本文選題：慢性應(yīng)激 + 束縛�。� 參考：《南方醫(yī)科大學(xué)》2015年碩士論文

【摘要】：背景慢性應(yīng)激(chronic stress)指的是機(jī)體長(zhǎng)時(shí)間持續(xù)或間斷暴露于應(yīng)激源中,也就是日常生活工作中所存在的壓力。長(zhǎng)期應(yīng)對(duì)壓力刺激將造成人體的失代償,全身功能失調(diào),特別是中樞神經(jīng)系統(tǒng)、神經(jīng)內(nèi)分泌系統(tǒng)、免疫系統(tǒng)功能低下,由此導(dǎo)致和(或)促進(jìn)多種疾病的發(fā)生,嚴(yán)重影響人們的生活及工作質(zhì)量。目前已經(jīng)明確,慢性應(yīng)激是多種心理疾病如抑郁癥及創(chuàng)傷后應(yīng)激障礙的病因之一,同時(shí)也是心腦血管疾病發(fā)生的一個(gè)中度危險(xiǎn)因素。據(jù)報(bào)道,在美國(guó)慢性應(yīng)激發(fā)生率約為50%,其中的25%為高強(qiáng)度的應(yīng)激。而國(guó)內(nèi)城市中慢性應(yīng)激的發(fā)生率為42%-46%。近些年我們收治了大量突發(fā)性耳聾、神經(jīng)性耳鳴、腦血管病的病人,其中有很大一部分存在生活、工作上的壓力。這些患者初期往往出現(xiàn)心率、血壓、血糖、血脂等指標(biāo)的變化,后期出現(xiàn)各種器質(zhì)性疾病,更有甚者發(fā)生嚴(yán)重的心、腦等嚴(yán)重不良事件。目前國(guó)內(nèi)外慢性應(yīng)激相關(guān)的研究大部分都集中在其致病機(jī)制上,而針對(duì)治療方面的研究則相對(duì)薄弱。目前臨床主要的干預(yù)方式為藥物及心理干預(yù)上,但是它們或是副作用或花費(fèi)過大等問題難以在大眾中推廣。高壓氧治療慢性器官或臟器缺血有特殊的效果,據(jù)白永杰等的介紹高壓氧治療的作用包括：提高人體內(nèi)的氧分壓、氧氣的彌散距離、刺激側(cè)枝循環(huán)的形成、抑制炎癥反應(yīng)等。在臨床中我們發(fā)現(xiàn),對(duì)于一些存在慢性應(yīng)激的病人,經(jīng)過一段時(shí)間的高壓氧治療后,發(fā)現(xiàn)這部分病人的主觀癥狀,特別是因壓力而來的輕度抑郁、焦慮狀態(tài)有所減輕。我們?cè)O(shè)想高壓氧治療可能具有緩解慢性應(yīng)激對(duì)機(jī)體損害的能力。為此我們?cè)O(shè)計(jì)了本實(shí)驗(yàn),通過觀察高壓氧治療對(duì)慢性應(yīng)激處理大鼠各種生理指標(biāo)的影響,探索高壓氧治療干預(yù)慢性應(yīng)激的可行性。目的通過觀察慢性應(yīng)激和高壓氧處理對(duì)Wistar大鼠體重、心率變異度、行為學(xué)、血清HPA軸激素水平、一氧化氮系統(tǒng)、TNF-α以及海馬糖皮質(zhì)激素受體表達(dá)的情況,探討：(1)高壓氧對(duì)動(dòng)物慢性應(yīng)激相關(guān)指標(biāo)的影響；(2)慢性束縛是否能模擬動(dòng)物慢性應(yīng)激的生理狀態(tài)；(3)高壓氧治療能否減輕慢性束縛對(duì)機(jī)體帶來的影響。為此我們?cè)O(shè)計(jì)了本課題,旨在為高壓氧治療作為慢性應(yīng)激的干預(yù)手段提供一定的實(shí)驗(yàn)依據(jù)。材料與方法1.實(shí)驗(yàn)材料1.1實(shí)驗(yàn)動(dòng)物：選取健康雄性8月齡Wistar大鼠60只,體重200-250g,大鼠分籠飼養(yǎng),5只/籠,飼養(yǎng)室內(nèi)溫度恒定于25-28℃,濕度50～60%,12h/d光照,給予自由進(jìn)食飲水,墊料隔日更換。實(shí)驗(yàn)前進(jìn)行適應(yīng)性飼養(yǎng)1周。1.2高壓氧治療設(shè)備：?jiǎn)稳酸t(yī)用高壓純氧艙,氧氣由海軍總醫(yī)院中心氣站提供。1.3十字高架迷宮材料：自制十字高架迷宮：高35cm,2個(gè)開臂及2個(gè)閉合臂寬10cm長(zhǎng)50crm,閉合臂壁高25cm,內(nèi)側(cè)及底部涂黑。十字高架迷宮上方1米處設(shè)有照明用500W燈及攝像頭。1.4曠場(chǎng)試驗(yàn)材料：曠場(chǎng)環(huán)境長(zhǎng)100cm,寬100cm,高35cm,四周及底部涂黑,底部用白線分成20cm×20cm小格,共25個(gè)。曠場(chǎng)上方lm處設(shè)有照明用500W光源及攝像頭。1.5免疫組織熒光材料：異硫氰酸熒光素(fluorescein isothiocyanate,FITC)標(biāo)記的羊抗兔IgG二抗,大鼠GR的單克隆抗體一抗。1.6心率變異度測(cè)量及計(jì)算：多導(dǎo)生理測(cè)量?jī)x1.7 血清學(xué)檢測(cè)：促腎上腺皮質(zhì)激素釋放激素(corticotropin releasinghormone, CRH)、促腎上腺皮質(zhì)激素(adrenocor ticotropic hormore, ATCH)、糖皮質(zhì)激素(glucocorticoids, GCS)、一氧化氮合酶(nitric oxide synthase,NOS)、一氧化氮(nitric oxide, NO)及腫瘤壞死因子-α(tumor necrosis factor alpha,TNF-a),使用MULTISKAN EX酶標(biāo)儀檢測(cè)。2.分組及實(shí)驗(yàn)方法2.1高壓氧對(duì)大鼠十字高架試驗(yàn)的影響將20只雄性Wistar大鼠隨機(jī)分成2組：?jiǎn)渭兏邏貉踅M和空白對(duì)照組,每組10只。單純高壓氧組給予2.0ATA高壓氧治療1次/d；空白對(duì)照組單純飼養(yǎng),共21d。2.2慢性束縛及高壓氧處理對(duì)大鼠心率變異度及曠場(chǎng)試驗(yàn)的影響將40只雄性Wistar大鼠隨機(jī)分成4組：?jiǎn)渭兪`組、單純高壓氧組、高壓氧聯(lián)合束縛組和空白對(duì)照組,每組10只。單純束縛組給予行為限制3h/d；單純高壓氧組給予2.0ATA高壓氧治療1次/d；高壓氧聯(lián)合束縛組,每天先后給予高壓氧處理與束縛處理；空白對(duì)照組單純飼養(yǎng),共21d�！