【作者】 李华；

【导师】 柳忠兰；

【作者基本信息】 中国医科大学 ， 神经病学， 2003， 硕士

【摘要】 前言 近年研究表明，吸烟与多种神经系统疾病有关。流行病学调查显示，吸烟是脑卒中，特别是缺血性脑卒中的重要危险因素。国外研究发现吸烟能加速脑萎缩和退化改变。 细胞凋亡是在生理或病理条件下细胞能按自身即定的程序主动结束生命的一种死亡形式，是一种多步骤发生的受基因调控的遗传机制。细胞凋亡在神经系统的正常发育及可塑性有重要作用。在一些因素刺激下，神经细胞可发生病理性凋亡，被认为与多种神经系统疾病有关，如神经退行性疾病，早老性痴呆和缺血性脑损伤等。细胞凋亡有复杂的分子调控机制，细胞凋亡的研究早期在细胞表面就发现一类可传递死亡信号的受体，即Fas(Apo-1或CD95)。各种离体和在体实验表明Fas可介导细胞凋亡，Fas/Fas配体(Fas Ligand，FasL)是一类重要的凋亡信号传导系统。 近年来研究发现，吸烟能诱导多种组织细胞发生凋亡，而吸烟能否诱导脑组织细胞凋亡国内外尚未见报道。本实验采用原位末端标记TUNEL法和免疫组织化学方法观察不同条件下吸烟大鼠脑组织细胞凋亡及Fas蛋白表达的情况，探讨其变化规律及可能的发生机制，为吸烟引起的脑损害及其防治提供理论依据。 实验材料方法 实验动物随机分为长期大量吸烟组、长期小量吸烟组、短期吸烟组、戒烟组和正常对照组。建立吸烟大鼠模型，标本制备，HE染色光镜观察。采用原位末端标记TUNEL法和免疫组织化学SABC法检测细胞凋亡及Fas蛋白表达。采用显微镜计数法，计数阳性细胞，SPSS10．0统计软件进行数据处理，各组间比较用方差分析。 实验结果 1．正常对照组有个别散在的凋亡细胞，少量Fs蛋白阳性表达。2．长期和短期吸烟组凋亡细胞及Fs蛋白阳性表达与对照组比有显著性增加沙＜0．01人3．戒烟组凋亡细胞及h蛋白表达与短期吸烟组比显著性减少k＜o对门，与对照组比元显著性差异口＞0．05八4．长期大量吸烟组凋亡细胞较长期小量及短期吸烟组显著增多（P＜o．01人5．长期大量吸烟组h蛋白阳性表达比短期组显著性增多K＜0刀＊，与长期小量组无显著差异u＞0．05）。 讨 论 细胞凋亡的概念于1972年被Kerr等人提出，近几年已逐渐成为各学科研究的热点。细胞凋亡与多种神经系统疾病有关，例如神经变性疾病，缺血性脑血管病等。许多因素如生长因子缺乏，辐射等都能诱导细胞凋亡。吸烟或烟雾暴露已被证明可诱导多种组织细胞发生凋亡。 本实验结果显示正常大鼠脑组织仅有个别散在的凋亡细胞，吸烟能引起显著的脑组织凋亡细胞增加，且凋亡细胞主要为神经细胞。随着吸烟的时间及每日烟量增加，脑组织凋亡细胞的数量随之增加。提示吸烟引起的脑组织细胞凋亡存在时间一效应和剂量一效应关系。戒烟组的凋亡细胞比短期组明显减少，与对照组无显著差别，提示戒烟可使吸烟引起的脑细胞损害恢复。在长期吸烟组，血管内皮细胞及脉络丛上皮细胞凋亡也有所增加，血管内 ·2·皮细胞凋亡具有促凝作用，能促发和加重动脉粥样硬化病变，这可能是吸烟促进脑动脉粥样硬化从而促进脑血栓形成的机制之一。 本实验发现，吸烟也能诱导大鼠脑组织细胞Fs蛋白表达增加，除戒烟组外各吸烟组与对照组比，Fs蛋白阳性表达均有显著性增加。长期大量吸烟组阳性表达比短期组显著增多而与长期小量组无显著差异，表明吸烟诱导的Fs蛋白表达与吸烟时间更相关，随吸烟时间增加，阳性表达随之增强。Fs抗原属于1型跨膜蛋白，膜内部分含有传递细胞死亡信号的同源功能区。Fas／FaSL是一类重要的凋亡信号传导系统。本实验结果显示Fs蛋白阳性表达主要分布于皮质，海马有部分表达，与细胞凋亡分布的区域相一致，Fas蛋白阳性表达增强时，细胞凋亡也相应增加，这表明Fs介导吸烟诱导的细胞凋亡的发生。 Fas介导的细胞凋亡受等多种因子的高度调节。目前已发现3个与 Fas直接联系的介导因子：Fs相关磷酸酯酶（FAPJ人受体相互反应蛋白（RIP）和Fs死亡域相关蛋白（FADD人FAP一互是一种重要的负性调节因子，FADD和RIP的过度表达可诱导细胞凋亡。此外，还发现Fs结合后另外一些信号传导，如刺激FaS，可激活膜鞘磷脂酶，后者可催化释放可溶性的神经酚胺，诱导细胞凋亡。 香烟烟雾中含有大量不同的成分，包括尼古了、一氧化碳、焦油、苯并志等，这些成分能引起、促进或扩大氧化损害。近年来研究表明，氧化与细胞凋亡关系密切。无论是香烟的气相或微粒（主要是焦油）均含有较高浓度的自由基。香烟烟雾中的氧化剂和自由基等通过反应性氧基（ROS）传人细胞，引起细胞脂质过氧化或与细胞凋亡有关的基因如 Fas的表达，通过一系列生化反应，最终发生细胞凋亡，这可能是吸烟诱导Fs表达而促进细胞凋亡的机制之一。 吸烟诱导的细胞凋亡还可能与缺血有关，推测引起缺血的因 ·3·素有：O）香烟中的尼古丁刺激交感神经，使缩血管物质分泌增加更多还原

