本文關(guān)鍵詞： 七氟醚 IL-18 NO 心肌凋亡　出處：《河南科技大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：背景:七氟醚是臨床麻醉中常用的吸入麻醉藥物,誘導(dǎo)快、可控性高是其得以廣泛應(yīng)用的前提,目前已被廣泛應(yīng)用于各種外科手術(shù)麻醉。因此關(guān)于七氟醚對各臟器功能的影響亦受到關(guān)注。圍術(shù)期應(yīng)激帶來的創(chuàng)傷對患者心肌損傷的研究一直是麻醉醫(yī)生關(guān)心的話題,通過低溫、控制性降壓等方法已經(jīng)取得良好效果,麻醉藥物本身是否還存在非麻醉效益值得探討研究。細胞凋亡是生物機體器官代謝啟動新陳代謝變化的細胞更新方式,與外界干預(yù)原因的死亡方式有著明顯的區(qū)別,是一種生物生理變化的過程,是細胞組織器官保持旺盛活力,但不是每個細胞的凋亡都是生理過程。正常生理狀態(tài)下細胞增殖以及細胞凋亡之間保持一個動態(tài)變化,對維持機體內(nèi)部一個相對動態(tài)穩(wěn)定有著重要的作用。以前有學(xué)者提出心肌細胞以及中樞神經(jīng)元細胞不會發(fā)生凋亡,但在上世紀九十年代初期,有學(xué)者已經(jīng)找到這些所謂機體形成過程中分化末期的細胞也可以有細胞的凋亡過程,心肌凋亡從一個嚴重心律失常重度心肌病患者心肌中找到證據(jù),證明心肌在一些特定情況下,包括藥物、環(huán)境等,可以出現(xiàn)心肌凋亡,從而找到答案。從越來越多的臨床以及基礎(chǔ)研究的專家研究的結(jié)果來看,心肌細胞肯定參與疾病的發(fā)生發(fā)展的過程,也在多種疾病的發(fā)生以及轉(zhuǎn)歸的過程扮演不同的角色,可能是多種疾病的變化過程的必要的細胞基礎(chǔ)之一;圍術(shù)期患者大多合并內(nèi)科疾病,心功能的關(guān)注和評估一直是重中之重,從心理應(yīng)激到創(chuàng)傷應(yīng)激,患者往往會反映在心血管系統(tǒng),以往的關(guān)于心肌凋亡損傷的研究相關(guān)機制、相關(guān)因子、基因均較多。因而,隨著國家經(jīng)濟發(fā)展,心血管病患病率也在逐步上升,開發(fā)減少凋亡的基因生物制品和抗凋亡的新藥,將會對心臟病的早期預(yù)防以及多種原因?qū)е碌男募∪毖俟嘧⒓靶难荑祿p傷有著重要的作用,具有重要的臨床意義。心肌細胞的細胞凋亡的微觀基礎(chǔ)研究,阻止或延緩心肌細胞凋亡很有價值,麻醉醫(yī)生找到專業(yè)切入點非常重要。目的:本研究在于建立慢性長期低濃度吸入七氟醚大鼠模型,本實驗采用三組不同濃度七氟醚建立大鼠模型,其濃度均高于實際工作中手術(shù)室暴露的七氟醚濃度,檢測大鼠各個時期段心肌細胞凋亡情況,檢測各個時間段大鼠心肌中IL-18、NO的表達,探討IL-18、NO在心肌凋亡過程中的作用及意義。方法:選2-4周齡健康雄性SD大鼠,體重80-120g。隨機分為四組,每組24只,空氣吸入組(對照組)、30ppm七氟醚組、100ppm七氟醚組、300ppm七氟醚組。七氟醚組每天吸入對應(yīng)濃度七氟醚6小時,連續(xù)吸入2周、4周、8周、12周,對照組吸入等量空氣,參考Richard I.Mazze等的方法在實驗開始前應(yīng)用公式濃度=質(zhì)量÷分子量×22.4×絕對溫度÷吸入染毒箱容積(其中濃度,質(zhì)量和容積的單位分別為ppm,mg,m3)向?qū)嶒炄径鞠鋬?nèi)注入計算所得的七氟醚的負荷量,以使箱內(nèi)七氟醚濃度迅速達到實驗所需濃度,并應(yīng)用氣相色譜檢測箱內(nèi)氣體濃度的穩(wěn)定。