奥允`及高壓氧處理對(duì)大鼠海馬區(qū)GR表達(dá)及血清HPA軸激素水平的影響”與“慢性束縛及高壓氧處理對(duì)大鼠一氧化氮系統(tǒng)及促炎癥因子表達(dá)的影響”分組方式同2.2。3.觀察指標(biāo)3.1高壓氧對(duì)大鼠十字高架試驗(yàn)的影響以適應(yīng)環(huán)境一周后的第1、7、14、21天對(duì)其進(jìn)行十字高架迷宮(elevated plus maze)試驗(yàn),觀察其開臂(Open Arm, OA)逗留時(shí)間,統(tǒng)計(jì)采用兩因素重復(fù)測(cè)量的方差分析。3.2慢性束縛及高壓氧處理對(duì)大鼠心率變異度及曠場(chǎng)試驗(yàn)的影響以適應(yīng)環(huán)境一周后觀察大鼠在第1d、第11d、第21d曠場(chǎng)試驗(yàn)中運(yùn)動(dòng)能力(水平得分)及探索行為(垂直得分)的變化,心電的檢測(cè)第21天大鼠自主神經(jīng)的變化。3.3慢性束縛及高壓氧處理對(duì)大鼠海馬區(qū)GR表達(dá)及血清HPA軸激素水平的影響。21天處理后,利用免疫熒光染色檢測(cè)大鼠海馬區(qū)GR的表達(dá)水平。利用酶聯(lián)免疫吸附測(cè)定法測(cè)定其血清中CRH、ACTH和GCS的含量。3.4慢性束縛及高壓氧處理對(duì)大鼠一氧化氮系統(tǒng)及促炎癥因子表達(dá)的影響利用酶聯(lián)免疫吸附測(cè)定法測(cè)定血清中NOS、NO及TNF-α的含量。4.統(tǒng)計(jì)學(xué)處理利用SPSS13.0軟件進(jìn)行統(tǒng)計(jì)學(xué)分析,計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,計(jì)數(shù)資料以秩來表示,連續(xù)時(shí)間點(diǎn)上行為學(xué)的差異比較采用重復(fù)測(cè)量的方差分析。組間差異以及高壓氧和束縛兩因素相互關(guān)系的分析采用兩因素析因分析設(shè)計(jì)資料的方差分析。組間兩兩比較采用LSD-t法。計(jì)數(shù)資料采用秩和檢驗(yàn),以P0.05表示差異有統(tǒng)計(jì)學(xué)意義。結(jié)果1.十字高架迷宮顯示：分組因素不具有統(tǒng)計(jì)學(xué)意義(P=0.450),高壓氧處理與空白對(duì)照組在焦慮程度上差異無統(tǒng)計(jì)學(xué)意義。2.曠場(chǎng)試驗(yàn)顯示：與空白對(duì)照組比較,單純束縛組在第11d水平得分(115.5±43.4)和垂直得分(23.9±10.1)均明顯升高(P0.001)；與單純束縛組比較,高壓氧聯(lián)合束縛組在第1d到第11d的得分升幅減小(P0.01)。3.心率變異度顯示：4組間LF/HF、交感及副交感活性差異無統(tǒng)計(jì)學(xué)意義。但仍能看出以下趨勢(shì)：束縛及高壓氧治療均可導(dǎo)致交感神經(jīng)張力下降,而高壓氧治療可引起副交感神經(jīng)張力上升。4.免疫組織熒光染色顯示：?jiǎn)渭兪`組海馬區(qū)GR表達(dá)(0.1546±0.0154)比空白對(duì)照組(0.2174±0.0123)減少(P0.001),高壓氧聯(lián)合束縛組與空白對(duì)照組比較無明顯差異(P0.05)。5.HPA軸激素顯示：4組血清CRH、ATCH及GCS差異無統(tǒng)計(jì)學(xué)意義。但可以得出以下趨勢(shì)：?jiǎn)渭兏邏貉踅M,單純束縛組,高壓氧聯(lián)合束縛組ACTH及GCS均較空白對(duì)照組有所升高,高壓氧聯(lián)合束縛組CRH水平較另三組下降。6.4組大鼠血清總NO、NOS及TNF-α差異無統(tǒng)計(jì)學(xué)意義。但仍能得出以下趨勢(shì)：?jiǎn)渭兪`組NOS水平較各組下降,束縛聯(lián)合高壓氧處理后發(fā)現(xiàn)血清TNF-α較單純束縛組下降。結(jié)論1.高壓氧處理后a.大鼠焦慮程度無明顯變化：b.大鼠出現(xiàn)輕度的交感神經(jīng)張力下降及副交感興奮；c.海馬區(qū)GR表達(dá)無明顯變化；d.大鼠血清HPA軸激素水平有所升高,而NO、NOS及TNF-α無明顯變化。提示高壓氧處理具有一般應(yīng)激的特征,但應(yīng)激強(qiáng)度不大。2.束縛處理后a.大鼠行為學(xué)試驗(yàn)表現(xiàn)出明顯的焦慮狀態(tài)；b.大鼠出現(xiàn)交感神經(jīng)張力下降；c.海馬區(qū)GR表達(dá)下降；d.大鼠血清GCS水平升高,但CRH及ACTH無明顯變化；e.大鼠NOS水平下降。提示慢性束縛在一定范圍內(nèi)模擬慢性應(yīng)激的生理狀態(tài)。3.高壓氧聯(lián)合束縛處理后a.大鼠的焦慮狀態(tài)較單純束縛有所減輕；b.大鼠出現(xiàn)交感神經(jīng)張力下降,但下降程度要小于單純束縛組；c.海馬區(qū)GR表達(dá)未見明顯下降；d.大鼠血清ACTH及GCS均較空白對(duì)照組有所升高,但高壓氧聯(lián)合束縛組CRH水平較另三組有所下降。e.束縛聯(lián)合高壓氧處理后發(fā)現(xiàn)血清TNF-α較單純束縛組下降。提示高壓氧能減緩束縛對(duì)大鼠相關(guān)生理指標(biāo)的影響。
[Abstract]:Background chronic stress (chronic stress) refers to the prolonged or intermittent exposure of the body to the stressor, which is the stress in the daily life. Long-term response to stress stimulates the body's decompensation and systemic dysfunction, especially the central nervous system, the neuroendocrine system, and the immune system, which leads to the low function of the immune system. It is now clear that chronic stress is one of the causes of many psychological diseases such as depression and post-traumatic stress disorder, and is also a moderate risk factor for the occurrence of cardiovascular and cerebrovascular diseases. It is reported that the incidence of chronic stress is about 50% in the United States. 