本文選題：腦出血 + 促紅細(xì)胞生成素(EPO)　； 參考：《西南醫(yī)科大學(xué)》2017年碩士論文

【摘要】：目的:腦出血(Intracerebral hemorrhage ICH)是急性腦血管疾病的一種,為神經(jīng)內(nèi)科多發(fā)疾病,因其死亡率及致殘率較高,嚴(yán)重危害著患者的生命健康及生活質(zhì)量。腦出血后血腫的占位效應(yīng)、血腫刺激周圍組織釋放的血管活性物質(zhì)和血腫的分解產(chǎn)物、凝血酶級聯(lián)放大反應(yīng)及補(bǔ)體系統(tǒng)的激活,這些因素相互作用直接或間接的引起腦組織損害,誘發(fā)炎癥反應(yīng)及細(xì)胞凋亡,導(dǎo)致神經(jīng)功能缺損;針對發(fā)病率及死亡率高的現(xiàn)狀,我國目前針對腦出血的治療方案尚無突破性進(jìn)展,目前的治療主要集中于降顱壓、清除氧自由基、營養(yǎng)神經(jīng)、調(diào)控血壓、血糖、維持水鹽電解質(zhì)平衡等對癥支持治療或外科手術(shù)治療,缺乏特異有效的治療方案,且就后期神經(jīng)功能的恢復(fù)方面的治療更是缺乏。神經(jīng)突觸重塑與ICH后神經(jīng)功能恢復(fù)密切相關(guān),較長時間內(nèi)都可受到調(diào)節(jié),有利于受損神經(jīng)功能的恢復(fù)。促紅細(xì)胞生成素(erythropoietin,EPO)是一種由腎臟分泌的相對分子量為34000的糖蛋白,以往認(rèn)為促紅細(xì)胞生成素(EPO)在體內(nèi)的主要作用是與紅系祖細(xì)胞表面的特異性促紅細(xì)胞生成素受體(erythropoietin receptor,EPOR)結(jié)合,從而刺激其增值分化,其在臨床上的應(yīng)用也局限于糾正慢性貧血。近年來大量研究表明腦組織中有促紅細(xì)胞生成素(EPO)和促紅細(xì)胞生成素受體(EPOR)的表達(dá);同時發(fā)現(xiàn)促紅細(xì)胞生成素(EPO)的生成量與腦組織的供血供氧情況密切相關(guān),當(dāng)腦組織缺血缺氧時促紅細(xì)胞生成素(EPO)的生成量成倍增加,對神經(jīng)元起保護(hù)作用,并且通過降低神經(jīng)元對缺血缺氧的敏感性,增強(qiáng)神經(jīng)元的存活能力,促進(jìn)神經(jīng)祖細(xì)胞的增值和抑制凋亡。但就促紅細(xì)胞生成素(EPO)是否能夠有助于神經(jīng)突觸重塑方面的研究尚不多,因此關(guān)于促紅細(xì)胞生成素(EPO)對腦出血后神經(jīng)突觸的影響方面的研究很有必要。本實(shí)驗(yàn)通過予以重組人促紅細(xì)胞生成素(rh EPO)干預(yù)后觀察ICH后血腫周圍突觸素(Synaptophysin,SYP)和突觸后致密物-95(Post synaptic density,PSD-95)蛋白的變化情況,探討促紅細(xì)胞生成素(EPO)對實(shí)驗(yàn)性腦出血大鼠神經(jīng)突觸重塑的影響。方法:1.在造模前根據(jù)Morris水迷宮視頻跟蹤分析系統(tǒng)訓(xùn)練預(yù)備大鼠,連續(xù)訓(xùn)練4天后測試,選取逃避潛伏期在10-40秒之間的大鼠進(jìn)入造模;2.根據(jù)《大鼠腦立體定向儀圖譜》以右側(cè)基底節(jié)區(qū)尾狀核(前囟前0.2mm,中線右側(cè)3.0mm)為造模注射點(diǎn),采用斷尾取自體血法制作ICH模型。按照Zea-longa評分標(biāo)準(zhǔn),將評分在1-3分之間的SD大鼠入選為實(shí)驗(yàn)大鼠;3.動物的分組及處理:將經(jīng)Morris水迷宮篩選出的105只健康雄性SD大鼠隨機(jī)分為三組:假手術(shù)組(Sham組)35只,腦出血組(ICH組)35只,腦出血+促紅細(xì)胞生成素治療組(EPO組)35只;將Sham組、ICH組、EPO組按時間點(diǎn)分為術(shù)后6h、24h、48h、72h、7d、14d、21d共七組,每組5只大鼠。實(shí)驗(yàn)大鼠的具體處理如下:(1)Sham組用微量注射器在右側(cè)基底節(jié)區(qū)尾狀核處定位并緩慢刺入,不注入自體血,其余步驟與ICH組一致;(2)ICH組用微量注射器在右側(cè)基底節(jié)區(qū)尾狀核處注入不抗凝自體血50μl,同時腹腔注射與EPO組所用EPO劑量相等的生理鹽水;(3)EPO組用微量注射器在右側(cè)基底節(jié)區(qū)尾狀核處注入不抗凝自體血50μl,同時按3000U/kg的劑量向腹腔注射EPO。4.標(biāo)本制備及檢測:將造模成功的大鼠于術(shù)后6h、24h、48h、72h、7d、14d、21d各對應(yīng)時間點(diǎn)先用Garcia神經(jīng)功能評分法評價神經(jīng)功能缺損情況后進(jìn)行Morris水迷宮測試,之后予以水合氯醛麻醉,麻醉后立即斷頭取腦,制作腦組織標(biāo)本,用干濕重法測定各組大鼠血腫周圍腦組織含水量變化,用HE染色觀察血腫周圍腦組織基本形態(tài)和結(jié)構(gòu),用免疫組化法檢測各時間點(diǎn)血腫周圍SYP、PSD-95蛋白表達(dá)。結(jié)果:1.腦組織含水量:6h、24h、48h、72h、7d各時間點(diǎn)ICH組和EPO組血腫周圍腦組織含水量均明顯高于Sham組(P0.05);6h時間點(diǎn)EPO組血腫周圍腦組織含水量與ICH組無差異(P0.05),但24h、48h、72h、7d各時間點(diǎn)EPO組血腫周圍腦組織含水量較ICH組明顯減少(P0.05)。2.HE染色觀察各組腦組織病理改變:(1)Sham組無血腫形成,各時間點(diǎn)腦組織結(jié)構(gòu)完整;(2)ICH組72h時間點(diǎn)血腫周圍可見大量紅細(xì)胞,腦組織結(jié)構(gòu)紊亂,炎癥細(xì)胞浸潤明顯,神經(jīng)細(xì)胞水腫嚴(yán)重甚至變性壞死,7d、14d時病灶周圍的血細(xì)胞逐漸減少,炎性細(xì)胞逐漸減少,神經(jīng)細(xì)胞水腫程度減輕,到21d時病灶周圍血細(xì)胞更少甚至消失、炎癥細(xì)胞幾乎消失,纖維組織、膠質(zhì)細(xì)胞增生,受損的組織結(jié)構(gòu)較前有所恢復(fù)。(3)與ICH相比,EPO組各時間點(diǎn)的水腫程度和神經(jīng)細(xì)胞變性壞死都有所減輕,受損組織的修復(fù)都較ICH組好。3.神經(jīng)功能評分:各實(shí)驗(yàn)組大鼠于術(shù)后6h、24h、48h、72h、7d、14d、21d各時間點(diǎn)予以神經(jīng)功能評分,其中(1)Sham組未見明顯神經(jīng)功能缺損情況;ICH組、EPO組各時間點(diǎn)神經(jīng)功能評分均低于Sham組,有統(tǒng)計學(xué)差異(P0.05);(2)各時間點(diǎn)EPO組神經(jīng)功能評分均高于ICH組,具有統(tǒng)計學(xué)差異(P0.05)。4.水迷宮測試結(jié)果:大鼠造模前進(jìn)行Morris水迷宮連續(xù)訓(xùn)練4天,每天4次,第5天測試逃避潛伏期、穿越平臺次數(shù)、平臺象限時間百分比和平臺象限路程百分比,各組無顯著差異(P0.05);造模后再次行水迷宮測試上述指標(biāo),Sham組各指標(biāo)較前均無明顯變化(P0.05),ICH組、EPO組的逃避潛伏期、穿越平臺次數(shù)、平臺象限時間百分比和平臺象限路程百分比等多種反應(yīng)學(xué)習(xí)記憶能力的指標(biāo),較前均有所下降,并隨著ICH后時間的延長各指標(biāo)逐漸上升,且EPO組的上升程度及速度要優(yōu)于ICH組(P0.05)。5.血腫周圍腦組織SYP蛋白含量測定:(1)Sham組僅有少量SYP蛋白表達(dá),各個時間點(diǎn)無明顯差異(P0.05);(2)在6h、24h、48h、72h時間點(diǎn)ICH組、EPO組僅有少量SYP蛋白表達(dá),與Sham組比較無明顯差異(P0.05);7d、14d、21d時間點(diǎn),ICH組、EPO組SYP蛋白表達(dá)明顯多于Sham組(P0.05);(3)7d、14d、21d時間點(diǎn),EPO組SYP蛋白表達(dá)明顯高于ICH組,具有統(tǒng)計學(xué)差異(P0.05);(4)ICH組、EPO組從72h后SYP蛋白開始增多,14d達(dá)峰值(P0.05),之后表達(dá)下降,但21d時血腫周圍仍有較多表達(dá)。