【作者】 姜彩肖；

【导师】 刘殿武；

【作者基本信息】 河北医科大学 ， 流行病与卫生统计学， 2008， 硕士

【摘要】 目的:血管性痴呆(Vascular Dementia,VD)是老年期痴呆的主要类型之一,它是由一系列脑血管因素(缺血、出血、急慢性缺氧性脑血管病等)导致脑组织损害引起的以认知功能障碍为特征的痴呆综合症,不仅给病人带来长期痛苦,严重影响其生活质量,而且给家庭、社会造成沉重负担,因此研究VD的发病机制以指导其防治具有很高的社会价值和现实意义。硫化氢(hydrogen sulfide,H2S)是“一种臭鸡蛋气味的气体”,H2S不仅有毒性作用,它还广泛参与机体多种生理和病理过程,因此被认为是继一氧化氮(nitric oxide,NO)、与一氧化碳(carbon monoxide, CO)后的第三类气体信号分子。H2S不仅在神经系统发挥重要的生理作用,而且还作为一种内源性的抗氧化剂,在氧化应激时能够清除羟自由基等活性氧簇,对神经细胞起保护作用。本实验以VD大鼠模型为研究对象,观察大脑缺血再灌注不同时间对大鼠学习记忆能力的损伤程度、血清中H2S含量的改变及海马CA1区神经元细胞凋亡率及凋亡调控蛋白Bcl-2和Bax的表达情况,从行为学、细胞凋亡及内源性H2S表达量及其相互关系等方面,探讨VD的发病机制,进一步认识H2S这个新的气体信号分子的作用,为VD的防治提供理论依据。方法:选用纯系健康雄性Wistar大鼠64只,将动物随机分为8组,正常组(Normal group),假手术组(Sham-operated group)和VD模型组(VD group),VD模型组又分为大脑缺血再灌注2小时组(IR 2h group),大脑缺血再灌注8小时组(IR 8h group),大脑缺血再灌注24小时组(IR 24h group),大脑缺血再灌注72小时组(IR 72h group),大脑缺血再灌注168小时组(IR 168h group)和大脑缺血再灌注720小时组(IR 720h group),每组8只动物,采用四血管阻断法(4-VO)制作VD大鼠模型,假手术组麻醉及手术过程与VD模型组相同,但不电凝双侧椎动脉,不阻断双侧颈总动脉。采用Morris水迷宫法测试各组大鼠的学习记忆能力,以逃避潜伏期为观测指标;制作大脑HE切片,采用光镜观察各组大鼠海马CA1区的形态学变化;采用免疫组织化学染色法检测凋亡调控蛋白Bcl-2及Bax的表达情况,采用Motic 6.0数码医学图象分析系统测量海马CA1区阳性目标平均光密度,进行统计分析;采用流式细胞术检测海马神经细胞的凋亡率;采用亚甲蓝比色法检测血清中H2S的含量,实验数据用SPSS13.0统计软件进行统计学分析。结果:⑴行为学:随着训练天数的增加,各组大鼠的逃避潜伏期均缩短,第二天各组大鼠的逃避潜伏期开始出现统计学差异,IR 720h组与正常组和假手术组比较逃避潜伏期明显延长(P<0.01);正常组和假手术组逃避潜伏期差异无统计学意义(P>0.05)。⑵形态学改变:假手术组大鼠海马CA1区神经元细胞完整,结构正常,胞浆丰富;细胞核呈圆形、椭圆形,无固缩或溶解现象;染色质在核内分布较均匀,间质无水肿表现。VD模型组大鼠海马CA1区可见大量神经元变性,体积变小,胞核与胞浆界限不清,核固缩成三角形或不规则形,而且随缺血再灌注时间的延长,变性神经元数目越多。⑶细胞凋亡蛋白表达:VD模型组缺血再灌注2小时后Bcl-2蛋白平均光密度值开始明显上调,缺血再灌注8小时达到高峰,之后开始下降,但IR 24h组仍高于假手术组(P<0.05);缺血再灌注早期Bax在海马CA1区的表达明显增强,随着再灌注时间的延长,其表达量不断增加,IR 72~168h达高峰(P<0.05);随缺血再灌注时间的延长,Bcl-2/Bax的比率逐渐降低。⑷海马神经细胞凋亡率:VD模型组与假手术组比较,海马神经细胞凋亡率均增加(P<0.01);VD模型组各组海马神经细胞凋亡率各不相同(P<0.01);随着缺血再灌注时间的延长,大鼠海马神经细胞凋亡率逐渐增高,海马神经细胞凋亡率呈现对数曲线上升趋势。⑸血清H2S含量: VD模型组与假手术组和正常组比较,血清中H2S含量减少(P<0.01);VD模型各组间血清H2S含量差异有显著性(P<0.01);随着缺血再灌注时间的延长,大鼠血清H2S含量逐渐降低,血清H2S含量呈现直线降低趋势。⑹血清H2S含量与海马神经细胞凋亡率相关分析:随着血清H2S含量的降低,海马神经细胞凋亡率逐渐升高,两者呈现明显的负相关,回归方程为:y =-0.135x + 12.068,相关系数r=-0.861(P<0.01),提示内源性H2S可能对海马神经细胞具有保护作用。结论:脑缺血再灌注损伤可导致VD大鼠学习记忆功能的减退,海马神经细胞凋亡程度增强和血清中H2S含量减少;血清H2S含量与海马神经细胞凋亡率呈负相关,海马神经细胞凋亡程度可能与内源性H2S生成减少有关。更多还原

