本文關(guān)鍵詞：膜聯(lián)蛋白A7在腦出血后繼發(fā)性腦損傷中作用的實驗研究　出處：《蘇州大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 腦出血 繼發(fā)性腦損傷 膜聯(lián)蛋白A7 突觸小體相關(guān)蛋白-23 25 興奮性氨基酸毒性

【摘要】：目的:腦出血(intracerebral hemorrhage ICH)是指腦組織中病變的血管突然破裂,血液流進(jìn)周圍的腦組織。ICH占所有中風(fēng)的10%-15%且擁有極高的致殘率和致死率。越來越多的研究證實ICH后繼發(fā)性腦損傷(secondary brain injury SBI)與患者神經(jīng)功能的恢復(fù)密切相關(guān)。興奮性氨基酸毒性在SBI的發(fā)生、發(fā)展過程中發(fā)揮著非常重要的病理作用。膜聯(lián)蛋白A7(annexin A7,ANXA7)是一種能促進(jìn)膜融合的鈣依賴性磷脂結(jié)合蛋白,近來研究表明其在多種腫瘤的發(fā)生、進(jìn)展、轉(zhuǎn)歸中起了非常重要作用。但有關(guān)ANXA7在ICH后SBI中的研究尚未涉及。本實驗通過觀察ANXA7在ICH后SBI中的時相變化,探討ICH后ANXA7的異常表達(dá)是否與神經(jīng)元的興奮性損傷有關(guān),并進(jìn)一步使用ANXA7的拮抗劑干預(yù)探討其是否對繼發(fā)性腦損傷有保護(hù)作用。方法:實驗1,42只健康成年雄性Sprague-Dawley(SD)大鼠隨機分為7組:假手術(shù)組(sham),ICH6h,12h,24h,48h,72h,1w組(n=6),使用基底節(jié)區(qū)注射膠原酶法建立活體ICH模型,分別在以上各時間點處死大鼠灌注并取腦,取基底節(jié)區(qū)血腫周圍的腦組織作為標(biāo)本。應(yīng)用免疫熒光(immunofluorescence IF)共染;半定量PCR(Semi-quantitative RT PCR)和蛋白質(zhì)印跡(Western bolt WB)來檢測血腫周圍腦組織中的ANXA7的表達(dá),干濕法檢測腦水腫;以滲出伊文思藍(lán)(Evans blue EB)的量來評價血腦屏障通透性的改變,進(jìn)而分析ANXA7的表達(dá)與ICH后繼發(fā)性腦損傷是否具有相關(guān)性。實驗2,在實驗1的基礎(chǔ)上,給予外源人重組ANXA7(rh ANXA7)和ANXA7的中和抗體處理,以增強和削弱ANXA7的作用,分析ANXA7在ICH后繼發(fā)性腦損傷發(fā)揮怎樣的作用。將48只健康成年雄性SD大鼠隨機分為sham組(n=12),ICH組(n=12),ICH+rh ANXA7組(n=12)和ICH+ANXA7抗體組(n=12)。在ICH后12h腦室注射rh ANXA7和ANXA7抗體,48小時后處死大鼠。干濕法測定腦水腫,以滲出EB的量來評價血腦屏障(Blood brain barrier BBB)通透性改變,采用末端標(biāo)記法(Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labelingtunel)染色與fluoro-jadeb熒光染色檢測神經(jīng)元細(xì)胞凋亡及壞死比率,高效液相色譜分析法(highperformanceliquidchromatographyhplc)測定氨基酸含量。實驗3,在實驗1和2的基礎(chǔ)上,分別利用高效液相色譜分析法(highperformanceliquidchromatographyhplc)測定腦脊液中谷氨酸含量和免疫沉淀法(immunoprecipitationip)檢測anxa7與snap23/snap25的相互作用,分析ich后以及rhanxa7和anxa7抗體處理對谷氨酸引起的神經(jīng)興奮性毒性和anxa7與snap23/snap25的相互作用的影響,進(jìn)而得出anxa7在ich后繼發(fā)性腦損傷中的可能作用機制。結(jié)果:實驗1,與sham組相比,anxa7的mrna水平在ich建立后早期(6h)即有顯著表達(dá),并在24小時到達(dá)高峰,48小時開始下降,但1w后仍有持續(xù)的高表達(dá);anxa7的蛋白水平在ich12h即有顯著的表達(dá),24小時達(dá)到高峰,1w后的表達(dá)仍高于正常sham組;同時,ich后腦組織水含量明顯增加,48h時水腫百分?jǐn)?shù)達(dá)到82.4±0.83%;ich6小時后血腦屏障的通透性開始增加,48小時達(dá)到高峰,局部eb含量達(dá)到3.8ug/g,1w后基本回到基線水平。綜上說明,伴隨著ich后繼發(fā)性腦損傷的發(fā)生,anxa7的表達(dá)水平上升,表明anxa7與ich后繼發(fā)性腦損傷可能具有相關(guān)性。實驗2,與ich組比較,應(yīng)用重組anxa7蛋白干預(yù)后,凋亡和壞死的比率較單一的ich組上升顯著,而應(yīng)用anxa7抗體干預(yù)時,神經(jīng)元凋亡和壞死的比率較ich組顯著下降。腦水腫含量和血管通透性在應(yīng)用anxa7蛋白后顯著加重,而應(yīng)用anxa7抗體干預(yù)時,腦水腫含量和血管通透性顯著改善。綜上說明,anxa7可能參與并促進(jìn)了ich后繼發(fā)性腦損傷的發(fā)生發(fā)展。實驗3,hplc結(jié)果顯示ich后腦脊液中的谷氨酸含量增高,應(yīng)用anxa7蛋白干預(yù)時,谷氨酸含量進(jìn)一步上升,而應(yīng)用anxa7抗體干預(yù)時,則腦脊液中的谷氨酸含量較ich組顯著下降。此外,ip結(jié)果顯示,sham組中anxa7與snap23/snap25相互作用水平較低,而ich組中結(jié)合活性較sham組顯著上升,rhanxa7進(jìn)一步促進(jìn)anxa7與snap23/snap25的相互作用,而應(yīng)用anxa7抗體干預(yù)時,則可以顯著抑制ich引起的anxa7與snap23/snap25的相互作用。結(jié)論:ich后,伴隨著繼發(fā)性腦損傷的發(fā)生,anxa7表達(dá)上升,且anxa7與snap23/snap25相互作用增多;rhanxa7可以促進(jìn)anxa7與snap23/snap25的相互作用,上調(diào)腦脊液中谷氨酸含量,并且加劇ich后繼發(fā)性腦損傷,anxa7中和抗體則發(fā)揮相反作用。綜上說明,ICH后,ANXA7通過與SNAP23/SNAP25相互作用參與到興奮性氨基酸毒性過程中,進(jìn)而加劇ICH后繼發(fā)性腦損傷。
