本文關鍵詞：早產(chǎn)兔生發(fā)基質(zhì)—腦室內(nèi)出血對海馬體的影響　出處：《河北醫(yī)科大學》2017年碩士論文　論文類型：學位論文

  更多相關文章： 早產(chǎn)兒 顱內(nèi)出血 生發(fā)基質(zhì)-腦室內(nèi)出血 海馬體 腫瘤壞死因子-α 天冬氨酸特異性半胱氨酸蛋白酶-3 去鐵胺

【摘要】：目的:顱內(nèi)出血(intracranial hemorrhage,ICH)是新生兒期常見病,與這一階段自身的解剖生理特點和多種圍產(chǎn)期高危因素有關,其中生發(fā)基質(zhì)-腦室內(nèi)出血(germinal matrix-intraventricular hemorrhage,GM-IVH)是早產(chǎn)兒最常見的ICH類型,胎齡愈小發(fā)病率越高,是影響早產(chǎn)兒救治,導致早產(chǎn)兒死亡,甚至存活者預后不良的主要原因之一。近年來,由于醫(yī)學技術(shù)的進步,尤其新生兒重癥監(jiān)護病房(NICU)設備與醫(yī)療護理水平的提高,早產(chǎn)兒GM-IVH的發(fā)生率已下降至約2.21%,但34周以下早產(chǎn)兒GM-IVH發(fā)生率仍高達24.1%,重度GM-IVH容易合并腦室周圍白質(zhì)損傷、腦室周出血性梗死等并發(fā)癥,往往遺留小兒神經(jīng)系統(tǒng)后遺癥,目前尚無有效治療方法。海馬體作為大腦邊緣系統(tǒng)的重要組成部分,與腦室系統(tǒng)密切相關,主要負責學習和記憶,是GM-IVH后潛在腦損傷部位,且海馬體病變與多種神經(jīng)精神疾病密切相關。目前大部分研究仍局限于腦室周圍白質(zhì)損傷,而鮮有報道GM-IVH是否對海馬體造成損傷。本文試圖通過動物模型探討:(1)早產(chǎn)兔GM-IVH后海馬體細胞凋亡相關蛋白的表達水平;(2)去鐵胺(Deferoxamin,DFX)干預治療對上述相關蛋白表達的影響;方法:1選擇孕29天孕兔進行剖宮產(chǎn)(足月為孕32天),迅速取幼兔,共36只,給予保溫、早產(chǎn)兒奶粉喂養(yǎng)。將所有幼兔隨機分為對照組、GM-IVH模型組(簡稱模型組)、GM-IVH模型+DFX干預組(簡稱治療組),每組各12只。參照腹腔注射丙三醇誘發(fā)GM-IVH的方法,模型制備方法如下:于生后3小時內(nèi)給早產(chǎn)兔腹腔注射50%丙三醇,劑量為10ml/kg。于注射丙三醇2小時后,治療組給予腹腔注射DFX,藥物濃度為50mg/ml,給藥總劑量為100mg/(kg·d),24小時內(nèi)分兩次給藥,其他兩組給予等量生理鹽水。24小時后行顱腦超聲篩查觀察出血情況,剔除模型組、治療組中未發(fā)生GM-IVH早產(chǎn)兔,和對照組中自發(fā)GM-IVH的早產(chǎn)兔。2將以上三組剩余早產(chǎn)兔(每組各10只)于生后48h腹腔注射2%戊巴比妥鈉(40mg/kg)麻醉,斷頭,于冰上迅速剝離腦組織。從前囟開始,剝離頭頂骨片,海馬體位于大腦皮層(cerebral cortex)底部,用直鑷小心地將大腦皮層撥開,分離出海馬體,用PBS(磷酸鹽緩沖液)洗凈,一半浸入4%多聚甲醛林中進行固定,用于HE染色、免疫組化,另一半置于-80℃冰箱冷凍,用于免疫印跡(Western Blot)檢測。3光鏡下觀察海馬CA1區(qū)神經(jīng)元的形態(tài)學變化;用免疫組織化學染色觀察腫瘤壞死因子-α(TNF-α)和天冬氨酸特異性半胱氨酸蛋白酶-3(caspase-3)的蛋白表達;Western Blot檢測TNF-α和caspase-3的蛋白表達水平。4統(tǒng)計方法:采用SPSS17.0統(tǒng)計學軟件、Microsoft Excel 2003處理數(shù)據(jù),計量資料若符合正態(tài)分布以均數(shù)±標準差表示。檢驗方差齊性,若方差齊,樣本均數(shù)間的兩兩比較比較采用LSD-t法檢驗;若方差不齊采用秩和檢驗,P0.05有統(tǒng)計學意義。結(jié)果:1成功復制了早產(chǎn)兔GM-IVH模型對孕29天的孕兔行剖宮產(chǎn),成功獲得早產(chǎn)兔。通過新生兔顱腦超聲和大體標本證實早產(chǎn)兔GM-IVH模型的成功建立。2各組早產(chǎn)兔海馬體CA1區(qū)神經(jīng)細胞HE染色結(jié)果對照組早產(chǎn)兔大腦海馬體CA1區(qū)錐體神經(jīng)細胞排列緊密,結(jié)構(gòu)清晰,胞體呈椎體,胞核大且圓,核仁明顯;模型組早產(chǎn)兔大腦海馬體CA1區(qū)錐體神經(jīng)細胞排列稀疏,細胞間隙擴大,胞體縮小,胞核濃染,部分細胞結(jié)構(gòu)破壞甚至崩解;治療組早產(chǎn)兔大腦海馬體CA1區(qū)錐體神經(jīng)細胞排列稍稀疏,部分細胞間隙稍有擴大,細胞數(shù)量介于對照組與模型組之間,細胞結(jié)構(gòu)基本正常。3免疫組織化學檢測海馬體CA1區(qū)TNF-α和caspase-3蛋白表達情況對照組早產(chǎn)兔海馬體CA1區(qū)可見豐富的神經(jīng)細胞,僅見少量的胞質(zhì)或胞核呈棕色的陽性細胞,染色程度較淺;與對照組相比,模型組早產(chǎn)兔海馬體CA1區(qū)神經(jīng)細胞數(shù)目明顯減少,排列稀疏,TNF-α和caspase-3陽性細胞的數(shù)量明顯增多,染色程度較深,為深棕色,差異有統(tǒng)計學意義(P0.05);與模型組相比,治療組早產(chǎn)兔海馬體CA1區(qū)TNF-α和caspase-3陽性細胞的數(shù)量減少,染色程度較模型組淺,差異有統(tǒng)計學意義(P0.05)。4 Western Blot檢測海馬體TNF-α和caspase-3蛋白表達水平對照組早產(chǎn)兔海馬體TNF-α、caspase-3蛋白表達較低;與對照組相比,模型組早產(chǎn)兔海馬體TNF-α、caspase-3蛋白表達于GM-IVH后升高,差異有統(tǒng)計學意義(P0.05);與模型組相比,治療組早產(chǎn)兔海馬體TNF-α、caspase-3蛋白的表達降低,差異有統(tǒng)計學意義(P0.05)。結(jié)論:1早產(chǎn)兔發(fā)生GM-IVH后,海馬體CA1區(qū)神經(jīng)細胞數(shù)量減少、細胞結(jié)構(gòu)破壞,提示早產(chǎn)兔GM-IVH后對海馬體造成損傷;2早產(chǎn)兔發(fā)生GM-IVH后,海馬體內(nèi)TNF-α、caspase-3表達量增加,差異有統(tǒng)計學意義(P0.05),提示細胞凋亡機制參與了GM-IVH后海馬體的損傷過程;3經(jīng)去鐵胺治療后,早產(chǎn)兔GM-IVH海馬體TNF-α、caspase-3蛋白表達水平下降,差異有統(tǒng)計學意義(P0.05),提示去鐵胺可以減輕GM-IVH后海馬體的損傷。
