【摘要】：目前,非酒精性脂肪性肝病(Non-alcoholic fatty liver disease,NAFLD)是全球重要的健康問(wèn)題之一,也是我國(guó)越來(lái)越受重視的慢性肝病問(wèn)題。它是一組由單純肝臟脂肪變性,發(fā)展到脂肪性肝炎(Non-alcoholic steatohepatitis,NASH)、肝硬化、肝癌的臨床癥候群。近年來(lái)隨著腸道微生態(tài)與NAFLD發(fā)病關(guān)系研究的深入,腸道菌群過(guò)度生長(zhǎng)、腸黏膜通透性增加所致的內(nèi)毒素血癥刺激機(jī)體炎癥反應(yīng)在NAFLD的發(fā)生發(fā)展中的作用得到越來(lái)越多的認(rèn)可。布拉氏酵母菌是目前唯一上市的真菌微生態(tài)制劑,它為腸道過(guò)路菌,在停藥一周后糞便中就不可檢測(cè)到該菌,不會(huì)影響腸道內(nèi)菌量�？诜蟛皇苣懼⑽杆岬绕茐�。它可與致病菌在腸上皮的黏附形成競(jìng)爭(zhēng),可以抑制炎癥信號(hào)的傳遞表達(dá)。我們通過(guò)飼以高脂飲食成功造模,研究布拉氏酵母菌對(duì)NAFLD大鼠腸黏膜屏障的保護(hù)作用,將有助于為NAFLD的治療及預(yù)防提供理論基礎(chǔ)。目的:觀(guān)察布拉氏酵母菌對(duì)NAFLD大鼠腸黏膜屏障的保護(hù)作用,并對(duì)其可能機(jī)制進(jìn)行探討。方法:實(shí)驗(yàn)用雄性健康Sprague-Dawley大鼠36只,體重為180±20g,適應(yīng)性喂養(yǎng)1周后隨機(jī)分為三組,即正常對(duì)照組(Control組,C組)、高脂模型組(Model組,M組)、高脂治療組(Treatment組,T組),每組12只;C組飼以基礎(chǔ)飼料(其熱量為碳水化合物65.5%,蛋白質(zhì)24.2%,脂肪10.3%)喂養(yǎng)12周;M組和T組飼以高脂飼料(碳水化合物和蛋白質(zhì)各20.1%,脂肪59.8%)喂養(yǎng)12周;每組各隨機(jī)處死2只,蘇丹黑染色觀(guān)察肝臟脂質(zhì)沉積程度確定造模成功。隨后T組開(kāi)始給予布拉氏酵母菌灌胃(75*108 CFU/kg?d-1),C、M組給予等容量的生理鹽水灌胃;治療過(guò)程中每周隨體重變化調(diào)整灌胃量。20周時(shí)在麻醉狀態(tài)下腹主動(dòng)脈取血處死大鼠,收集血液測(cè)定丙氨酸氨基轉(zhuǎn)移酶(ALT)、天冬氨酸氨基轉(zhuǎn)移酶(AST)、甘油三酯(TG)、腸型脂肪酸結(jié)合蛋白(IFABP)、腫瘤壞死因子-α(TNF-α)、內(nèi)毒素水平;觀(guān)察肝臟的大體情況,稱(chēng)肝臟濕重,采用蘇丹黑染色觀(guān)察肝臟病理變化;腸組織行HE染色觀(guān)察腸絨毛完整程度,免疫組化染色檢測(cè)腸黏膜occludin蛋白的表達(dá)情況。留取大鼠盲腸內(nèi)糞便,用于糞便大腸桿菌、擬桿菌定量檢測(cè)。結(jié)果:1大鼠體重(g)、肝重(g)和肝指數(shù)(肝重/體重×100%):M組大鼠的體重(548.7±16.1)、肝重(17.516±1.112)、肝指數(shù)(3.191±0.170)均明顯高于C組的體重(423.2±19.2)、肝重(9.493±0.739)及肝指數(shù)(2.241±0.112),差異有統(tǒng)計(jì)學(xué)意義(P0.05)。T組體重(499.1±18.1)、肝重(13.062±1.307)和肝指數(shù)(2.616±0.230)與M組相比均明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2血清TG(mmol/l)、ALT(u/l)、AST(u/l)水平:M組TG(0.585±0.057)、ALT(78.75±10.92)、AST(205.74±10.05)較C組TG(0.373±0.081)、ALT(30.99±8.59)、AST(108.04±11.60)顯著增高,差異有統(tǒng)計(jì)學(xué)意義(P㩳0.05);T組的AST(194.42±13.74)與M組相比明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05);T組TG(0.541±0.072)、ALT(72.36±9.87)與M組相比稍有下降,無(wú)統(tǒng)計(jì)學(xué)差異(P0.05)。3血清內(nèi)毒素(EU/ml)及TNF-α(ng/ml)水平:M組內(nèi)毒素(0.306±0.054)、TNF-α(1.160±0.129)較C組內(nèi)毒素(0.201±0.047)、TNF-α(0.917±0.115)顯著升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05);T組內(nèi)毒素(0.249±0.026)、TNF-α(1.042±0.078)與M組相比均明顯降低,有顯著性差異(P0.05)。4血清IFABP(pg/ml)水平:M組IFABP(288.76±59.17)較C組IFABP(137.19±39.12)明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05);T組IFABP(193.02±27.13)與M組相比明顯降低,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。5組織病理結(jié)果:(1)在12周時(shí)蘇丹黑染色顯示M、T組肝細(xì)胞脂肪變明顯增加,證實(shí)造模成功。20周時(shí)肝組織蘇丹黑染色提示M組肝細(xì)胞脂肪變(2.90±0.32)較C組(0.00±0.00)明顯增加(P0.05);治療后,T組肝細(xì)胞脂肪變(1.80±0.79)較M組明顯減少(P0.05)。(2)腸黏膜組織HE染色結(jié)果:光鏡下,C組腸絨毛排列整齊,結(jié)構(gòu)完整;M組腸絨毛變短、局部有絨毛脫落,排列欠整齊,可見(jiàn)炎性細(xì)胞浸潤(rùn);與M組相比,T組腸絨毛輕度水腫,排列較整齊,炎性細(xì)胞浸潤(rùn)較少。6免疫組織化學(xué)結(jié)果:M組的腸黏膜occludin蛋白(0.042±0.006)較C組(0.177±0.007)明顯減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05);T組(0.049±0.010)較M組表達(dá)稍有增加,但無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。7糞便PCR結(jié)果(log10(CFU/g)):(1)M組的大腸桿菌(E.Coli)數(shù)量(5.98±0.08)較C組(4.00±0.09)明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05),與M組相比,T組大腸桿菌數(shù)量(4.92±0.09)明顯下降,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。(2)M組的擬桿菌數(shù)量(8.04±0.17)較C組(9.47±0.09)明顯減少,差異有統(tǒng)計(jì)學(xué)意義(P0.05),與M組相比,T組的擬桿菌數(shù)量(8.89±0.10)明顯增加,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1高脂飲食可成功誘導(dǎo)大鼠NAFLD,經(jīng)布拉氏酵母菌干預(yù)可以起到減輕體重和改善肝細(xì)胞脂肪變的作用。2布拉氏酵母菌能通過(guò)減輕NAFLD大鼠內(nèi)毒素血癥,減輕機(jī)體炎癥反應(yīng)。3布拉氏酵母菌能調(diào)節(jié)NAFLD大鼠腸道大腸桿菌和擬桿菌的比例。
