本文選題：RGMa + 6FNIII　； 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：背景與目的:腦血管病是對人類健康極具威脅的常見病,是目前人類三大致死率最高的疾病之一,而缺血性腦血管病是其主要類型。腦組織缺血將會導(dǎo)致腦組織損害,而腦缺血后再灌注過程往往加重這一損害。自噬(autophagy)是真核細胞中一種高度保守的,形成囊泡并與溶酶體結(jié)合后降解長效蛋白、受損蛋白及受損細胞器,為細胞的代謝、發(fā)育提供養(yǎng)分和能量,維持細胞內(nèi)穩(wěn)態(tài)的過程。大量研究發(fā)現(xiàn),腦缺血再灌注可誘發(fā)細胞自噬,且自噬過程基本發(fā)生于神經(jīng)元,主要與饑餓、生長因子缺乏、病原體感染等有關(guān),是腦缺血再灌注損傷的重要環(huán)節(jié),此過程受一系列復(fù)雜的信號分子調(diào)控,其中Beclin1分子是自噬調(diào)控通路中最主要的分子之一,其與PIK3C3結(jié)合形成Beclin1-PIK3C3復(fù)合物,廣泛參與自噬體的形成與成熟。不少研究者發(fā)現(xiàn),其目的蛋白通過作用于Beclin1-PIK3C3復(fù)合物影響自噬過程,此過程稱為“Beclin1依賴性自噬”,而部分研究者發(fā)現(xiàn)其目的蛋白不通過Beclin1作用影響自噬,此過程稱為“Beclin1非依賴性自噬”。近年來有研究發(fā)現(xiàn)腦缺血再灌注損傷后可導(dǎo)致的神經(jīng)元自噬過程,而Beclin1非依賴性自噬與促進凋亡水平、抑制腦功能恢復(fù)有關(guān)。因此,自噬可能成為未來治療腦缺血性疾病的重要靶點。排斥導(dǎo)向分子a(Repulsive guidance molecule a,RGMa)是一種排斥性軸突導(dǎo)向分子,大量研究發(fā)現(xiàn),腦缺血再灌注后RGMa表達增多,正是由于RGMa等一系列軸突生長抑制因子的存在,使得中樞神經(jīng)系統(tǒng)(CNS)損傷后神經(jīng)元修復(fù)及神經(jīng)功能恢復(fù)極為困難。我們之前的研究發(fā)現(xiàn),在大鼠腦缺血損傷后干預(yù)RGMa表達,可顯著改善缺血再灌注腦損傷軸突的生長情況并有效促進神經(jīng)功能恢復(fù)。6FNIII為RGMa受體neogenin分子中6個纖維蛋白Ⅲ樣結(jié)構(gòu)域,外源性6FNIII在體內(nèi)可以和RGMa分子結(jié)合而抑制其生物學作用的發(fā)揮。本實驗利用6FNIII對RGMa/Neogenin通路進行干預(yù)以及重組腺病毒抑制RGMa蛋白表達兩種干預(yù)手段探討RGMa對大鼠大腦皮質(zhì)神經(jīng)元缺血再灌注急性期引發(fā)的自噬以及神經(jīng)功能恢復(fù)情況的影響,同時對RGMa影響自噬的機制作初步探索。方法:1.將30只雄性成年SD大鼠隨機分為5組:假手術(shù)組(sham)、腦缺血再灌注組(I/R)、腦缺血再灌注加低濃度6FNIII干預(yù)組(I/R+L-6FNIII)、腦缺血再灌注加中濃度6FNIII干預(yù)組(I/R+M-6FNIII)、腦缺血再灌注加高濃度6FNIII干預(yù)組(I/R+H-6FNIII),立體定位側(cè)腦室注射等體積不同濃度6FNⅢ后立即建立大腦中動脈阻塞(MCAO)模型,所有組于再灌注后24h,采用Western-blot檢測RGMa下游分子CRMP-2蛋白表達水平,確定最佳6FNIII干預(yù)濃度。2.將108只雄性成年SD大鼠隨機分成6組:假手術(shù)組(sham)、腦缺血再灌注組(I/R)、腦缺血再灌注加生理鹽水注射組(I/R+NS)、腦缺血再灌注加6FNIII干預(yù)組(I/R+6FNIII)、腦缺血再灌注加空載體腺病毒注射組(I/R+r Ad-HK)及腦缺血再灌注加RGMa重組腺病毒干預(yù)組(I/R+r Ad-sh RGMa)。6FNIII干預(yù)組及NS注射組于6FNIII或NS側(cè)腦室注射后立即建立MCAO模型,腺病毒干預(yù)組于RGMa病毒大腦皮質(zhì)注射后3天建立MCAO模型,所有組于再灌注后24h,采用Western blot檢測腦組織自噬相關(guān)蛋白LC3II/I表達水平;免疫熒光雙標觀察LC3與神經(jīng)元的共表達情況;透射電鏡觀察自噬體的形成情況;神經(jīng)功能缺損評分估計神經(jīng)功能恢復(fù)情況,TTC染色法檢測腦梗死體積。然后采用Co-IP檢測RGMa干預(yù)后Beclin1與PIK3C3結(jié)合有無改變;Co-IP檢測缺血再灌注后RGMa是否與Beclin1或PIK3C3結(jié)合。結(jié)果:1.大鼠腦缺血再灌注后24h,I/R組、I/R+NS組及I/R+r Ad-HK組較sham組自噬相關(guān)蛋白LC3II/I水平升高(P0.05)、自噬體形成增多、腦梗死體積擴大(P0.05)、神經(jīng)功能評分降低(P0.05)、;I/R+6FNIII組較I/R組、I/R+NS組自噬相關(guān)蛋白LC3II/I水平降低(P0.05)、自噬體形成減少、腦梗死體積縮小(P0.05)、神經(jīng)功能評分增高(P0.05);I/R+r Ad-sh RGMa組較I/R組、I/R+NS組、I/R+r Ad-HK組自噬相關(guān)蛋白LC3II/I水平降低(P0.05)、自噬體形成減少、腦梗死體積縮小(P0.05)、神經(jīng)功能評分增高(P0.05);而I/R+NS組、I/R+r Ad-HK組較I/R組以上各項均無統(tǒng)計學意義(P0.05)。2.大鼠腦缺血再灌注后24h,I/R組RGMa與Beclin1及PIK3C3均無結(jié)合;I/R+6FNIII組可見Beclin1與PIK3C3結(jié)合。結(jié)論:在缺血再灌注急性期,外源性6FNIII可有效抑制RGMa生物學作用的發(fā)揮。RGMa干預(yù)后自噬水平降低、促進神經(jīng)功能恢復(fù),提示腦梗死急性期RGMa可能通過促進自噬而參與抑制神經(jīng)功能恢復(fù)的過程。缺血再灌注后RGMa與Beclin1及PIK3C3均無結(jié)合,RGMa干預(yù)后仍可見Beclin1與PIK3C3結(jié)合,提示腦梗死急性期RGMa可能通過Beclin1非依耐途徑參與自噬的調(diào)控。
