發(fā)布時(shí)間：2018-07-31 10:55

【摘要】： 背景與目的：參加1990～1991年海灣戰(zhàn)爭(zhēng)的近70萬美國(guó)軍人中,至少四分之一在戰(zhàn)后出現(xiàn)了持續(xù)的、難以處理的健康問題,包括認(rèn)知困難、持續(xù)廣泛的疼痛、疲勞、頭痛、慢性腹瀉、皮疹等,被稱為海灣戰(zhàn)爭(zhēng)綜合征(Gulf War Syndrome,GWS)或海灣戰(zhàn)爭(zhēng)疾病。GWS的病因可能包括貧鈾、油井火焰煙霧、神經(jīng)毒氣、溴化吡啶斯的明(PB)及殺蟲劑(DEET)、疫苗接種、心理應(yīng)激、感染性疾病等多種因素。海灣戰(zhàn)爭(zhēng)流行病學(xué)研究一致性發(fā)現(xiàn),溴化吡啶斯的明及殺蟲劑是與GWS明顯有關(guān)的唯一兩種接觸,較長(zhǎng)時(shí)間服用PB或多量使用殺蟲劑者,GWS發(fā)生率增高。有關(guān)研究表明,GWS患者大腦海馬、扣帶回等腦區(qū)體積縮小。動(dòng)物實(shí)驗(yàn)也提示海馬神經(jīng)細(xì)胞死亡。米諾環(huán)素(鹽酸二甲胺四環(huán)素,minoeyeline hydroehloride)為第二代人工半合成四環(huán)類抗生素。研究表明,米諾環(huán)素是一種Caspase-3抑制劑,具有神經(jīng)保護(hù)作用。本實(shí)驗(yàn)?zāi)康?聯(lián)合應(yīng)用PB及DEET建立海灣戰(zhàn)爭(zhēng)大鼠模型,觀察海馬CA1區(qū)神經(jīng)細(xì)胞凋亡、凋亡蛋白酶激活因子-1(Apaf-1)及Caspase-3的表達(dá),并探討米諾環(huán)素的神經(jīng)保護(hù)機(jī)制。 方法：采用Abdel-Rahman法建立海灣戰(zhàn)爭(zhēng)綜合征大鼠模型。實(shí)驗(yàn)設(shè)立對(duì)照組、束縛組、PB/DEET組、模型組、低劑量米諾環(huán)素組、高劑量米諾環(huán)素組。束縛組大鼠給與固定束縛,置于暗環(huán)境中,每次20分鐘。模型組：參照Abdel-Rahman文獻(xiàn)建立海灣戰(zhàn)爭(zhēng)大鼠模型,大鼠每天給與溴化吡啶斯的明(1.3mg/kg/d)口服,DEET (40mg/kg/d)以70%乙醇皮下注射,暗環(huán)境固定束縛20分鐘。PB/DEET組：每天給予PB及DEET皮下注射。低劑量米諾環(huán)素組：米諾環(huán)素15mg/kg/d,腹腔注射,余同模型組。高劑量米諾環(huán)素組：米諾環(huán)素45mg/kg/d,腹腔注射,余同模型組。對(duì)照組大鼠自由取水進(jìn)食,給與等量的酒精溶液皮下注射。實(shí)驗(yàn)周期為28天。在0天、14天及28天測(cè)定大鼠體重。實(shí)驗(yàn)結(jié)束后,行多聚甲醛灌流固定、取腦、石蠟包埋、切片、采用免疫組化檢測(cè)大鼠海馬CA1區(qū)神經(jīng)細(xì)胞Apaf-1及Caspase-3的表達(dá),采用TUNEL法檢測(cè)海馬CA1區(qū)神經(jīng)細(xì)胞的凋亡情況。 結(jié)果：各組大鼠體重均有不同程度的增加,4周后對(duì)照組大鼠體重增加了79%,束縛組為75%,PB/DEET組為49%,模型組為47%,低高劑量米諾環(huán)素組為70%,高劑量米諾環(huán)素組為71%。PB/DEET組與對(duì)照組、模型組與束縛組相比,體重增幅減少,P0.01,有統(tǒng)計(jì)學(xué)差異；束縛組與對(duì)照組相比,模型組與PB/DEET組比較,體重增加相似,P0.05,無統(tǒng)計(jì)學(xué)差異；低、高劑量米諾環(huán)素組與模型組相比較,體重增加明顯,P0.01,有統(tǒng)計(jì)學(xué)差異。 束縛組與對(duì)照組相比較以及PB/DEET組與模型組相比較,海馬CA1區(qū)神經(jīng)細(xì)胞凋亡數(shù)量、Caspase-3及Apaf-1積分光密度無統(tǒng)計(jì)學(xué)意義(P0.05)。模型組與對(duì)照組相比,海馬CA1區(qū)凋亡細(xì)胞數(shù)增多,Caspase-3及Apaf-1積分光密度明顯增高,統(tǒng)計(jì)學(xué)差異非常顯著(P0.01)。低、高劑量米諾環(huán)素組與對(duì)照組比較,海馬CA1區(qū)凋亡細(xì)胞數(shù)增多,Caspase-3及Apaf-1積分光密度明顯增高,統(tǒng)計(jì)學(xué)差異非常顯著(P0.01)。低、高劑量米諾環(huán)素組與模型組比較,海馬CA1區(qū)凋亡細(xì)胞數(shù)減少,Caspase-3積分光密度降低,統(tǒng)計(jì)學(xué)差異非常顯著(P0.01)。兩個(gè)劑量米諾環(huán)素對(duì)海馬CA1區(qū)神經(jīng)細(xì)胞凋亡數(shù)目、Caspase-3及Apaf-1積分光密度的影響并未顯示明顯差異(P0.05)。 結(jié)論：1、束縛應(yīng)激對(duì)大鼠體重增幅無明顯影響,對(duì)海馬CA1區(qū)神經(jīng)細(xì)胞未見明顯損害。2、海灣戰(zhàn)爭(zhēng)綜合征大鼠模型體重增幅減少,海馬CA1區(qū)神經(jīng)細(xì)胞凋亡增多,Caspase-3及Apaf-1表達(dá)增加,考慮與PB及DEET有關(guān)。3、米諾環(huán)素通過抑制Caspase-3的表達(dá),減少神經(jīng)細(xì)胞凋亡,對(duì)海馬CA1區(qū)神經(jīng)細(xì)胞具有神經(jīng)保護(hù)作用。4、低、高劑量米諾環(huán)素對(duì)海灣戰(zhàn)爭(zhēng)大鼠模型的海馬神經(jīng)細(xì)胞保護(hù)作用未見明顯區(qū)別。
[Abstract]:Background and purpose: of the nearly 700 thousand American soldiers who participated in the 1990~1991 year Gulf War, at least 1/4 of the post-war health problems, including cognitive difficulties, persistent and extensive pain, fatigue, headache, chronic diarrhea, rash, are known as the Gulf War Syndrome (GWS) or the Gulf War. The causes of disease.GWS may include depleted uranium, oil well flame smoke, nerve gas, PB and DEET, vaccination, psychological stress, infectious diseases and many other factors. The consensus of the Gulf War epidemiological studies found that pyridine bromide and insecticides are the only two contacts associated with GWS The incidence of GWS in the brain hippocampus and cingulate gyrus in GWS patients was reduced. The animal experiments also suggested that the hippocampal neurons died. Minocycline (two methylamine tetracycline hydrochloride, minoeyeline hydroehloride) was the second generation of artificial semi synthetic four ring antibiotics. Minocycline (minocycline) is a kind of Caspase-3 inhibitor, which has neuroprotective effect. The aim of this experiment was to establish a rat model of Gulf War with PB and DEET, to observe the apoptosis of neurons in the hippocampus CA1, the expression of -1 (Apaf-1) and Caspase-3, and to explore the neuroprotective mechanism of minocycline.