[Abstract]:Objective: intracranial hemorrhage (intracranial hemorrhage ICH) is a common disease in neonatal period, and this stage of its anatomy and physiology characteristics and various perinatal risk factors, including germinal matrix intraventricular hemorrhage (germinal matrix-intraventricular hemorrhage, GM-IVH) is the most common type of ICH in preterm infants, gestational age less the higher incidence effect of treatment, is the leading cause of premature death, and even the main reason of poor prognosis of survivors. In recent years, due to advances in medical technology, especially in the neonatal intensive care unit (NICU) equipment and medical care of preterm infants, the incidence of GM-IVH has decreased to about 2.21%, but 34 weeks following the incidence of GM-IVH is still as high as 24.1%, with severe GM-IVH to periventricular white matter injury, the complications of periventricular hemorrhagic infarct, often left with neurological sequelae, there is no The effective method of treatment. The hippocampus is an important part of the limbic system, and is closely related to the ventricular system, mainly responsible for learning and memory, is a potential region of brain injury after GM-IVH, and hippocampal lesions and various neuropsychiatric disorders are closely related. Most of the research is still limited to the periventricular white matter injury, but rarely reported whether GM-IVH in the hippocampus damage. This paper attempts to explore the animal model: (1) the expression level of apoptosis in premature rabbit GM-IVH Houhai equine somatic cell related proteins; (2) deferoxamine (Deferoxamin, DFX) the intervention effect on the expression of related proteins; methods: 1 pregnant 29 days pregnant rabbits for cesarean section (for full-term pregnant 32 days), quickly take the rabbit, a total of 36, giving the insulation, milk feeding in premature infants. All the rabbits were randomly divided into control group, GM-IVH model group (model group), GM-IVH model +DFX treatment group (treatment Group), 12 rats in each group. Referring to the method of intraperitoneal injection of glycerol induced GM-IVH model, the preparation method is as follows: within 3 hours after birth to premature rabbits intraperitoneal injection of 50% glycerol, the dose of 10ml/kg. in 2 hours after the injection of glycerol, the treatment group was given intraperitoneal injection of DFX, the drug concentration is 50mg/ml, the total dose of drug 100mg/ (kg - D), 24 hours two times to medicine, the other two groups were given saline.24 hours after craniocerebral ultrasound screening observed bleeding, excluding the model group, GM-IVH treatment group did not occur in premature rabbits, premature rabbit.2 spontaneous GM-IVH and control group in the above three groups of premature rabbits (residual n = 10) at 48h after intraperitoneal injection of 2% pentobarbital anesthesia (40mg/kg), decapitated, on ice to quickly separate the brain. Once started off the head bone skull, hippocampus, cerebral cortex (cerebral cortex) is located at the bottom, with straight tweezers carefully to the cerebral cortex The separation layer off, sea horse, PBS (phosphate buffer) wash, half immersed in 4% paraformaldehyde in the forest were fixed for HE staining, immunohistochemistry, the other half is placed in the freezer for -80 C, immunoblotting (Western Blot) to detect and observe the morphological changes of neurons in hippocampal CA1 area of.3 under the light microscope we used immunohistochemistry staining; tumor necrosis factor alpha (TNF- alpha) and aspartate specific cysteine protease -3 (caspase-3) protein expression; the expression of Western Blot detection of TNF- alpha and caspase-3 protein level of.4 statistical methods: SPSS17.0 statistical software, Microsoft Excel 2003 data processing of measurement data, if accord with