长期低浓度吸入七氟醚大鼠心肌中IL-18、NO表达以及与心肌凋亡的关系

发布时间：2018-01-24 02:13

本文关键词： 七氟醚 IL-18 NO 心肌凋亡　出处：《河南科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：背景:七氟醚是临床麻醉中常用的吸入麻醉药物,诱导快、可控性高是其得以广泛应用的前提,目前已被广泛应用于各种外科手术麻醉。因此关于七氟醚对各脏器功能的影响亦受到关注。围术期应激带来的创伤对患者心肌损伤的研究一直是麻醉医生关心的话题,通过低温、控制性降压等方法已经取得良好效果,麻醉药物本身是否还存在非麻醉效益值得探讨研究。细胞凋亡是生物机体器官代谢启动新陈代谢变化的细胞更新方式,与外界干预原因的死亡方式有着明显的区别,是一种生物生理变化的过程,是细胞组织器官保持旺盛活力,但不是每个细胞的凋亡都是生理过程。正常生理状态下细胞增殖以及细胞凋亡之间保持一个动态变化,对维持机体内部一个相对动态稳定有着重要的作用。以前有学者提出心肌细胞以及中枢神经元细胞不会发生凋亡,但在上世纪九十年代初期,有学者已经找到这些所谓机体形成过程中分化末期的细胞也可以有细胞的凋亡过程,心肌凋亡从一个严重心律失常重度心肌病患者心肌中找到证据,证明心肌在一些特定情况下,包括药物、环境等,可以出现心肌凋亡,从而找到答案。从越来越多的临床以及基础研究的专家研究的结果来看,心肌细胞肯定参与疾病的发生发展的过程,也在多种疾病的发生以及转归的过程扮演不同的角色,可能是多种疾病的变化过程的必要的细胞基础之一;围术期患者大多合并内科疾病,心功能的关注和评估一直是重中之重,从心理应激到创伤应激,患者往往会反映在心血管系统,以往的关于心肌凋亡损伤的研究相关机制、相关因子、基因均较多。因而,随着国家经济发展,心血管病患病率也在逐步上升,开发减少凋亡的基因生物制品和抗凋亡的新药,将会对心脏病的早期预防以及多种原因导致的心肌缺血再灌注及心血管璧损伤有着重要的作用,具有重要的临床意义。心肌细胞的细胞凋亡的微观基础研究,阻止或延缓心肌细胞凋亡很有价值,麻醉医生找到专业切入点非常重要。目的:本研究在于建立慢性长期低浓度吸入七氟醚大鼠模型,本实验采用三组不同浓度七氟醚建立大鼠模型,其浓度均高于实际工作中手术室暴露的七氟醚浓度,检测大鼠各个时期段心肌细胞凋亡情况,检测各个时间段大鼠心肌中IL-18、NO的表达,探讨IL-18、NO在心肌凋亡过程中的作用及意义。方法:选2-4周龄健康雄性SD大鼠,体重80-120g。随机分为四组,每组24只,空气吸入组(对照组)、30ppm七氟醚组、100ppm七氟醚组、300ppm七氟醚组。七氟醚组每天吸入对应浓度七氟醚6小时,连续吸入2周、4周、8周、12周,对照组吸入等量空气,参考Richard I.Mazze等的方法在实验开始前应用公式浓度=质量÷分子量×22.4×绝对温度÷吸入染毒箱容积(其中浓度,质量和容积的单位分别为ppm,mg,m3)向实验染毒箱内注入计算所得的七氟醚的负荷量,以使箱内七氟醚浓度迅速达到实验所需浓度,并应用气相色谱检测箱内气体浓度的稳定。对照组同等条件下仅给予医用压缩空气。4组动物每天连续吸入相应气体6小时,持续2周、4周、8周以及12周。分别检测不同时期心电图变化,并留取心脏标本。he染色确定心肌细胞数量的变化;免疫组化检测心脏组织il-18、no蛋白表达;应用反转录酶联集合反应以及免疫印迹综合分析在模型大鼠心肌中il-18、no基因及蛋白的表达。结果:1.细胞形态学观察,在高倍光镜下,心肌细胞清晰可见,各组细胞表达有明显不同,空气吸入组四个时间点均未出现凋亡情况,30ppm七氟醚组中,心肌细胞形态学无明显变化,四个时间点(2周、4周、8周、12周)均未出现明显细胞凋亡情况;100ppm七氟醚组中,2周、4周、8周未出现细胞凋亡情况,第12周心肌细胞出现凋亡,统计学分析,有统计学意义,p=0.038;300ppm七氟醚组,大鼠2周、4周时未出现明显细胞凋亡现象,8周以及12周时细胞出现凋亡,组间以及组内比较均有统计学意义,p0.01。2.在大鼠心电图对比中,100ppm浓度下12周大鼠心电图发现不同程度异常,而在300ppm浓度下,大鼠8周后也出现心电图异常情况,且部分心电图为心肌缺血表现,而在对于30ppm浓度暴露环境下,大鼠心电图也未出现异常。3.在对心肌细胞进行免疫组化检测il-18、no在两种组织中表达有明显差异,30ppm浓度下早期,大鼠心肌组织的分子生物学检测,il-18略低于正常对照组,而no浓度略高于正常组,有统计学意义,p0.01,4周后无特殊改变,100ppm组中,12周实验时,il-18明显高于其他三个时间点,组间比较有统计学意义,no明显低于其他三个时间段,组间比较均有统计学意义;在300ppm组中,il-18阳性细胞表达率8周、12周明显高于4周、2周两个时间点,组间及组内比较均有统计学意义,p0.05,no表达在第8周及第12周,表达明显下降,组内及组间比较均有统计学意义,p0.05;对照组即空气吸入组il-18、no表达无差异。4.在对心肌组织进行rt-pcr以及westernblot检测结果又不同程度变化,结果与免疫组化、he染色结果基本趋向一致。结论:长期低浓度吸入七氟醚大鼠心肌细胞出现凋亡现象,随着吸入浓度增大,时间的延长,表现越明显;短时间,小剂量七氟醚对大鼠心脏有可能起到保护作用;长期低浓度吸入七氟醚大鼠心肌组织中il-18、no表达有明显差异,是大鼠心肌凋亡相关重要因子之一。
[Abstract]:Background: sevoflurane is commonly used in clinical anesthesia inhalation anesthetics, induced by fast, high controllability is the premise of the wide application, it has been widely used in various surgical anesthesia. So the effect of sevoflurane on the organ is also concerned. The perioperative period should bowel trauma to patients with myocardial damage caused has been a topic of concern by the anesthesiologist, low temperature, has achieved good results of controlled hypotension and other methods, the anesthetic itself whether there are benefits worthy of study. Non narcotic apoptosis is way to update the organism metabolism change and start The new supersedes the old. cells, cause of death outside intervention has obvious difference, is a physiological change process, is the organ cell maintain strong vitality, but not every cell apoptosis is a physiological process. The normal physiological state Maintain a dynamic changes of cell apoptosis and cell proliferation, the organism to maintain a relatively stable internal dynamics plays an important role. Some scholars put forward