6.血腫周圍腦組織PSD-95蛋白含量測定:(1)Sham組僅有少量PSD-95蛋白表達(dá),各個時間點(diǎn)無明顯差異(P0.05);(2)在6h、24h、48h、72h時ICH組、EPO組僅有少量PSD-95蛋白表達(dá),與Sham組比較無明顯差異(P0.05),在7d、14d、21d時ICH組、EPO組PSD-95蛋白表達(dá)明顯多于Sham組(P0.05);(3)在7d、14d、21d時間點(diǎn),EPO組PSD-95蛋白表達(dá)明顯高于ICH組,具有統(tǒng)計學(xué)差異(P0.05);(4)ICH組、EPO組從72h后PSD-95蛋白開始增多,14d達(dá)峰值(P0.05),之后表達(dá)下降,但21d時血腫周圍仍有較多表達(dá)。7.各組大鼠神經(jīng)功能評分與SYP、PSD-95蛋白含量行相關(guān)性分析:(1)在14d時間點(diǎn),ICH組、EPO組的SYP蛋白含量與相應(yīng)實(shí)驗(yàn)大鼠神經(jīng)功能評分呈正相關(guān)(r=0.940,P0.001);(2)在14d時間點(diǎn),ICH組、EPO組的PSD-95蛋白含量與相應(yīng)實(shí)驗(yàn)大鼠神經(jīng)功能評分呈正相關(guān)(r=0.848,P0.001)。結(jié)論:1、采取不抗凝自體血注入法制作大鼠ICH模型方法較簡單,易于操作,且與臨床上人體自發(fā)性ICH的病理變化過程相似,是比較理想的研究ICH的動物模型;2、ICH后血腫周圍SYP、PSD-95蛋白表達(dá)增多,利于神經(jīng)功能恢復(fù);3、促紅細(xì)胞生成素(EPO)能上調(diào)血腫周圍SYP、PSD-95蛋白表達(dá),促進(jìn)ICH后神經(jīng)突觸重塑;4、促紅細(xì)胞生成素(EPO)能促進(jìn)ICH大鼠肢體功能及學(xué)習(xí)記憶能力的恢復(fù)。
[Abstract]:Objective: Intracerebral hemorrhage ICH is a kind of acute cerebrovascular disease, which is a multiple disease of Neurology. The mortality and disability rate are high, which seriously harm the life health and quality of life of the patients. The occupying effect of hematoma after intracerebral hemorrhage and the decomposition of vasoactive substances and hematoma released in the surrounding tissues by hematoma. The product, the cascade amplification reaction of thrombin and the activation of the complement system, these factors interact directly or indirectly to the damage of brain tissue, induce the inflammatory reaction and cell apoptosis, and lead to the nerve function defect. In view of the current situation of high morbidity and mortality, there is no breakthrough progress in the treatment of cerebral hemorrhage at present in China. The treatment is mainly focused on craniofacial pressure, oxygen free radical scavenging, nourishment nerve, regulation of blood pressure, blood sugar, maintenance of water and salt electrolyte balance and other symptomatic support treatment or surgical treatment, lack of specific and effective treatment, and lack of treatment for the recovery of neural function later. Neural synaptic remodeling is closely related to the recovery of nerve function after ICH. Erythropoietin (EPO) is a glycoprotein of the relative molecular weight of 34000, which was secreted by the kidney. It was previously thought that the main use of erythropoietin (EPO) in the body is specific erythropoiesis with the surface of the erythroid progenitor cells. The combination of erythropoietin receptor (EPOR) stimulates its value-added differentiation, and its clinical application is limited to the correction of chronic anemia. In recent years, a large number of studies have shown that the expression of erythropoietin (EPO) and erythropoietin receptor (EPOR) in the brain tissue; and the formation of erythropoietin (EPO) and the brain in the brain The blood supply and supply of oxygen in the tissue is closely related. When cerebral ischemia and hypoxia, the production of erythropoietin (EPO) increases exponentially, protects the neurons, and enhances the viability of neurons by reducing the sensitivity of neurons to ischemia and anoxia, and promotes the increment and inhibition of apoptosis of the progenitor cells of the deity. It is necessary to study the effect of erythropoietin (EPO) on neural synapses after intracerebral hemorrhage, and the study of the effect of erythropoietin (RH EPO) on the posthematoma peripheral synaptophysin (Synaptophysin, SYP) after ICH is necessary for the study of whether EPO can contribute to neural synapse remodeling. The changes of -95 (Post synaptic density, PSD-95) protein