[Abstract]:Background and purpose: cerebrovascular disease is a common disease which is very dangerous to human health. It is one of the three most fatal diseases of human being, and ischemic cerebrovascular disease is the main type. Cerebral ischemia will cause brain tissue damage, and cerebral ischemia reperfusion process often aggravates this damage. Autophagy (autophagy) is a eukaryotic cell. A highly conserved cell that forms vesicles and combines with lysosomes to degrade long acting proteins, damaged proteins and damaged organelles, the metabolism of cells, the development of nutrients and energy, and the process of maintaining intracellular homeostasis. A large number of studies have found that cerebral ischemia reperfusion can induce autophagy, and the process of autophagy mainly occurs in neurons, mainly with hunger. Starvation, lack of growth factor and infection of pathogens are important links of cerebral ischemia reperfusion injury. This process is regulated by a series of complex signal molecules, in which Beclin1 is one of the most important molecules in autophagy regulation pathway. It is combined with PIK3C3 to form Beclin1-PIK3C3 complex, and is widely involved in the formation and maturation of autophagic. Few researchers have found that the target protein affects autophagy by acting on the Beclin1-PIK3C3 complex, known as "Beclin1 dependent autophagy," and some researchers found that the target protein does not affect autophagy through the action of Beclin1. This process is called "Beclin1 independent autophagy." in recent years there has been a study of cerebral ischemia and reperfusion. Beclin1 non dependent autophagy may be associated with the level of apoptosis and the inhibition of the recovery of brain function. Therefore, autophagy may become an important target for the treatment of cerebral ischemic disease in the future. The rejection guide molecule a (Repulsive guidance molecule a, RGMa) is a kind of repellent axon guiding molecule, and a large number of studies have been made. Now, the expression of RGMa increases after cerebral ischemia and reperfusion, which is due to the existence of a series of axon growth inhibitors such as RGMa, which makes the neuron repair and neural function recovery of the central nervous system (CNS) damaged extremely difficult. Our previous study found that the intervention of RGMa expression after cerebral ischemia injury in rats could significantly improve the cerebral ischemia reperfusion injury. The growth of the axon injury and the effective promotion of neural function recovery.6FNIII are 6 fibrin III like domains of the RGMa receptor neogenin molecule. Exogenous 6FNIII can combine with RGMa molecules in vivo to inhibit its biological function. This experiment uses 6FNIII to interfere with RGMa/Neogenin pathway and recombinant adenovirus to inhibit RGMa The effect of RGMa on the autophagy and the recovery of neural function induced by cerebral cortical neuron ischemia reperfusion in rats was investigated by two means of protein expression. At the same time, preliminary exploration was made on the mechanism of RGMa affecting autophagy. Method: 1. 30 adult male adult SD rats were randomly divided into 5 groups: sham operation group (sham), cerebral ischemia reperfusion group (I/R), Cerebral ischemia reperfusion and low concentration 6FNIII intervention group (I/R+L-6FNIII), cerebral ischemia reperfusion and middle concentration 6FNIII intervention group (I/R+M-6FNIII), cerebral ischemia reperfusion and high concentration 6FNIII intervention group (I/R+H-6FNIII), stereotactic lateral ventricle injection of 6FN III of different concentration of 6FN III, immediately established the middle cerebral artery occlusion (MCAO) model, all groups were re