the previous myocardial cells and central neuronal cell apoptosis but not, in the early 90s, the process of apoptosis forming process scholars have found these so-called body terminally differentiated cells can also have cells, myocardial apoptosis from a serious arrhythmia in patients with severe myocardial cardiomyopathy found evidence that myocardial in some specific cases, including medicine, environment and so on, can appear myocardial apoptosis, so as to find the answer. More and more results from clinical and basic research on expert's point of view, myocardial cells must participate in the development of the disease process, but also play different roles in the process of many diseases and the outcome may be more. One of the cellular basis of necessary change process of disease; perioperative period of most patients with internal disease, attention and assessment of heart function is always a priority among priorities, stress from mental stress to patients with trauma, often reflected in the cardiovascular system and related factors on myocardial apoptosis damage mechanism research, the past. Genes are more. Therefore, with the development of the national economy, the prevalence rate of cardiovascular disease is gradually rising, the development of reducing gene biological products apoptosis and anti apoptosis drugs, will be on the early prevention of heart disease and a variety of reasons caused by myocardial ischemia reperfusion injury and cardiovascular wall plays an important role, has important clinical significance the study on the micro foundation. Apoptosis of myocardial cells, prevent or delay the apoptosis of myocardial cells is very valuable, anesthesiologists find professional entry point is very important. Objective: the purpose of this study is that The establishment of chronic inhalation of low concentrations of sevoflurane in model rats, three groups of different concentrations of sevoflurane to establish a rat model of this experiment, the concentrations were higher in the operation room exposure to sevoflurane concentration in the practical work, to detect cell apoptosis in rats Duan Xinji each time, each time the detection in myocardium of rats with IL-18, the expression of NO on IL-18. The significance of NO in myocardial apoptosis. Methods: 2-4 week old healthy male SD rats weighing 80-120g. were randomly divided into four groups, 24 rats in each group, air inhalation group (control group), 30ppm sevoflurane group, 100ppm sevoflurane group, 300ppm sevoflurane group. Sevoflurane group daily inhaled sevoflurane corresponding to 6 hours, continuous inhalation for 2 weeks, 4 weeks, 8 weeks, 12 weeks, the control group inhaled air equivalent method, reference Richard I.Mazze etc. before the start of the experiment using formula concentration = quality / molecular weight * 22.4 * absolute temperature, inhalation Tank capacity (including concentration, quality and volume of the unit are respectively ppm, Mg, m3) load into sevoflurane calculated to experimental exposure box, in order to achieve the experimental concentration in rapid sevoflurane concentration, and the application of gas chromatography gas concentration detection box is stable. The control group was given the same conditions medical compressed air.4 group animal day continuous inhalation of corresponding gas for 6 hours, 2 weeks, 4 weeks, 8 weeks and 12 weeks respectively. Detection of ECG changes in