[Abstract]:Objective: intracerebral hemorrhage (intracerebral hemorrhage ICH) refers to the lesions in the brain tissue of vascular rupture suddenly, blood flowing to the brain tissue around.ICH% of all stroke 10%-15% and have high morbidity and mortality. More and more studies have confirmed that the secondary brain injury after ICH (secondary brain injury SBI) and neurological function recovery is closely related. The toxicity of excitatory amino acids in SBI, plays a very important role in the development of pathological process. Annexin A7 (annexin A7 ANXA7) is a kind of membrane fusion can promote calcium dependent phospholipid binding protein, recent studies show that the in a variety of tumor genesis, progress, play a very important role in the outcome. But the study of ICH SBI in the ANXA7 is not involved. This experiment by observing changes in ANXA7 in SBI after ICH, to investigate the abnormal expression of ICH after ANXA7 and whether neurons Xing The excitatory injury, the use of ANXA7 antagonists and further explore whether intervention on secondary brain injury. Methods: the 1,42 male Sprague-Dawley (SD) rats were randomly divided into 7 groups: sham operation group (sham), ICH6h, 12h, 24h, 48h, 72h, 1W group (n=6), established in vivo ICH model using basal ganglia collagenase injection method, respectively, at each time point the rats were sacrificed and brain perfusion, the basal ganglia hematoma surrounding brain tissue as samples. By immunofluorescence (immunofluorescence IF) were stained; semi quantitative PCR (Semi-quantitative RT PCR) and Western blot (Western bolt WB) expression of brain tissue around the hematoma in the detection of ANXA7, detection of dry and wet brain edema; the exudation of Evans blue (Evans blue EB) to evaluate the amount of blood brain barrier permeability changes, and analysis of secondary brain injury and expression of ICH and ANXA7 after The relationship of Experiment 2, based on 1 experiments, exogenous recombinant human ANXA7 (RH ANXA7) and ANXA7 neutralizing antibody treatment, in order to strengthen and weaken the role of ANXA7, analysis of ANXA7 in ICH after secondary brain injury. How to play the role of 48 healthy adult male SD rats were randomly divided into sham group (n=12), ICH group (n=12), ICH+rh ANXA7 group (n=12) and ICH+ANXA7 antibody group (n=12). After ICH 12h and ANXA7 ANXA7 after injection of RH antibody, 48 hours after the rats were sacrificed. The dry and wet determination of brain edema, exudation by EB to evaluate the amount of blood brain barrier (Blood brain barrier BBB) the changes of permeability by TUNEL techniques (Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labelingtunel) staining and fluoro-jadeb staining were used to detect neuronal apoptosis and necrosis rate, high performance liquid chromatography (highperformanceliquidchromatographyhplc) determination of amino Acid. In Experiment 3, in Experiment 1 and 2 respectively on the basis of using high performance liquid chromatography (highperformanceliquidchromatographyhplc) method for the determination of the content of glutamic acid and immunoprecipitation in cerebrospinal fluid (immunoprecipitationip) interaction detection of anxa7 and snap23/snap25, ICH, rhanxa7 and anxa7 after the analysis of the antibody interaction on glutamate induced excitotoxicity