25% of them were high stress. And the incidence of chronic stress in the domestic cities was 42%-46%. in recent years. We treated a large number of patients with sudden deafness, neurogenic tinnitus, and cerebrovascular disease, of which a large portion of these patients had life and stress. These patients were at the initial stage of heart rate, blood pressure, blood sugar, blood lipid and other indicators. There are various organic diseases in the later period and serious adverse events such as heart and brain. Most of the research on chronic stress at home and abroad is concentrated on its pathogenesis, but the research on treatment is relatively weak. The main clinical intervention methods are drug and psychological intervention, but they are the main methods of intervention. The effects of hyperbaric oxygen therapy on chronic organ or organ ischemia have special effects. The effect of hyperbaric oxygen therapy, such as Bai Yongjie and so on, includes improving the oxygen partial pressure in the body, the diffusion distance of oxygen, stimulating the formation of collateral branches and inhibiting the inflammatory reaction, and so on. We found that for some patients with chronic stress, after a period of hyperbaric oxygen therapy, we found that the subjective symptoms of the patients, especially the mild depression caused by pressure, were relieved. We conceive that hyperbaric oxygen therapy may have the ability to alleviate the damage to the body by chronic stress. The experiment was to observe the effect of hyperbaric oxygen therapy on the physiological indexes of rats treated with chronic stress, and to explore the feasibility of hyperbaric oxygen therapy to intervene chronic stress. Objective To observe the weight, heart rate variability, behavior, serum HPA axis hormone level, nitric oxide system, TNF- A and hippocampus of Wistar rats. The expression of glucocorticoid receptor: (1) the effect of hyperbaric oxygen on the related indicators of chronic stress in animals; (2) whether chronic restraint can simulate the physiological state of chronic stress in animals; (3) whether hyperbaric oxygen therapy can reduce the effect of chronic restraint on the body. The intervention means of chronic stress provide certain experimental basis. Material and methods 1. experimental materials 1.1 experimental animals: 60 healthy male 8 month old Wistar rats, weight 200-250g, cage rearing, 5 cage, feeding room temperature constant at 25-28, humidity 50 to 60%, 12h/ D light, free feeding drinking water, mat material daily replacement. .1.2 hyperbaric oxygen treatment equipment for 1 weeks before examination: single