【Abstract】 Objective: Vascular Dementia is one of the main types of geriatric dementia, and it is caused by a series of cerebral vessels factor, such as ischemic, sanguinis profluvium and hypoxia cerebrovascular disease. Vascular Dementia is a kind of dementia syndrome with recognition functional disturbance. It not only takes long-term anguish to the patient, but also poses heavy burden on the family and society. So the studies of pathogenesis for Vascular Dementia have very important social value and practical importance. Hydrogen sulfide (H2S) is a kind of gas with brom-egg taste. It has toxic action, but it also participate many physiological and path process in organism, so it is regard as the third kind of gas signaling molecule after nitric oxide and carbon monoxide. Hydrogen sulfide can play an important role in nervous system, and it also can be an endogenic anti-oxidant to remove active oxygen for example hydroxyls radical and protect the nerve cells. The research observed the capability of learning and memory, contents of hydrogen sulfide in blood serum and nerve cells apoptosis in CA1 region of hippocampus in Vascular Dementia rats of cerebral ischemia reperfusion different time, investigated the pathogenesis of Vascular Dementia, knowed the effect of H2S as a new gas signaling molecule and provided theory evidence for Vascular Dementia’s prevention and cure.Methods: 64 healthy male Wister rats were randomly divided into 8 groups: Normal group, Sham-operated group and IR groups, IR group were divided into IR 2h group, IR 8h group, IR 24h group, IR 72h group, IR 168h group and IR 720h group (n=8 each). Using the method of 4-VO, the animal Vascular Dementia models were established. The capability of learning and memory were tested by Morris water maze, cerebrum morphology were observed by light microscope, the expression of Bcl-2 and Bax were detected by immunohistochemistry staining method, and the average optical density values of masc. target in CA1 region of hippocampus were measured by Motic6.0 medical image analysis system. The apoptosis rates of nerve cells in hippocampus were detected by flow cytometry, and the contents of hydrogen sulfide in blood serum were detected by methylene blue chromatometry. All the experimental data used SPSS13.0 software for statistics analysis.Results: 1. Behavior science: All rats shortened their escape latency following with the increasing of training time, and statistics difference began to appear in sequenti luce of training. Escape latency was significantly longer in the IR 720h group compared to Normal group and Sham-operated group (P<0.01), escape latency was not statistically significant in Normal group compared to Sham-operated group (P>0.05). 2. Morphology: Nerve cells in CA1 region of hippocampus of Sham-operated group were integrity and normal with abundant endochylema, cell nucleus presented round or ellipse, no cell nucleus pyknosis or dissolving emerged, chromatin distributed uniformity, and interstitial substance had no dropsy. Neuronal degeneration phenomenon could be seen in the model group. Nerve cells volume shrinked, nucleus and endochylema had no clear margin and nerve cells emerged karyopycnosis. The amounts of degeneration nerve cells were more following with the extending time of ischemic reperfusion. 3. Expression of apoptosis protein: The average optical density values of both the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were up-regulated, during reperfusion after 2h of ischemia, the expression of Bcl-2 was induced with peak expression at 8h, and then decreased, but the expression of Bcl-2 was higher in the reperfusion after 24h of ischemia compared to Sham-operated group (P<0.05). The average optical density values of Bax increased with time after reperfusion with peak expression at 72~168h (P<0.05). The ratios of Bcl-2/Bax decreased following with the extending time of ischemic reperfusion. 4. The apoptosis rates of nerve cells in hippocampus: The apoptosis rates of nerve cells in hippocampus were up-regulated in the model groups compared to Sham-operated group (P<0.01), and the apoptosis rates of nerve cells in hippocampus increased following with the extending time of ischemic reperfusion and presented an increasing tendency of logarithmic curve. 5. Contents of hydrogen sulfide in blood serum: The contents of hydrogen sulfide in blood serum of the model groups reduced compared to Normal group and Sham-operated group (P<0.01), and decreased straightly following with the extending time of ischemic reperfusion. 6. The correlation analysis for contents of serum hydrogen sulfide and apoptosis rates of hippocampus nerve cells: The apoptosis rates of hippocampus nerve cells heightened following with the decreasing of the contents of serum hydrogen sulfide, they presented linearity correlation relation and they might be the cues of endogenous hydrogen sulfide protecting nerve cells from apoptosis.Conclusion: Cerebral ischemia reperfusion injury resulted in the impairment of learning and memory, apoptosis of nerve cells in hippocampus and reducing of hydrogen sulfide contents in blood serum. The apoptosis level of hippocampus nerve cells might be concern with the decreasing of endogenous hydrogen sulfide.更多还原