[Abstract]:Nowadays, non-alcoholic fatty liver disease (NAFLD) is one of the most important health problems in the world, and it is also a chronic liver disease that has been paid more and more attention in China. In recent years, with the deepening of the relationship between intestinal microecology and the pathogenesis of NAFLD, intestinal flora overgrowth and intestinal mucosal permeability increase caused by endotoxemia stimulate the role of inflammation in the occurrence and development of NAFLD has been more and more recognized. Passage-passing bacteria can not be detected in the stool after a week of drug withdrawal and do not affect the amount of bacteria in the intestine.They are not damaged by bile and gastric acid after oral administration.They can compete with pathogenic bacteria in the intestinal epithelium and inhibit the transmission and expression of inflammation signals.We successfully established a model by feeding high-fat diet to study the effect of Saccharomyces brasiliensis on NAF. OBJECTIVE: To observe the protective effect of Saccharomyces brasiliensis on intestinal mucosal barrier in rats with NAFLD and to explore its possible mechanism. Then they were randomly divided into three groups: normal control group (Control group, C group), high-fat model group (Model group, M group), high-fat treatment group (Treatment group, T group), 12 rats in each group; group C was fed with basic diet (65.5% carbohydrate, 24.2% protein, 10.3% fat) for 12 weeks; group M and group T were fed with high-fat diet (20.1% carbohydrate and 20.1% protein, respectively). Fat 59.8% was fed for 12 weeks; 2 rats in each group were sacrificed randomly, and Sudan black staining was used to observe the degree of liver lipid deposition to determine the success of modeling. Rats were sacrificed by taking blood from the lower abdominal aorta and blood samples were collected to determine the levels of ALT, AST, TG, IFABP, TNF - alpha and endotoxin. HE staining was used to observe intestinal villi integrity and immunohistochemical staining was used to detect occludin protein expression in intestinal mucosa. The body weight, liver index (3.191 (+0.170)) and liver index (9.493 (+0.739) of group C were significantly higher than those of group C (423.2 (+19.2)), and liver index (2.241 (+0.112)). The body weight, liver weight (13.062 (+1.307) and liver index (2.616 (+0.230)) of group T were significantly lower than those of group M (P 0.05). Mmol / l, ALT (u / l), ALT (u / l) and AST (u / l) levels: TG (0.585 (+ 0.057), ALT (78.75 (+ 10.92), ALT (78.75 (+ 10.92), AST (205.74 (+ 10.05) (205.74 (+ 10.74 (+ 10.05) TG (0.373 [(0.373 +0.081), ALT (30.373 [(30.99 [8.99 [.59]), ALT (30.99 [(30.99 [8.59]]), AST (108.04 [(108.04 [11.04 [11.60] 0.05), AST A The levels of endotoxin (EU/ml) and TNF-alpha (ng/ml) in serum of group M were significantly higher than those of group C in endotoxin (0.306+0.054), TNF-alpha (1.160+0.129) and TNF-alpha (0.201+0.047) and TNF-alpha (0.917+0.115), respectively (P 0.05). The levels of serum IFABP (pg/ml) in group M (288.76+59.17) were significantly higher than those in group C (137.19+39.12) (P 0.05); IFABP (193.02+27.13) in group T (193.02+27.13) was significantly lower than that in group M (P 0.05). 