞超激化具有減少鈣離子通過電壓門控通道內(nèi)流的重要作用,但如果過度開放,ATP耗竭引起神經(jīng)元能量代謝紊亂并減慢小膠質(zhì)細胞對外界刺激的反應速度。最近格列本脲通過阻斷磺酰脲類受體1在缺血性卒中、脊髓損傷、顱內(nèi)轉(zhuǎn)移瘤、蛛網(wǎng)膜下腔出血等中樞神經(jīng)系統(tǒng)疾病模型研究中發(fā)揮神經(jīng)保護效應而被廣泛關注,糖尿病合并卒中患者回顧性研究分析發(fā)現(xiàn):卒中后磺酰脲類藥物的使用可獲得更好的預后。課題組前期研究成果及相關研究報道基質(zhì)金屬蛋白酶(metalloproteinases,MMPs)在腦出血后血腦屏障完整性破壞中起重要作用,格列本脲能減少基質(zhì)金屬蛋白酶的表達或降低其活性。然而,格列本脲在腦出血中的作用目前仍不清楚。本課題假設格列本脲能通過阻斷Sur1-Trpm4通道抑制基質(zhì)金屬蛋白酶表達、保護血腦屏障緊密連接蛋白從而發(fā)揮潛在的神經(jīng)保護效應;課題選用大鼠自體血注射腦出血模型進行實驗研究,觀察腦出后神經(jīng)功能缺損、血腦屏障破壞、腦水腫的變化特征;并以血腫周圍腦組織Sur1表達上調(diào)為前提,觀察探索格列苯脲對大鼠腦出血后腦水腫、血腦屏障、神經(jīng)功能的干預效應和相關機制。為此,將課題分成了兩個研究部分來驗證上述假設:第一部分,建立成年SD大鼠腦出血模型,觀察出血損傷對大鼠腦組織磺酰脲類受體1表達的影響;第二部分,建立大鼠腦出血模型后,給予格列本脲干預,觀察格列本脲對腦出血模型后腦水含量、神經(jīng)功能恢復、血腦屏障完整性、基質(zhì)金屬蛋白酶表達的影響。一、大鼠腦出血后血腫周圍Sur1表達目的通過自體血注入方法建立大鼠ICH模型,動態(tài)觀察出血后Sur1及相關離子通道表達變化,評估出血損傷對血腫周圍腦組織Sur1表達的調(diào)節(jié)。方法實驗動物隨機分成ICH組和Sham組;ICH組將自體血(取自實驗大鼠股動脈)100μL立即緩慢(約10μL/min)注入右側(cè)基底神經(jīng)節(jié);對照組與ICH組進行同樣的手術過程,但不注入自體血液。于術后12h、24h、48h收集腦標本行PCR,術后24h收集腦標本行Western blot檢測、免疫熒光染色。結(jié)果大鼠實驗性自體血腦出血模型血腫周圍腦組織Sur1表達明顯上調(diào),但Kir6.2通道蛋白未探及表達增加。因此推斷:出血性損傷后,血腫周圍腦組織Sur1表達上調(diào)主要是伴隨Sur1-Trpm4通道的表達上調(diào)。結(jié)論出血性損傷后,血腫周圍腦組織Sur1-Trpm4通道表達上調(diào)。二、大鼠腦出血后格列本脲的干預效應目的建立成年SD大鼠腦出血模型,給予格列本脲干預;觀察大鼠ICH后的神經(jīng)功能缺損、腦水腫等情況以及運用格列本脲干預的神經(jīng)保護效應,探討格列本脲神經(jīng)保護效應的可能機制。方法將實驗動物隨機分為對照組(Sham組)、ICH+Vehicle(Vehicle組)、ICH+GLC(GLC組);大鼠腦出血模型建立后CLC組立即給予格列本脲10μg/kg腹腔注射,并皮下植入微量泵以200ng/h的速度持續(xù)泵入(建模后15分鐘內(nèi)完成給藥);Vehicle組用相同方式給予相同劑量的溶劑。在建模后24小時、72小時測定腦組織水含量;建模后每天行改良神經(jīng)功能評分(m NSS);建模后23天開始行水迷宮測試訓練;建模后24小時行Western blotting免疫印跡分析MMP9表達情況,72小時行Western blotting免疫印跡分析ZO-1、occludin表達情況;建模72小時切片行免疫熒光觀察血腦屏障完整性、MMP9的表達;建模后72小時切片觀察伊文思藍(Evans-blue)滲漏;建模后12小時、24小時、48小時收集標本行PCR檢測MMP2、MMP9、MMP12 m RNA表達變化。結(jié)果1.格列本脲干預能減輕ICH后腦水含量,促進ICH后神經(jīng)功能(改良的神經(jīng)功能評分)恢復。2.格列本脲干預減少ICH后血腫周圍腦組織伊文思藍滲漏。3.格列本脲干預能改善ICH模型實驗大鼠空間學習記憶能力(通過水迷宮實驗測試)中的表現(xiàn)。4.格列本脲干預保護ICH后血腦屏障的完整性。5.格列本脲干預能減少ICH后血腫周圍腦組織MMPs的表達。結(jié)論在大鼠腦出血模型實驗中觀察到:格列本脲能保護血腦屏障、改善腦水腫、改善神經(jīng)功能,并且這種神經(jīng)保護作用可能與格列本脲減少MMPs的表達有關。
[Abstract]:Cerebral edema is the key link of brain injury after intracerebral hemorrhage (ICH), and the destruction of blood-brain barrier (BBB) is an important starting factor for the cerebral edema after ICH. The effective measures to protect the integrity of the blood brain barrier are of great significance to reduce the secondary brain injury after clinical cerebral hemorrhage. Glibenclamide, GLC) is a sulfonylurea drug that has been clinically used for oral hypoglycemic for decades; sulfonylurea has been involved in pharmacological action by blocking sulfonylurea receptor 1 (sulfonylurea receptor 1, Sur1). Sulfonylurea receptor 1 participates in the formation and regulation of Sur1-Kir6.2 and Sur1-Trpm4 two ion channels.Sur1-Kir6.2 channels and Sur1-Trpm The 4 channel has the opposite physiological function, the Sur1-Kir6.2 channel open cell overintensification, the Sur1-Trpm4 channel opening when the opening is open, the cell excates, the cell overintensification and the intensification participates in the different physiological processes; the