After perfusion 24h, Western-blot was used to detect the expression of CRMP-2 protein in the downstream of RGMa, and the best 6FNIII intervention concentration.2. was determined to divide 108 adult male adult SD rats into 6 groups randomly: sham operation group (sham), cerebral ischemia reperfusion group (I/R), cerebral ischemia reperfusion plus physiological saline injection group (I/R+NS), cerebral ischemia-reperfusion plus 6FNIII intervention group (I/R+6FNI) II), cerebral ischemia reperfusion and adenovirus injection group (I/R+r Ad-HK) and cerebral ischemia reperfusion plus RGMa recombinant adenovirus intervention group (I/R+r Ad-sh RGMa).6FNIII intervention group and NS injection group were injected immediately after the 6FNIII or NS lateral ventricle injection, and the adenovirus intervention group established the model 3 days after the injection of the cerebral cortex of the virus. After reperfusion 24h, Western blot was used to detect the expression of autophagy related protein LC3II/I expression in brain tissue; the co expression of LC3 and neurons was observed by double immunofluorescence; transmission electron microscopy was used to observe the formation of autophagic body; neural function defect score was used to estimate the recovery of nerve function, and TTC staining method was used to detect the volume of cerebral infarction. Then Co-I was used to detect the volume of cerebral infarction. Then Co-I was used to detect the volume of cerebral infarction. P detected the combination of Beclin1 and PIK3C3 after RGMa intervention; Co-IP was used to detect the combination of RGMa with Beclin1 or PIK3C3 after ischemia-reperfusion. Results: the levels of autophagic related proteins in the 1. rats were higher than those in the group of 24h, I/R, I/R+NS and I/R+r. The neurological function score was reduced (P0.05), and in the group I/R+6FNIII, the LC3II/I level of autophagic related proteins decreased (P0.05), the formation of autophagic bodies decreased, the volume of cerebral infarction decreased (P0.05) and the neurological function score increased (P0.05) in the group I/R+NS, and the I/R+r Ad-sh RGMa group was lower than that of the I/R group. The volume of body formation decreased, the volume of cerebral infarction was reduced (P0.05), and the score of nerve function increased (P0.05), while in group I/R+NS and I/R+r Ad-HK, there was no statistical significance in group I/R above group I/R (P0.05), and 24h in.2. rats after cerebral ischemia-reperfusion. During the sexual phase, exogenous 6FNIII could effectively inhibit the biological action of RGMa, and the level of autophagy decreased after.RGMa intervention, promoting the recovery of nerve function, suggesting that RGMa may participate in the process of inhibiting the recovery of nerve function by promoting autophagy at acute stage of cerebral infarction. RGMa and Beclin1 and PIK3C3 are not combined after ischemia-reperfusion, and Becli still can be seen after RGMa intervention. The combination of N1 and PIK3C3 suggests that RGMa may participate in the regulation of autophagy through the Beclin1 non dependent pathway in acute phase of cerebral infarction.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743

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