本文選題：OSAHS + SiRNA　； 參考：《天津醫(yī)科大學》2017年碩士論文

【摘要】：目的阻塞性睡眠呼吸暫停低通氣綜合征(OSAHS)可導致或加重認知功能障礙,其機制與間歇低氧(IH)所致海馬神經(jīng)細胞凋亡有關(guān)。我們前期研究初步發(fā)現(xiàn)間歇低氧可能是通過HIF-1α-BNIP3-Beclin1介導自噬發(fā)生,且自噬激活加重了海馬神經(jīng)細胞凋亡。但前期研究主要依賴于免疫印跡、體外藥物干預的方法去探討,由于任何單一一種檢測自噬方法均不能準確的反映細胞自噬水平,應用多種實驗方法檢測自噬發(fā)生及其分子機制是對整個研究的進一步完善和升華。因此,本研究通過建立OSAHS體外細胞模型,應用免疫熒光和透射電子顯微鏡觀察的實驗方法從形態(tài)學角度檢測間歇低氧條件下自噬的表達,通過基因沉默和免疫共沉淀的實驗方法進一步明確間歇低氧條件下HIF-1α介導自噬發(fā)生的具體機制,并應用基因沉默和藥物干預的實驗方法初步探討自噬激活加重海馬神經(jīng)細胞損傷的分子機制。方法(1)研究對象:購買出生24小時之內(nèi)的SD乳鼠,培養(yǎng)原代海馬神經(jīng)元7-9天。(2)建立OSAHS細胞模型并根據(jù)實驗不同條件進行分組:正常對照組(NC)、不同時間段的間歇低氧(IH)組。檢測指標:免疫熒光技術(shù)檢測自噬標記蛋白LC3-II的表達;透射電子顯微鏡下觀察雙層膜結(jié)構(gòu)的自噬體的形成;(3)Si RNA轉(zhuǎn)染與分組:給予Si RNA干預HIF-1α(Beclin1)表達,設(shè)陰性對照組、IH組、HIF-1αSi RNA(Beclin1 Si RNA)干預組、IH+HIF-1αSi RNA(Beclin1 Si RNA)干預組。選取HIF-1α及自噬表達變化最明顯的間歇低氧時間點12h進行處理,HIF-1α(Beclin1)si RNA預處理方法:細胞培養(yǎng)7天給予HIF-1α(Beclin1)si RNA轉(zhuǎn)染處理48h。檢測指標:疫印跡技術(shù)檢測各組海馬神經(jīng)細胞LC3-II、cleaved-caspase-3、HIF-1α-BNIP3-Beclin1信號通路相關(guān)蛋白的表達。(4)免疫共沉淀技術(shù)檢測Beclin1-Bcl2的結(jié)合狀態(tài)。(5)給予自噬抑制劑和激活劑干預已間歇低氧處理的海馬神經(jīng)細胞。分組為:正常對照組、間歇低氧組、藥物預處理組、單獨給藥組。檢測指標:免疫印跡技術(shù)檢測各組海馬神經(jīng)細胞cleaved-caspase-3和XIAP蛋白的表達變化。結(jié)果(1)免疫熒光檢測自噬的表達:隨著間歇低氧時間延長,IH組LC3-II熒光點表達增多,且在IH12h組LC3-II表達最多,與NC組相比,差異具有統(tǒng)計學意義(P0.05)。(2)透射電子顯微鏡觀察自噬體的形成:與NC組相比,IH組雙層膜結(jié)構(gòu)的自噬溶酶體明顯增多。(3)Si RNA干預HIF-1α表達對海馬神經(jīng)元的自噬水平影響:免疫印跡結(jié)果顯示,與NC組相比,IH組HIF-1α、LC3-II蛋白表達增多(P0.05);Si RNA+IH組HIF-1α和LC3-II表達較IH組明顯減少(P0.05)。(4)Si RNA干預海馬神經(jīng)元HIF-1α表達后,檢測HIF-1α、BNIP3和Beclin1蛋白的表達。免疫印跡結(jié)果顯示,IH組HIF-1α、BNIP3、Beclin1蛋白表達較NC組明顯增高(P0.05);Si RNA+IH組HIF-1α、BNIP3、Beclin1蛋白表達較IH組明顯減少(P0.05)。(5)免疫共沉淀檢測Bcl2和Beclin1的結(jié)合狀態(tài):NC組Beclin1和Bcl2處于結(jié)合狀態(tài),IH和EBSS組Beclin1和Bcl2處于分離狀態(tài)。(6)間歇低氧對XIAP表達的影響:隨著間歇低氧時間延長,IH組LC3-II和cleaved caspase-3蛋白表達呈增多趨勢,而XIAP蛋白表達減少;IH12h組LC3-II和cleaved caspase-3蛋白表達最多而XIAP蛋白表達最少,與NC組相比,差異具有統(tǒng)計學意義(P0.05);(7)藥物干預自噬后對XIAP蛋白和凋亡表達的影響:與IH組相比,RAP+IH組XIAP的表達明顯減少,而LC3-II和cleaved caspase-3表達顯著高于IH組(P0.05);CQ+IH組與IH組相比,在LC3-II和cleaved caspase-3表達減少的同時,XIAP表達增多,且差異均具有統(tǒng)計學意義(P0.05)。結(jié)論本研究證實:(1)從形態(tài)學角度進一步證明間歇低氧處理后海馬神經(jīng)細胞自噬表達增多;(2)間歇低氧狀態(tài)下通過si RNA抑制HIF-1α的表達后,自噬標記蛋白LC3-II表達也降低;(3)間歇低氧狀態(tài)下通過si RNA抑制HIF-1α的表達后HIF-1α-BNIP3-Beclin1信號通路蛋白表達均降低。(4)常氧狀態(tài)下Beclin1以Beclin1-Bcl2復合體的形式存在于細胞中,而間歇低氧可促使Beclin1-Bcl2的分離釋放出游離Beclin1以促進自噬的發(fā)生。(5)間歇低氧狀態(tài)下通過Si RNA抑制Beclin1的表達后,促凋亡蛋白cleaved caspase-3表達也降低。(6)隨著間歇低氧時間延長,自噬和凋亡增多,而凋亡抑制劑XIAP的表達減少。(7)通過藥物抑制(促進)自噬表達后,在促凋亡蛋白cleaved caspase-3表達減少(增多)的同時,凋亡抑制劑XIAP蛋白的表達增多(減少)。綜上所述,我們通過免疫熒光、透射電子顯微鏡、基因沉默、免疫共沉淀等實驗方法對前期研究進行了完善和補充,結(jié)果證實:間歇低氧條件下HIF-1α通過激活其下游靶蛋白BNIP3促進了Beclin1-Bcl2的分離,所釋放出的游離的Beclin1激活自噬并可能通過過度清除XIAP加重了海馬神經(jīng)細胞凋亡。
[Abstract]:Objective obstructive sleep apnea hypopnea syndrome (OSAHS) can lead to or aggravate cognitive dysfunction. Its mechanism is related to the apoptosis of hippocampal neurons induced by intermittent hypoxia (IH). Our preliminary study preliminarily found that intermittent hypoxia may be mediated by HIF-1 alpha -BNIP3-Beclin1, and autophagy activates the hippocampal neurons. But the previous study mainly depends on the immunoblotting and the methods of drug intervention in vitro. Because any single method of autophagy can not accurately reflect the level of autophagy, the application