different periods, and take the heart specimens.He staining to determine the changes of myocardial cell number; immunohistochemical detection of heart tissue IL-18, no protein expression by reverse transcription; ELISA and Western blot analysis of assembly reaction of IL-18 in myocardial model rats, the expression of no mRNA and protein. Results: 1. observation of cell morphology, in high power microscope, myocardial cells were clearly visible, the expression of Ming Dynasty Significantly different, air inhaled apoptosis were not seen in four time points, 30ppm sevoflurane group, the myocardial cell morphology had no obvious change, four time points (2 weeks, 4 weeks, 8 weeks, 12 weeks) showed no obvious cell apoptosis; 100ppm sevoflurane group, 2 weeks, 4 weeks. 8 weeks of cell apoptosis, myocardial cell apoptosis occurred in twelfth weeks, statistical analysis, statistical significance, p=0.038; 300ppm sevoflurane group, rats for 2 weeks, there was no obvious cell apoptosis at 4 weeks, 8 weeks and 12 weeks of apoptosis between groups and within group comparison was statistically significant, p0.01.2. in the electrocardiogram of rats in comparison, the concentration of 100ppm for 12 weeks in rats of different abnormal ECG findings, and at the concentration of 300ppm, rats after 8 weeks also appeared abnormal electrocardiogram, electrocardiogram and myocardial ischemia as part of performance, and in the 30ppm exposure environment, electrocardiogram of rats No abnormal changes in.3. on myocardial cells by immunohistochemical detection of IL-18, no were differentially expressed in two tissues, 30ppm concentration of early molecular biology in myocardial tissue of rats, IL-18 was slightly lower than the normal control group, while the NO concentration is higher than that of normal group, there is statistical significance, no special change after p0.01,4 weeks, the 100ppm group, the experiment of 12 weeks, IL-18 was significantly higher than that of the other three time points, the difference was statistically significant, no was significantly lower than that of the other three time periods, comparison between groups were statistically significant; in group 300ppm, IL-18 positive expression rate of 8 weeks, 12 weeks was significantly higher than that of 4 week 2, week two time points between groups and within groups were statistically significant, P0.05, no expression in eighth weeks and 12 weeks, the expression decreased obviously, the intra and inter group comparisons were statistically significant, P0.05; control group air inhalation group IL-18, no.4. expression was no significant difference in the heart Muscle RT-PCR and Westernblot test results and different degrees of change, and the results of immunohistochemistry, he staining results tend to be consistent. Conclusion: the long-term inhalation of low concentrations of sevoflurane in rats of myocardial cell apoptosis phenomenon, with the increase of the concentration of inhalation, the extension of time, the more obvious the phenomenon; short time, small dose of sevoflurane may the rat heart has protective effect; long-term inhalation of low concentrations of IL-18 in myocardial tissue of sevoflurane rats, no expression was significantly different, is one of the important factors related to myocardial apoptosis in rats.

【学位授予单位】：河南科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R614

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