medical high pressure pure oxygen tank, oxygen by Navy General Hospital central gas station.1.3 cross elevated maze material: self-made cross elevated maze: high 35cm, 2 open arms and 2 closed arm width 10cm long 50crm, close arm wall high 25cm, inside and bottom black. Cross viaduct maze 1 meters above the top 1 meters with lighting and camera.1.4 open field test materials: open field environment long 100cm, wide 100cm, high 35cm, black around and bottom, the bottom is divided into 20cm x 20cm small lattice, a total of 25. Above the open field, there is a 500W light source and a camera.1.5 immunofluorescence material above the open field: fluorescein isothiocyanate (fluorescein I) Sothiocyanate, FITC) labeled Sheep anti rabbit IgG two resistance, rat GR monoclonal antibody one anti.1.6 heart rate variability measurement and calculation: multi lead physiological measuring instrument 1.7 serological detection: adrenocorticotropin releasing hormone (corticotropin releasinghormone, CRH), suprarenal cortical hormone (adrenocor ticotropic hormore, ATCH), sugar Corticosteroids (glucocorticoids, GCS), nitric oxide synthase (nitric oxide synthase, NOS), nitric oxide (nitric oxide, NO) and tumor necrosis factor - alpha (tumor necrosis factor). The effect of 2.1 hyperbaric oxygen on the cross viaduct test in rats was detected by the enzyme labeling method and the effect of hyperbaric oxygen on the cross Viaduct of rats. Rats were randomly divided into 2 groups: simple hyperbaric oxygen group and blank control group, 10 rats in each group. Pure hyperbaric oxygen group was given 2.0ATA hyperbaric oxygen therapy 1 times /d; blank control group was fed alone. The effect of chronic constriction of 21d.2.2 and hyperbaric oxygen treatment on heart rate variability and open field test in rats was divided into 4 groups of 40 male Wistar rats randomly: simple binding Group, simple hyperbaric oxygen group, hyperbaric oxygen binding group and blank control group, each group was 10. The simple binding group was given the behavior restriction 3h/d; the simple hyperbaric oxygen group was given the 2.0ATA hyperbaric oxygen therapy 1 times /d; the hyperbaric oxygen combined binding group was given hyperbaric oxygen treatment and binding treatment every day; the blank control group was simply fed, a total of 21d. "chronic shackles. Effect of hyperbaric oxygen treatment on the expression of GR and serum HPA axis hormone levels in the hippocampus of rats "and the effect of" chronic binding and hyperbaric oxygen treatment on the expression of nitric oxide and proinflammatory factors in rats "with 2.2.3. observation index 3.1 the effect of hyperbaric oxygen on the cross elevated test in rats to adapt to the environment one week after the 1,7,14 On the 21 day, the elevated plus maze test was carried out to observe the duration of the opening arm (Open Arm, OA), and the statistical analysis of the variance of the two factor repeated measurements was used to analyze the effect of.3.2 chronic binding and hyperbaric oxygen treatment on the heart rate variability and open field test in rats to observe the rats in the 1D, 11d, 21d open field after a week. Changes of exercise ability (level score) and exploratory behavior (vertical score) in the test, changes of electrocardiogram (vertical score), changes of autonomic nerve in twenty-first days of rats,.3.3 chronic binding and the effect of hyperbaric oxygen treatment on GR expression and serum HPA axis hormone levels in the hippocampus of rats.21 days after.21 day treatment, the expression level of GR in the hippocampus of rats was detected by immunofluorescence staining. Determination of CRH, ACTH and GCS in serum by enzyme linked immunosorbent assay the effect of.3.4 chronic binding and hyperbaric oxygen treatment on the expression of nitric oxide and proinflammatory cytokines in rats by using enzyme linked immunosorbent assay to determine the content of NOS, NO and TNF- alpha in serum.4. statistical treatment using SPSS13.0 software for statistical analysis The measurement data were expressed in the mean number of standard deviation (x + s), the counting data were expressed as rank, and the difference of behavior in the continuous time point was compared with the variance analysis of repeated measurements. The analysis of the interrelationship between the groups and the two factors of the hyperbaric oxygen and the binding were analyzed by the analysis of the variance of the two factors analysis of the factorial analysis. 22 of the groups were compared with LSD- T method. The count data were measured by the rank sum test, and the difference was statistically significant by P0.05. Results 1. the cross viaduct labyrinth showed that the group factors were not statistically significant (P=0.450). There was no significant difference between the hyperbaric oxygen treatment and the blank control group in the anxiety level. The.2. open field test showed that the simple binding group was compared with the blank control group. The score of 11d level (115.5 + 43.4) and vertical score (23.9 + 10.1) were significantly higher (P0.001). Compared with the simple binding group, the score of the HBO group from 1D to 11d decreased (P0.01).3. heart rate variability, and there was no statistical difference between the 4 groups of LF/HF, and there was no significant difference between the sympathetic and the parasympathetic activity. However, the following trend was still seen. The binding and hyperbaric oxygen therapy could lead to the decrease of sympathetic nerve tension, and hyperbaric oxygen therapy could cause the increase of parasympathetic nervous tension in.4. immunofluorescence staining. The expression of GR in the