Sur1-Trpm4 channel depolarization can reduce the role of the pathological calcium ion through the voltage gated channel, but if the normal regulation is lost, it is excessive. Open cations can cause cytotoxic edema and cell necrosis through the flow of the channel. The overactivation of Sur1-Kir6.2 channel open cells has an important role in reducing the flow of calcium ions through the voltage-gated channel. However, if overopen, the depletion of ATP causes the energy metabolism disorder of neurons and slows the response of microglia to external stimuli. Neuroprotective effects of sulfonylurea receptor 1 in ischemic stroke, spinal cord injury, intracranial metastases, subarachnoid hemorrhage and other central nervous system disease models are widely concerned. A retrospective study of diabetes combined with stroke patients has been found: the use of sulfonylureas after stroke Metalloproteinases (MMPs) plays an important role in the integrity destruction of the blood brain barrier after intracerebral hemorrhage. Glibenclamide can reduce the expression of matrix metalloproteinase or reduce its viability. However, the role of glibenclamide in cerebral hemorrhage is present. It is still unclear. It is hypothesize that glibenclamide can inhibit the expression of matrix metalloproteinase by blocking the Sur1-Trpm4 channel and protect the close connexin of blood brain barrier and thus play a potential neuroprotective effect. The changes of brain edema and the up-regulation of Sur1 expression in the brain tissue around the hematoma were used to observe the intervention effects and related mechanisms of glibenclamide on cerebral edema, blood brain barrier and nerve function in rats after intracerebral hemorrhage. To this end, the subjects were divided into two parts to verify the above hypothesis: the first part was to establish the brain of adult SD rats. Blood model, observe the effect of bleeding injury on the expression of sulfonylureas receptor 1 in rat brain tissue; the second part, after establishing rat model of cerebral hemorrhage, the effect of glibenclamide on brain water content, nerve function recovery, blood brain barrier integrity and expression of matrix metalloproteinase after cerebral hemorrhage model. The expression of Sur1 in the posterior hematoma was established by autologous blood injection to establish the rat ICH model. The changes of Sur1 and related ion channel expression after bleeding were dynamically observed and the expression of Sur1 in the brain tissue around hematoma was regulated by bleeding injury. Methods the experimental animals were randomly divided into ICH and Sham groups, and the ICH group took the autologous blood (from the femoral artery of experimental rats) 100 L was injected into the right basal ganglia immediately and slowly (about 10 L/min); the control group performed the same procedure with the ICH group, but did not infuse the autologous blood. After the operation, 12h, 24h, 48h collected the brain line PCR, and the 24h collection was collected by Western blot