toxicity and anxa7 and snap23/snap25 effects, and then draw the possible mechanism of anxa7 in secondary brain injury after ICH. Results: 1, compared with the sham group, the anxa7 level of mRNA in ICH after the establishment of early (6h) is a significant expression, and reached the peak in 24 hours, 48 hours began to decline. But 1W still expressed continuously; the protein level of anxa7 is significantly expressed in ich12h, peaked at 24 hours after 1W, the expression is still higher than the normal sham group; at the same time, after ICH The water content increased significantly, 48h edema percentage reached 82.4 + 0.83%; ich6 hours after the blood brain barrier permeability began to increase, reached a peak at 48 hour, local EB content reached 3.8ug/g, after 1W returned to baseline. In conclusion, accompanied by the occurrence of secondary brain injury after ICH, the expression level of anxa7 increased. Anxa7 and ICH showed that after secondary brain injury may be related. In Experiment 2, compared with the ICH group, the application of recombinant anxa7 protein intervention, ICH group the ratio of apoptosis and necrosis increased significantly than the single, and the application of anxa7 antibody intervention, the ratio of neuron apoptosis and necrosis was significantly lower than that in ICH group. The content of brain edema and increased vascular permeability significantly in the application of anxa7 protein, and the application of anxa7 antibody intervention, brain edema and vascular permeability were significantly improved. In conclusion, anxa7 may participate in and contribute to secondary brain injury after ICH The occurrence and development of HPLC. In Experiment 3, results showed that ICH increased the content of glutamic acid in cerebrospinal fluid after intervention, application of anxa7 protein, glutamate levels rise further, while the application of anxa7 antibody intervention, then the content of glutamic acid in cerebrospinal fluid was significantly lower than that in ICH group. In addition, the results of IP showed that the level of anxa7 in group sham and snap23/snap25 interaction the lower, while the ICH group with activity was significantly higher than that in sham group, rhanxa7 further promote the interaction between anxa7 and snap23/snap25, and the application of anxa7 antibody intervention, the interaction between anxa7 and snap23/snap25 can significantly inhibit ICH induced. Conclusion: after ICH, accompanied by the occurrence of secondary brain injury, the expression of anxa7 increased. And the anxa7 and snap23/snap25 interaction increased; rhanxa7 can promote the interaction between anxa7 and snap23/snap25, the content of glutamic acid in cerebrospinal fluid increase, secondary brain injury and increased after ich, Anxa7 neutralizing antibody plays the opposite role. In conclusion, after ICH, ANXA7 participates in the excitatory amino acid toxicity through interaction with SNAP23/SNAP25, and then aggravates secondary brain injury after ICH.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R743.34

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