hippocampal area in the simple binding group was less than that of the blank control group (0.2174 + 0.0123) (P0.001), and the combination of hyperbaric oxygen binding group and blank control group was no more than that of the blank control group (P0.001). The significant difference (P0.05).5.HPA axis hormone showed that there was no statistical difference between the 4 groups of serum CRH, ATCH and GCS, but the following trend was found: simple hyperbaric oxygen group, simple binding group, hyperbaric oxygen binding group ACTH and GCS were higher than that of the blank control group, and the level of CRH in hyperbaric oxygen binding group was lower than that of the other three groups. There was no significant difference in O, NOS and TNF- alpha. However, the following trend was still found: the level of NOS in the simple binding group was lower than that of each group. The serum TNF- alpha was lower than that of the simple binding group after the binding combined hyperbaric oxygen treatment. Conclusion there was no significant change in the anxiety level of A. rats after hyperbaric oxygen treatment (1.): there was a slight decrease of sympathetic nervous tension and a pair of pairs in B. rats. There was no obvious change in the expression of GR in the hippocampal region of C.; the level of serum HPA axis hormone in D. rats increased, but NO, NOS and TNF- alpha had no obvious changes. It suggested that hyperbaric oxygen treatment had general stress characteristics, but the stress intensity was not significant in the behavior test of a. rats after the restraint of.2.; B. rats appeared sympathetic God. The tension decreased, the expression of GR in the C. hippocampus decreased, the level of GCS in the serum of D. rats increased, but there was no obvious change in the CRH and ACTH, and the level of NOS in E. rats decreased. It suggested that the chronic restraint in a certain range simulated the physiological state of chronic stress and the anxiety state of A. rats was less than that of the pure binding after the combined binding treatment of the.3. hyperbaric oxygen (HHP); B. rats The level of sympathetic nervous tension decreased, but the degree of decrease was less than that of the simple binding group; the expression of GR in the C. hippocampus was not significantly decreased; the serum ACTH and GCS in D. rats were higher than that in the blank control group, but the CRH level of the combined binding group of the hyperbaric oxygen group was lower than the other three groups and the serum TNF- a was found to be more than the simple binding group after.E. binding hyperbaric oxygen treatment. This indicates that hyperbaric oxygen can slow down the influence of restraint on Physiological Indexes in rats.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R459.6;R749

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