and the effect of erythropoietin (EPO) on neural synapse remodeling in experimental intracerebral hemorrhage rats were investigated. Methods: 1. before modeling, the rats were trained on the basis of the Morris water maze video tracking analysis system for 4 days after continuous training, and the escape latency was 1. The rats between 0-40 seconds entered the model; 2. according to the atlas of the rat brain stereotactic apparatus, the injection point was made by the caudate nucleus of the right basal ganglia region (0.2mm of the anterior fontanelle, the right 3.0mm of the middle line), and the autologous blood method was used to make the ICH model. In accordance with the Zea-longa score standard, the rats of 1-3 scores of SD were selected as experimental rats; 3. animals were selected. Group and treatment: 105 healthy male SD rats were randomly divided into three groups: 35 sham operation group (group Sham), 35 brain hemorrhage group (group ICH), cerebral hemorrhage + erythropoietin group (group EPO) 35, Sham group, ICH group and EPO group were divided into 6h, 24h, 48h, 72h, 72h, seven groups, 5 rats in each group. The specific treatment of rats was as follows: (1) group Sham was located at the caudate nucleus of the right basal ganglia with a micro injector and slowly pricking, and the other steps were consistent with the ICH group. (2) group ICH was injected with an anticoagulant autologous blood 50 mu l at the caudate nucleus of the right basal ganglia region, and the EPO agent used in the EPO group was injected into the abdominal cavity. Equal amount of physiological saline; (3) group EPO was injected with an anticoagulant autologous blood of 50 mu by injecting a micro injector to the right basal ganglia caudate nucleus and injected into the abdominal cavity at the dose of 3000U/kg to prepare and detect the EPO.4. specimens. The rats with the successful model were evaluated by the Garcia neural functional scoring method for 6h, 24h, 48h, 72h, 7d, 14d, 21d at the corresponding time points after the operation. After the Morris water maze test was carried out, chloral hydrate was followed by anaesthesia. After anesthesia, the brain tissue was cut off immediately after anesthesia and the brain tissue was made. The changes of water content around the hematoma around the hematoma were measured by dry wet weight method. The basic morphology and structure of the brain tissue around hematoma were observed by HE staining, and the time was detected by immunohistochemical method. SYP, PSD-95 protein expression around the hematoma. Results: 1. the water content of brain tissue: 6h, 24h, 48h, 72h, the water content around the hematoma in ICH group and EPO group of EPO group was significantly higher than that of Sham group (P0.05), and there was no difference in the brain tissue around the hematoma around the hematoma at the 6h time point. Compared with group ICH, the pathological changes of brain tissue were observed in group ICH (P0.05): (1) no hematoma was formed in group Sham, and the structure of brain tissue was complete at all time points. (2) a large number of red blood cells were seen around hematoma at 72h time point in group ICH, brain tissue structure disorder, inflammatory cells infiltrating obvious, nerve cell edema and even degeneration and necrosis, 7d, 14d disease The blood cells around the focal point decreased gradually, the inflammatory cells gradually decreased, the degree of edema of the nerve cells decreased, and the blood cells around the focus were less or even disappeared at 21d, the inflammatory cells almost disappeared, fibrous tissue, glial cells proliferated, and the damaged tissue was restored. (3) the