【Abstract】 Recently, Some studies have shown that cigarette smoking is related to some diseases of nervous system. Epidemiological study has shown that cigarette smoking is a major risk factor for stroke, especially for ischemic stroke. The research in foreign country found that smoking could accelerate the brain atrophy and degeneration.Apoptosis is a kind of death form that cell can end its life actively according to some established program under the physiologic or pathologic conditions, and it is a multistage genetic mechanism under the gene control. Apoptosis is important to the compliance and normal development of nervous system. Pathologic apoptosis can occur in nerve cell under some stimulus, and it can consider to be associated with some kinds of nervous system diseases, such as presenile dementia, ischemic brain damage, nervous degenerative disease, etc. Apoptosis has complex molecular control mechanism, and Fas ( Apo - 1 or CD95 ) is a kind of receptor at the surface of the cell that can transmit death signal. A lot of experiment both in vivo and in vitro have demonstrated that Fas can mediate apoptosis. Fas/Fas ligand is an important kind of apoptosis signal transmitting system.Some studies have shown that cigarette smoking can induce kinds of tissues apoptosis, but whether it can induce nerve cell apoptosis have not been reported. This study used terminal in situ nick end la-beling TUNEL and immunohistochemical methods to observe the smoking - induced apoptosis and Fas protein expression in rat brain, also discussed the variation pattern and possible mechanism in order to provide the theoretic foundation to smoking - induced brain damage and its therapy.Materials and methodsLaboratory animals were distributed randomly into 5 groups -chronic and large - dose smoking group, chronic and small - dose smoking group, short - term smoking group, anti - smoking group and control group. Animal model of smoking and Specimen were made, HE dying and observing with light microscope. Used terminal in situ nick end labeling TUNEL and immunohistochemical SABC methods to detect apoptosis and Fas protein expression. Used microscope counting method to count the positive cells, and used SPSS 10. 0 software to process data. Analysis of variance was used to compare the data of different groups.Results1. There were few apoptotic cells and small amount of Fas protein in control group. 2. Compared with the control group, apoptosis and Fas protein expression in chronic and short - term smoking group were significantly increased (P<0.01). 3. Apoptosis and Fas protein expression in anti - smoking group were significantly decreased compared with short - term group ( P < 0. 01) , there was no significant difference between anti - smoking group and control group (P > 0.05 ). 4. Apoptosis in chronic and large - dose group was significantlyincreased compared with the chronic and small - dose group and short- term groups (P<0.01). 5. Fas protein expression in chronic andlarge - dose group was significantly increased compared with the short- term groups ( P < 0. 01) , and there was no significant difference between it and chronic and small - dose group ( P > 0.05).DiscussionApoptosis was brought forward in 1972 by Kerr, and then became the research hotpot of each subject. Apoptosis is associated with some nervous system diseases, such as ischemic brain damage, nervous degenerative disease, etc. Diverse factors can induce apoptosis such as deficiency of growth factor, radiation, etc. It has been demonstrated that cigarette smoking and cigarette smoke exposure can induce apoptosis in different tissues.This study showed that there were few apoptotic cells in normal rat brain, and cigarette smoking could significantly elevate apoptosis in brain tissue, most of apoptotic cells were nerve cells. The number of apoptotic cell in brain tissue was increased with the increasing of smoking time and daily smoke dose. It suggested that there was time- effect and dose - effect relationship between cigarette更多还原