normal the distribution of mean standard deviation. Test for homogeneity of variance, if homogeneity of variance, the sample mean between 22 compared to using the method of LSD-t test; if the missingvariance Wilcoxon test was used, P0.05 was statistically significant results: 1 the success of preterm GM-IVH rabbit model of pregnant 29 day pregnant rabbits underwent cesarean section, premature rabbit successfully. By newborn rabbit cranial ultrasound with specimen confirmed premature rabbit GM-IVH model was successfully established in.2 group premature rabbit hippocampus CA1 neurons HE stainingin control CA1 group premature rabbit hippocampus pyramidal cells arranged closely, clear structure, the cell body showed vertebral body, large and round nucleus, obvious nucleolus; model group premature rabbit hippocampal pyramidal neurons of CA1 area are sparse, cell gap widened, cell body shrinkage, nuclear stained, and even disintegration part of cell structure; treatment of CA1 group premature rabbit hippocampus pyramidal cells arranged a little sparse, part of the cell gap slightly expanded, between the number of cells between the control group and model group. The cell structure is normal.3 immunohistochemical detection of TNF- in hippocampus of CA1 alpha and caspase-3 The expression of the control CA1 group premature rabbit hippocampus showed rich nerve cells, only a small amount of cytoplasm or nucleus Brown positive cells, staining shallow degree; compared with the control group, model group, premature rabbit hippocampus CA1 neurons significantly reduce the number of sparse arrangement, the number of TNF- alpha and caspase-3 the positive cells increased significantly, the degree of staining deep, dark brown, the difference was statistically significant (P0.05); compared with the model group, the number of treatment group premature rabbit hippocampal TNF- CA1 alpha and caspase-3 positive cells decreased, the degree of staining compared with the model group, the difference was statistically significant (P0.05).4 Western Blot detection of hippocampus TNF- alpha and caspase-3 protein expression in control group premature rabbit hippocampus TNF- alpha, caspase-3 protein expression was lower; compared with the control group, model group, premature rabbit hippocampus TNF- alpha, caspase-3 protein expression increased at GM-IVH, the difference There was statistical significance (P0.05); compared with the model group, the treatment group premature rabbit hippocampus TNF- alpha, caspase-3 protein expression decreased, the difference was statistically significant (P0.05). Conclusion: 1 premature rabbits after GM-IVH, the number of hippocampal CA1 neurons decreased, the cell structure was destroyed, prompting premature rabbits after GM-IVH of hippocampus body damage; 2 premature rabbits after GM-IVH in the hippocampus of TNF- alpha, caspase-3 expression was increased, the difference was statistically significant (P0.05), suggesting that the mechanism of cell apoptosis is involved in the damage process of GM-IVH Houhai horse; 3 after deferoxamine treatment, premature rabbit hippocampus GM-IVH TNF- alpha, caspase-3 protein expression level decreased, the difference was statistically significant (P0.05), suggesting that deferoxamine can reduce the GM-IVH of Houhai horse body damage.

【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R743.34

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