degree of edema and nerve finer at each time point in group EPO compared with ICH The degeneration and necrosis of the cells were all relieved, and the repair of the damaged tissues was better than the ICH group.3. neural function score: the rats in the experimental group were given neurological function score at 6h, 24h, 48h, 72h, 7d, 14d, 21d at all time points after the operation, and (1) there was no obvious nerve function defect in the Sham group. Study difference (P0.05); (2) the score of EPO group in each time point was higher than that of group ICH, with statistical difference (P0.05).4. water maze test results: 4 days of continuous training for the rats before modeling, 4 times a day, fifth days to test the escape latency, the times of flat platform crossing, the percentage of platform quadrant time and the percentage of platform quadrant distance, There was no significant difference in each group (P0.05). The index of water maze was tested again after the model, and there was no significant change in each index in group Sham (P0.05). The escape latency, the number of crossing platform, the percentage of platform quadrant time, the percentage of platform quadrant and the percentage of platform quadrant were decreased in group ICH and EPO. With the prolongation of ICH, the index of each index increased gradually, and the level and speed of EPO group was better than that of SYP protein content in the brain tissue around.5. hematoma in group ICH (P0.05): (1) only a small amount of SYP protein was expressed in the Sham group, and there was no significant difference at every time point (P0.05); (2) there were only a small amount of protein expression in 6h, 24h, 48h, and time points. There was no significant difference in Sham group (P0.05); 7d, 14d, 21d time point, ICH group, SYP protein expression in group EPO was more than Sham group (P0.05). (3) 7d, 14d, and time points were significantly higher than those in the group. (4) There was still more expression of PSD-95 protein content around.6. hematoma around hematoma: (1) only a small amount of PSD-95 protein was expressed in group Sham, and there was no significant difference at each time point (P0.05). (2) there was only a small amount of PSD-95 protein expression in 6h, 24h, 48H, 72h, and there was no significant difference between the EPO group and the group. The expression of protein was significantly more than that of Sham group (P0.05); (3) the expression of PSD-95 protein in group EPO was significantly higher than that in ICH group at 7d, 14d and 21d, and there was a statistical difference (P0.05). (4) ICH group, EPO group began to increase from 72h. Correlation analysis with SYP and PSD-95 protein content: (1) at 14d time point, ICH group, EPO group SYP protein content was positively correlated with the corresponding experimental rat neural function score (r=0.940, P0.001). (2) in 14d time point, ICH group, EPO group PSD-95 protein content was positively correlated with the corresponding test of rat neural function score. Conclusion: 1, take The method of making ICH model of rats with anticoagulant autologous blood injection is simple and easy to operate, and it is similar to the pathological process of spontaneous ICH in clinic. It is an ideal animal model for studying ICH. 2, SYP, PSD-95 protein expression is increased around hematoma after ICH, and it is beneficial to the restoration of the function of the God, and 3, erythropoietin (EPO) can up the hematoma. The expression of SYP and PSD-95 protein promotes the synaptic remodeling after ICH. 4, erythropoietin (EPO) can promote the recovery of limb function and learning and memory ability of ICH rats.