Methods: the rat model of Gulf War syndrome was established by Abdel-Rahman. The experiment set up the control group, the binding group, the PB/DEET group, the model group, the low dose minocycline group and the high dose minocycline group. The bound group rats were given the fixed shackles and placed in the dark environment for 20 minutes each time. The model group established the Gulf War with reference to the Abdel-Rahman literature. Rat model, rats were given oral 1.3mg/kg/d per day, DEET (40mg/kg/d) was subcutaneously injected with 70% ethanol, and dark environment was bound for 20 minutes in group.PB/DEET: PB and DEET were given subcutaneously every day. Low dose minocycline group: minocycline 15mg/kg/d, abdominal cavity injection, residual model group. High dose minocycline group: Mino 45mg/kg/d, intraperitoneal injection, and Yu Tong model group. The rats in the control group were free to take water to feed and give the same amount of alcohol solution subcutaneously. The experimental period was 28 days. The rats' weight was measured at 0 days, 14 days and 28 days. After the experiment, the paraformaldehyde was perfused and fixed, the brain, paraffin embedded and sliced, and the hippocampus CA1 region nerve was detected by immunohistochemistry. The expression of Apaf-1 and Caspase-3 was detected by TUNEL, and the apoptosis of CA1 neurons in hippocampus was detected.
Results: the weight of the rats in each group increased in different degrees. After 4 weeks, the weight of the control group increased by 79%, the bound group was 75%, the PB/DEET group was 49%, the model group was 47%, the low dose minocycline group was 70%, the high dose minocycline group was 71%.PB/DEET and the control group. The model group was compared with the restraint group, the weight gain decreased, P0.01, statistically significant. Compared with the control group, the model group and the control group compared with the PB/DEET group, the weight increased similar, P0.05, no statistical difference, low, high dose minocycline group compared with the model group, the weight increased significantly, P0.01, there was statistical difference.
Compared with the control group and the control group, the number of apoptotic neurons in the hippocampal CA1 region, the Caspase-3 and the Apaf-1 integral light density were not statistically significant (P0.05). Compared with the control group, the number of apoptotic cells in the hippocampal CA1 area increased and the integral light density of Caspase-3 and Apaf-1 increased significantly, and the statistical difference was very significant. (P0.01) low, high dose minocycline group compared with the control group, the number of apoptotic cells in the hippocampal CA1 area increased, the Caspase-3 and Apaf-1 integral light density increased significantly, the statistical difference was very significant (P0.01). Low dose minocycline group compared with the model group, the number of apoptotic cells in the hippocampus CA1 area decreased, the Caspase-3 integral light density decreased, statistical difference. It was very significant (P0.01). The effect of two doses of minocycline on the number of apoptotic neurons in the hippocampal CA1 region, the effect of Caspase-3 and Apaf-1 integral light density did not show significant difference (P0.05).
Conclusion: 1, there is no obvious effect of restraint stress on the weight gain of rats, no significant damage to.2 in the CA1 region of the hippocampus, the decrease of the weight gain of the rat model of the Bay War syndrome, the increase of neuronal apoptosis in the hippocampal CA1 region, the increase of the expression of Caspase-3 and Apaf-1, considering the.3 of PB and DEET, and the expression of minocycline by inhibiting the expression of Caspase-3, The neuronal apoptosis was reduced and the neuroprotective effect of the hippocampal CA1 region nerve cells was.4, low, and the protective effect of high dose of minocycline on the hippocampal neurons in the rat model of the Gulf War was not significantly different.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R82

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