of a variety of experimental methods to detect the occurrence of autophagy and its molecular mechanism is the further improvement and sublimation of the whole research. The expression of autophagy was detected by the experimental method of immunofluorescence and transmission electron microscopy. The specific mechanism of HIF-1 alpha mediated autophagy under intermittent hypoxia was further clarified by the experimental method of immunofluorescence and transmission electron microscopy. The specific mechanism of autophagy mediated by OSAHS was further clarified. The molecular mechanism of autophagy activation aggravated the damage of hippocampal neurons was preliminarily investigated by the experimental methods of silence and drug intervention. Methods (1) the study object: to buy SD milk rats within 24 hours of birth and to cultivate the primary hippocampal neurons for 7-9 days. (2) the OSAHS cell model was established and grouped according to the different conditions: normal control group (NC), not simultaneously. Interintermittent hypoxia (IH) group. Detection index: the expression of autophagy protein LC3-II was detected by immunofluorescence; the formation of autophagic in the double layer membrane structure was observed under transmission electron microscope; (3) Si RNA transfection and grouping: Si RNA intervention was given to HIF-1 alpha (Beclin1) expression, negative control group, IH group, HIF-1 alpha Si RNA group intervention group, +HIF-1 alpha Si RNA (Beclin1 Si RNA) intervention group. Selected HIF-1 alpha and the most obvious intermittent hypoxia time point 12h to be treated, HIF-1 alpha (Beclin1) Si RNA preconditioning method: cell culture for 7 days. Se-3, HIF-1 alpha -BNIP3-Beclin1 signaling pathway related protein expression. (4) immuno coprecipitation technique to detect the binding state of Beclin1-Bcl2. (5) the intervention of autophagic inhibitors and activators to interfere with the intermittent hypoxia treatment of hippocampal neurons. Group: normal control, intermittent hypoxia group, drug preconditioning group, individual drug delivery group. The expression of cleaved-caspase-3 and XIAP protein in the hippocampal neurons of each group was detected by Western blot. Results (1) the expression of autophagy detected by immunofluorescence: the expression of LC3-II fluorescence points in group IH increased with the prolongation of intermittent hypoxia time, and the most expression in IH12h group LC3-II, compared with the NC group, the difference was statistically significant (P0.05). (2) transmission electron microscopy The autophagosome formation was observed by microscope: compared with the NC group, the autophagic lysosomes in the IH group were significantly increased. (3) the effect of HIF-1 alpha expression on the autophagy level of the hippocampal neurons was influenced by the intervention of Si RNA. The result of immunoblotting showed that the expression of HIF-1 A and LC3-II protein in the IH group increased (P0.05) compared with the NC group, and the Si RNA+IH group was significantly lower than that of the NC group. (4) (4) (4) Si RNA interfered with the expression of HIF-1 alpha in hippocampal neurons, and the expression of HIF-1, BNIP3 and Beclin1 protein was