膜联蛋白A7在脑出血后继发性脑损伤中作用的实验研究

发布时间：2018-01-07 15:28

本文关键词：膜联蛋白A7在脑出血后继发性脑损伤中作用的实验研究　出处：《苏州大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:脑出血(intracerebral hemorrhage ICH)是指脑组织中病变的血管突然破裂,血液流进周围的脑组织。ICH占所有中风的10%-15%且拥有极高的致残率和致死率。越来越多的研究证实ICH后继发性脑损伤(secondary brain injury SBI)与患者神经功能的恢复密切相关。兴奋性氨基酸毒性在SBI的发生、发展过程中发挥着非常重要的病理作用。膜联蛋白A7(annexin A7,ANXA7)是一种能促进膜融合的钙依赖性磷脂结合蛋白,近来研究表明其在多种肿瘤的发生、进展、转归中起了非常重要作用。但有关ANXA7在ICH后SBI中的研究尚未涉及。本实验通过观察ANXA7在ICH后SBI中的时相变化,探讨ICH后ANXA7的异常表达是否与神经元的兴奋性损伤有关,并进一步使用ANXA7的拮抗剂干预探讨其是否对继发性脑损伤有保护作用。方法:实验1,42只健康成年雄性Sprague-Dawley(SD)大鼠随机分为7组:假手术组(sham),ICH6h,12h,24h,48h,72h,1w组(n=6),使用基底节区注射胶原酶法建立活体ICH模型,分别在以上各时间点处死大鼠灌注并取脑,取基底节区血肿周围的脑组织作为标本。应用免疫荧光(immunofluorescence IF)共染;半定量PCR(Semi-quantitative RT PCR)和蛋白质印迹(Western bolt WB)来检测血肿周围脑组织中的ANXA7的表达,干湿法检测脑水肿;以渗出伊文思蓝(Evans blue EB)的量来评价血脑屏障通透性的改变,进而分析ANXA7的表达与ICH后继发性脑损伤是否具有相关性。实验2,在实验1的基础上,给予外源人重组ANXA7(rh ANXA7)和ANXA7的中和抗体处理,以增强和削弱ANXA7的作用,分析ANXA7在ICH后继发性脑损伤发挥怎样的作用。将48只健康成年雄性SD大鼠随机分为sham组(n=12),ICH组(n=12),ICH+rh ANXA7组(n=12)和ICH+ANXA7抗体组(n=12)。在ICH后12h脑室注射rh ANXA7和ANXA7抗体,48小时后处死大鼠。干湿法测定脑水肿,以渗出EB的量来评价血脑屏障(Blood brain barrier BBB)通透性改变,采用末端标记法(Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labelingtunel)染色与fluoro-jadeb荧光染色检测神经元细胞凋亡及坏死比率,高效液相色谱分析法(highperformanceliquidchromatographyhplc)测定氨基酸含量。实验3,在实验1和2的基础上,分别利用高效液相色谱分析法(highperformanceliquidchromatographyhplc)测定脑脊液中谷氨酸含量和免疫沉淀法(immunoprecipitationip)检测anxa7与snap23/snap25的相互作用,分析ich后以及rhanxa7和anxa7抗体处理对谷氨酸引起的神经兴奋性毒性和anxa7与snap23/snap25的相互作用的影响,进而得出anxa7在ich后继发性脑损伤中的可能作用机制。结果:实验1,与sham组相比,anxa7的mrna水平在ich建立后早期(6h)即有显著表达,并在24小时到达高峰,48小时开始下降,但1w后仍有持续的高表达;anxa7的蛋白水平在ich12h即有显著的表达,24小时达到高峰,1w后的表达仍高于正常sham组;同时,ich后脑组织水含量明显增加,48h时水肿百分数达到82.4±0.83%;ich6小时后血脑屏障的通透性开始增加,48小时达到高峰,局部eb含量达到3.8ug/g,1w后基本回到基线水平。综上说明,伴随着ich后继发性脑损伤的发生,anxa7的表达水平上升,表明anxa7与ich后继发性脑损伤可能具有相关性。实验2,与ich组比较,应用重组anxa7蛋白干预后,凋亡和坏死的比率较单一的ich组上升显著,而应用anxa7抗体干预时,神经元凋亡和坏死的比率较ich组显著下降。脑水肿含量和血管通透性在应用anxa7蛋白后显著加重,而应用anxa7抗体干预时,脑水肿含量和血管通透性显著改善。综上说明,anxa7可能参与并促进了ich后继发性脑损伤的发生发展。实验3,hplc结果显示ich后脑脊液中的谷氨酸含量增高,应用anxa7蛋白干预时,谷氨酸含量进一步上升,而应用anxa7抗体干预时,则脑脊液中的谷氨酸含量较ich组显著下降。此外,ip结果显示,sham组中anxa7与snap23/snap25相互作用水平较低,而ich组中结合活性较sham组显著上升,rhanxa7进一步促进anxa7与snap23/snap25的相互作用,而应用anxa7抗体干预时,则可以显著抑制ich引起的anxa7与snap23/snap25的相互作用。结论:ich后,伴随着继发性脑损伤的发生,anxa7表达上升,且anxa7与snap23/snap25相互作用增多;rhanxa7可以促进anxa7与snap23/snap25的相互作用,上调脑脊液中谷氨酸含量,并且加剧ich后继发性脑损伤,anxa7中和抗体则发挥相反作用。综上说明,ICH后,ANXA7通过与SNAP23/SNAP25相互作用参与到兴奋性氨基酸毒性过程中,进而加剧ICH后继发性脑损伤。
[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.

【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R743.34

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