【學(xué)位授予單位】：西南醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R743.34

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相關(guān)期刊論文 前10條

1 謝巍;張波;宋世賓;吉蒙;邵丹丹;;大鼠腦出血后AQP-4、IL-6及MMP-9的表達(dá)與腦水腫的相關(guān)性研究[J];貴州醫(yī)藥;2015年08期

2 何雨;李彤;胡艷兵;;血管性癡呆大鼠PSD-95變化機(jī)制的研究[J];中國實(shí)驗(yàn)診斷學(xué);2010年12期

3 周元成;吳新貴;;腦神經(jīng)可塑性的研究進(jìn)展[J];廣西中醫(yī)學(xué)院學(xué)報;2010年02期

4 楊燕;;腦出血臨床表現(xiàn)與預(yù)后的關(guān)系及其護(hù)理[J];實(shí)用臨床醫(yī)藥雜志;2007年04期

5 李新娟;葛治國;李東亮;李慶崗;李明陽;秦宇;趙明虎;;牛磺酸對大鼠腦損傷后認(rèn)知功能及海馬凋亡相關(guān)蛋白表達(dá)的影響[J];新鄉(xiāng)醫(yī)學(xué)院學(xué)報;2006年06期

6 田梅,趙紅崗,李東亮,張耀東,毛會麗,李東飛;缺氧預(yù)處理抑制新生鼠缺氧缺血性腦損傷細(xì)胞凋亡的觀察[J];新鄉(xiāng)醫(yī)學(xué)院學(xué)報;2005年06期

7 時嵐,陳俊濤,李平法;低氧預(yù)適應(yīng)對大鼠腦缺血再灌注神經(jīng)細(xì)胞凋亡和Caspase-3活性的影響[J];新鄉(xiāng)醫(yī)學(xué)院學(xué)報;2005年04期

8 張淑玲,常全忠,李銀生,郭學(xué)鵬;缺氧缺血性腦損傷大鼠腦內(nèi)bcl-2基因表達(dá)與細(xì)胞凋亡的關(guān)系[J];實(shí)用兒科臨床雜志;2004年05期

9 朱鏞連;腦卒中康復(fù)與神經(jīng)康復(fù)機(jī)制[J];中國康復(fù)理論與實(shí)踐;2003年03期

10 劉勝達(dá),周志明,吳家冪;立體定向技術(shù)制作大鼠腦出血模型評價[J];中國航天醫(yī)藥雜志;2003年01期

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