5 histopathological results were statistically significant: (1) At 12 weeks, IFABP in group M (288.76+59.17) was significantly higher than that in group C (137.19+39.12) (P 0.05). Sudan black staining showed that hepatocyte steatosis in group M and T was significantly increased, which confirmed the success of modeling. At 20 weeks, Sudan black staining showed that hepatocyte steatosis in group M (2.90.32) was significantly increased compared with group C (0.00.00) (P 0.05); after treatment, hepatocyte steatosis in group T (1.80.79) was significantly decreased compared with group M (P 0.05). (2) HE staining of intestinal mucosa The intestinal villi of group C were arranged neatly and structurally intact; the intestinal villi of group M were shortened, some villi were shedding off, and irregular arrangement, and inflammatory cell infiltration was observed; compared with group M, the intestinal villi of group T were slightly edematous, arranged neatly, and inflammatory cell infiltration was less.6 Immunohistochemical results showed that occludin protein (0.042 + 0.006) of intestinal mucosa of group M was higher than that of group C (0.177 + 0.007). The expression of E. coli in group T (0.049.010) was slightly higher than that in group M (P 0.05), but there was no significant difference (P 0.05). 7 Fecal PCR results (log10 (CFU / g): (1) The number of E. coli in group M (5.98.08) was significantly higher than that in group C (4.00.09), and the number of E. coli in group T (4.05) was significantly higher than that in group M (P 0.05). (2) The number of Bacteroides in group M (8.04 + 0.17) was significantly lower than that in group C (9.47 + 0.09), and the difference was statistically significant (P 0.05). Compared with group M, the number of Bacteroides in group T (8.89 + 0.10) was significantly increased, and the difference was statistically significant (P 0.05). The intervention of Brucella yeast can reduce body weight and improve hepatic steatosis. 2 Brucella yeast can alleviate inflammation by reducing endotoxemia in NAFLD rats. 3 Brucella yeast can regulate the proportion of enteric E. coli and Bacteroides in NAFLD rats.
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R575.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 荀運(yùn)浩;施軍平;;2014年非酒精性脂肪性肝病診治進(jìn)展[J];實(shí)用肝臟病雜志;2015年01期

2 Ke-Xin Gan;Chao Wang;Jin-Hu Chen;Chun-Jing Zhu;Guang-Yao Song;;Mitofusin-2 ameliorates high-fat diet-induced insulin resistance in liver of rats[J];World Journal of Gastroenterology;2013年10期

3 楊林輝;郭華;蔡俊;蔡曉偉;劉貴倫;陳東風(fēng);;枯草桿菌腸球菌二聯(lián)活菌膠囊對(duì)非酒精性脂肪性肝炎腸道菌群失調(diào)的干預(yù)作用[J];世界華人消化雜志;2012年20期

4 郭亞瓊;;布拉酵母菌治療嬰幼兒抗生素相關(guān)性腹瀉的臨床研究[J];中國(guó)醫(yī)藥指南;2012年18期

5 張華玲;譚周進(jìn);蔡光先;蔡瑩;;腸道菌群失調(diào)調(diào)控技術(shù)研究進(jìn)展[J];中國(guó)微生態(tài)學(xué)雜志;2011年11期

6 張學(xué)娜;金世祿;;腸道菌群失調(diào)與腸易激綜合征[J];中華臨床醫(yī)師雜志(電子版);2011年22期

，

本文編號(hào)：2206679