detected. The results of immunoblotting showed that the expression of HIF-1 alpha, BNIP3, Beclin1 protein in IH group was significantly higher than that of NC group. The binding state of eclin1: the NC group Beclin1 and Bcl2 are in a binding state, Beclin1 and Bcl2 in IH and EBSS groups are in separate state. (6) the effect of intermittent hypoxia on XIAP expression: with the prolongation of intermittent hypoxia, the expression of LC3-II and cleaved proteins in IH group increased and the expression of proteins decreased. The expression of XIAP protein was the least, and the difference was statistically significant compared with the NC group (P0.05). (7) the effect of the drug intervention on the expression of XIAP protein and apoptosis after autophagy: compared with the IH group, the expression of XIAP in the RAP+IH group was significantly reduced, and the expression of LC3-II and cleaved caspase-3 was significantly higher than that in the IH group (P0.05). The expression of ved caspase-3 was decreased and the expression of XIAP increased, and the difference was statistically significant (P0.05). Conclusion: (1) further evidence of the increase of autophagy expression in hippocampal neurons after intermittent hypoxia treatment was confirmed from the morphological point of view; (2) the expression of autophagy protein LC3-II table in the intermittent hypoxic state after the expression of HIF-1 alpha by Si RNA (3) the expression of HIF-1 alpha -BNIP3-Beclin1 signaling protein was reduced after Si RNA inhibition of HIF-1 alpha in intermittent hypoxia. (4) Beclin1 was in the form of Beclin1-Bcl2 complex in the normal oxygen state, and intermittent hypoxia could induce the separation and release of Beclin1-Bcl2 to promote the occurrence of autophagy. 5) the expression of apoptotic protein cleaved caspase-3 decreased after Si RNA inhibited Beclin1 expression in intermittent hypoxia. (6) the expression of autophagy and apoptosis increased with the prolongation of intermittent hypoxia, and the expression of apoptosis inhibitor XIAP decreased. (7) the expression of apoptotic protein cleaved caspase-3 decreased by drug inhibition (promoting) autophagic expression. At the same time, the expression of apoptosis inhibitor XIAP protein increased (decrease). In summary, we perfected and supplemented the previous studies by immunofluorescence, transmission electron microscopy, gene silencing, immunoprecipitation and other experimental methods. The results confirmed that HIF-1 alpha under intermittent hypoxia activates its downstream target protein BNIP3 to promote Beclin1- The free Beclin1 released by Bcl2 activates autophagy and may aggravate hippocampal neuronal apoptosis through overcleaning XIAP.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R766

【參考文獻】

相關(guān)期刊論文 前3條

1 MU WenLi;ZHANG QingJun;TANG XiaoQiang;FU WenYan;ZHENG Wei;LU YunBiao;LI HongLiang;WEI YuSheng;LI Li;SHE ZhiGang;CHEN HouZao;LIU DePei;;Overexpression of a dominant-negative mutant of SIRT1 in mouse heart causes cardiomyocyte apoptosis and early-onset heart failure[J];Science China(Life Sciences);2014年09期

2 陳曦;梁文穎;熊靜波;;自噬的檢測方法及評述[J];現(xiàn)代生物醫(yī)學進展;2012年09期

3 許德暉;黃辰;劉利英;宋土生;;高效siRNA設(shè)計的研究進展[J];遺傳;2006年11期

，

本文編號：2003441