對照組同等條件下僅給予醫(yī)用壓縮空氣。4組動物每天連續(xù)吸入相應(yīng)氣體6小時,持續(xù)2周、4周、8周以及12周。分別檢測不同時期心電圖變化,并留取心臟標本。he染色確定心肌細胞數(shù)量的變化;免疫組化檢測心臟組織il-18、no蛋白表達;應(yīng)用反轉(zhuǎn)錄酶聯(lián)集合反應(yīng)以及免疫印跡綜合分析在模型大鼠心肌中il-18、no基因及蛋白的表達。結(jié)果:1.細胞形態(tài)學(xué)觀察,在高倍光鏡下,心肌細胞清晰可見,各組細胞表達有明顯不同,空氣吸入組四個時間點均未出現(xiàn)凋亡情況,30ppm七氟醚組中,心肌細胞形態(tài)學(xué)無明顯變化,四個時間點(2周、4周、8周、12周)均未出現(xiàn)明顯細胞凋亡情況;100ppm七氟醚組中,2周、4周、8周未出現(xiàn)細胞凋亡情況,第12周心肌細胞出現(xiàn)凋亡,統(tǒng)計學(xué)分析,有統(tǒng)計學(xué)意義,p=0.038;300ppm七氟醚組,大鼠2周、4周時未出現(xiàn)明顯細胞凋亡現(xiàn)象,8周以及12周時細胞出現(xiàn)凋亡,組間以及組內(nèi)比較均有統(tǒng)計學(xué)意義,p0.01。2.在大鼠心電圖對比中,100ppm濃度下12周大鼠心電圖發(fā)現(xiàn)不同程度異常,而在300ppm濃度下,大鼠8周后也出現(xiàn)心電圖異常情況,且部分心電圖為心肌缺血表現(xiàn),而在對于30ppm濃度暴露環(huán)境下,大鼠心電圖也未出現(xiàn)異常。3.在對心肌細胞進行免疫組化檢測il-18、no在兩種組織中表達有明顯差異,30ppm濃度下早期,大鼠心肌組織的分子生物學(xué)檢測,il-18略低于正常對照組,而no濃度略高于正常組,有統(tǒng)計學(xué)意義,p0.01,4周后無特殊改變,100ppm組中,12周實驗時,il-18明顯高于其他三個時間點,組間比較有統(tǒng)計學(xué)意義,no明顯低于其他三個時間段,組間比較均有統(tǒng)計學(xué)意義;在300ppm組中,il-18陽性細胞表達率8周、12周明顯高于4周、2周兩個時間點,組間及組內(nèi)比較均有統(tǒng)計學(xué)意義,p0.05,no表達在第8周及第12周,表達明顯下降,組內(nèi)及組間比較均有統(tǒng)計學(xué)意義,p0.05;對照組即空氣吸入組il-18、no表達無差異。4.在對心肌組織進行rt-pcr以及westernblot檢測結(jié)果又不同程度變化,結(jié)果與免疫組化、he染色結(jié)果基本趨向一致。結(jié)論:長期低濃度吸入七氟醚大鼠心肌細胞出現(xiàn)凋亡現(xiàn)象,隨著吸入濃度增大,時間的延長,表現(xiàn)越明顯;短時間,小劑量七氟醚對大鼠心臟有可能起到保護作用;長期低濃度吸入七氟醚大鼠心肌組織中il-18、no表達有明顯差異,是大鼠心肌凋亡相關(guān)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.

【學(xué)位授予單位】：河南科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R614

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