detection, immunofluorescence staining. The expression of Sur1 was obviously up-regulated, but the expression of Kir6.2 channel protein was not detected and expressed. Therefore, after hemorrhagic injury, the up regulation of Sur1 expression around the hematoma was mainly associated with the up regulation of the expression of the Sur1-Trpm4 channel. Conclusion the expression of Sur1-Trpm4 channel in the brain tissue around hematoma is up to up after hemorrhagic injury. Two, glibenclamide after intracerebral hemorrhage in rats. Objective to establish the cerebral hemorrhage model of adult SD rats and give glibenclamide intervention, observe the nerve function defect, brain edema after ICH and the neuroprotective effect of glibenclamide, and explore the possible mechanism of neuroprotective effect of glibenclamide. Methods the experimental animals were randomly divided into control group (group Sham), ICH+Ve Hicle (group Vehicle), ICH+GLC (group GLC); after the rat model of cerebral hemorrhage was established, the CLC group was given an injection of glipezourea 10 u g/kg, and the micropump was implanted subcutaneously for 200ng/h at the rate of 200ng/h, and the Vehicle group was given the same dose of solvent in the same way. The brain group was determined at 24 hours after modeling and 72 hours after modeling. Water content was improved after modeling (m NSS) every day after modeling; water maze test training was conducted 23 days after modeling. MMP9 expression was analyzed by Western blotting immunoblotting at 24 hours after modeling, ZO-1 with Western blotting immunoblotting was performed at 72 hours, and occludin table was reached, and the blood brain was observed by immunofluorescence for 72 hours of modeling. Barrier integrity, MMP9 expression, 72 hours after modeling, observation of Evans blue (Evans-blue) leakage; after modeling 12 hours, 24 hours, 48 hours, collection of specimens, PCR MMP2, MMP9, MMP12 m RNA expression changes. Results 1. glibenclamide intervention can reduce the content of the brain water after ICH, promote ICH neural function (improved neural function score) recovery.2. The intervention of glibenclamide intervention to reduce the eevith blue leakage of the peripheral brain tissue around the hematoma after ICH to improve the spatial learning and memory ability of the ICH model rats (through the water maze test) the performance of.4. glibenclamide interfered with the integrity of the blood brain barrier after the protection of ICH by.5. glibenclamide intervention to reduce the brain tissue around the hematoma after ICH. MMPs expression. Conclusion glibenclamide can protect the blood brain barrier, improve brain edema and improve the function of nerve, and this neuroprotective effect may be related to the expression of glibenclamide to reduce the expression of MMPs.
【學位授予單位】：第三軍醫(yī)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743.34

【參考文獻】

相關期刊論文 前2條

1 閆峰;吉訓明;羅玉敏;;多種實驗動物腦出血模型的制作[J];實驗動物科學;2009年01期

2 張潔茵;關健偉;;實驗性腦出血動物模型的研究現(xiàn)狀[J];廣東醫(yī)學院學報;2008年01期

，

本文編號：1999485