早产兔生发基质—脑室内出血对海马体的影响

发布时间：2018-01-13 11:51

本文关键词：早产兔生发基质—脑室内出血对海马体的影响　出处：《河北医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:颅内出血(intracranial hemorrhage,ICH)是新生儿期常见病,与这一阶段自身的解剖生理特点和多种围产期高危因素有关,其中生发基质-脑室内出血(germinal matrix-intraventricular hemorrhage,GM-IVH)是早产儿最常见的ICH类型,胎龄愈小发病率越高,是影响早产儿救治,导致早产儿死亡,甚至存活者预后不良的主要原因之一。近年来,由于医学技术的进步,尤其新生儿重症监护病房(NICU)设备与医疗护理水平的提高,早产儿GM-IVH的发生率已下降至约2.21%,但34周以下早产儿GM-IVH发生率仍高达24.1%,重度GM-IVH容易合并脑室周围白质损伤、脑室周出血性梗死等并发症,往往遗留小儿神经系统后遗症,目前尚无有效治疗方法。海马体作为大脑边缘系统的重要组成部分,与脑室系统密切相关,主要负责学习和记忆,是GM-IVH后潜在脑损伤部位,且海马体病变与多种神经精神疾病密切相关。目前大部分研究仍局限于脑室周围白质损伤,而鲜有报道GM-IVH是否对海马体造成损伤。本文试图通过动物模型探讨:(1)早产兔GM-IVH后海马体细胞凋亡相关蛋白的表达水平;(2)去铁胺(Deferoxamin,DFX)干预治疗对上述相关蛋白表达的影响;方法:1选择孕29天孕兔进行剖宫产(足月为孕32天),迅速取幼兔,共36只,给予保温、早产儿奶粉喂养。将所有幼兔随机分为对照组、GM-IVH模型组(简称模型组)、GM-IVH模型+DFX干预组(简称治疗组),每组各12只。参照腹腔注射丙三醇诱发GM-IVH的方法,模型制备方法如下:于生后3小时内给早产兔腹腔注射50%丙三醇,剂量为10ml/kg。于注射丙三醇2小时后,治疗组给予腹腔注射DFX,药物浓度为50mg/ml,给药总剂量为100mg/(kg·d),24小时内分两次给药,其他两组给予等量生理盐水。24小时后行颅脑超声筛查观察出血情况,剔除模型组、治疗组中未发生GM-IVH早产兔,和对照组中自发GM-IVH的早产兔。2将以上三组剩余早产兔(每组各10只)于生后48h腹腔注射2%戊巴比妥钠(40mg/kg)麻醉,断头,于冰上迅速剥离脑组织。从前囟开始,剥离头顶骨片,海马体位于大脑皮层(cerebral cortex)底部,用直镊小心地将大脑皮层拨开,分离出海马体,用PBS(磷酸盐缓冲液)洗净,一半浸入4%多聚甲醛林中进行固定,用于HE染色、免疫组化,另一半置于-80℃冰箱冷冻,用于免疫印迹(Western Blot)检测。3光镜下观察海马CA1区神经元的形态学变化;用免疫组织化学染色观察肿瘤坏死因子-α(TNF-α)和天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)的蛋白表达;Western Blot检测TNF-α和caspase-3的蛋白表达水平。4统计方法:采用SPSS17.0统计学软件、Microsoft Excel 2003处理数据,计量资料若符合正态分布以均数±标准差表示。检验方差齐性,若方差齐,样本均数间的两两比较比较采用LSD-t法检验;若方差不齐采用秩和检验,P0.05有统计学意义。结果:1成功复制了早产兔GM-IVH模型对孕29天的孕兔行剖宫产,成功获得早产兔。通过新生兔颅脑超声和大体标本证实早产兔GM-IVH模型的成功建立。2各组早产兔海马体CA1区神经细胞HE染色结果对照组早产兔大脑海马体CA1区锥体神经细胞排列紧密,结构清晰,胞体呈椎体,胞核大且圆,核仁明显;模型组早产兔大脑海马体CA1区锥体神经细胞排列稀疏,细胞间隙扩大,胞体缩小,胞核浓染,部分细胞结构破坏甚至崩解;治疗组早产兔大脑海马体CA1区锥体神经细胞排列稍稀疏,部分细胞间隙稍有扩大,细胞数量介于对照组与模型组之间,细胞结构基本正常。3免疫组织化学检测海马体CA1区TNF-α和caspase-3蛋白表达情况对照组早产兔海马体CA1区可见丰富的神经细胞,仅见少量的胞质或胞核呈棕色的阳性细胞,染色程度较浅;与对照组相比,模型组早产兔海马体CA1区神经细胞数目明显减少,排列稀疏,TNF-α和caspase-3阳性细胞的数量明显增多,染色程度较深,为深棕色,差异有统计学意义(P0.05);与模型组相比,治疗组早产兔海马体CA1区TNF-α和caspase-3阳性细胞的数量减少,染色程度较模型组浅,差异有统计学意义(P0.05)。4 Western Blot检测海马体TNF-α和caspase-3蛋白表达水平对照组早产兔海马体TNF-α、caspase-3蛋白表达较低;与对照组相比,模型组早产兔海马体TNF-α、caspase-3蛋白表达于GM-IVH后升高,差异有统计学意义(P0.05);与模型组相比,治疗组早产兔海马体TNF-α、caspase-3蛋白的表达降低,差异有统计学意义(P0.05)。结论:1早产兔发生GM-IVH后,海马体CA1区神经细胞数量减少、细胞结构破坏,提示早产兔GM-IVH后对海马体造成损伤;2早产兔发生GM-IVH后,海马体内TNF-α、caspase-3表达量增加,差异有统计学意义(P0.05),提示细胞凋亡机制参与了GM-IVH后海马体的损伤过程;3经去铁胺治疗后,早产兔GM-IVH海马体TNF-α、caspase-3蛋白表达水平下降,差异有统计学意义(P0.05),提示去铁胺可以减轻GM-IVH后海马体的损伤。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R743.34

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