發(fā)布時間：2018-04-16 01:29

  本文選題：非酒精性脂肪性肝病 + 脂多糖��； 參考：《河北醫(yī)科大學》2017年碩士論文

【摘要】：目的:西方飲食建立非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)小鼠模型,觀察血清內(nèi)毒素水平,肝臟早期生長反應因子-1(early growth response-1,Egr-1)、腫瘤壞死因子-α(tumor necrosis factor-α,TNF-α)表達及腸黏膜機械屏障變化情況,進而探討布拉氏酵母菌干預后的影響,初步探討該藥物對NAFLD的療效以及相關(guān)作用機制。方法:將30只6-8周齡的清潔級雄性C57BL/6小鼠適應性喂養(yǎng)1周后應用隨機數(shù)字表將其隨機分為正常對照組(CG,n=12)和模型組(MG,n=18)。正常對照組給予正常對照飲食(AIN93M對照飼料+正常飲用水)喂養(yǎng),模型組給予西方飲食(高脂飼料+高糖飲用水)喂養(yǎng)。于喂養(yǎng)的24周末隨機處死各組6只小鼠,留取相應標本,并進行肝臟HE染色,病理改變證實NAFLD小鼠模型成功。自第25周起,將模型組剩余12只小鼠隨機分為布拉氏酵母菌灌胃組(TB,n=6)和模型灌胃對照組(TD,n=6),繼續(xù)西方飲食喂養(yǎng)。正常對照組剩余6只小鼠為正常灌胃對照組(NG,n=6),繼續(xù)給予正常對照飲食。TB組給予布拉氏酵母菌(5.98×108 CFU/kg/day)灌胃,TD組及NG組同時開始給予布拉氏酵母菌相應輔料果糖(284mg/kg/day)+乳糖(19mg/kg/day)溶于蒸餾水中灌胃,每日灌胃1次,共6周,6周后處死全部小鼠,留取標本進行相應檢測。觀察小鼠的毛色、行為、精神狀態(tài)等情況,同時每周測定小鼠體重。處死小鼠后,觀察肝臟及回腸的大體情況,并完整切除肝臟,留取末端回腸組織,稱肝臟濕重;采用全自動生化分析儀檢測血清丙氨酸氨基轉(zhuǎn)氨酶(alanine aminotransferase,ALT)、門冬氨酸氨基轉(zhuǎn)氨酶(aspartate aminotransferase,AST)、甘油三酯(triglyceride,TG)、總膽固醇(total cholesterol,TC)水平;采用鱟試劑法測定血清脂多糖(lipopolysaccharide,LPS)水平;HE染色觀察肝臟及末端回腸黏膜病理變化;透射電鏡觀察回腸組織超微結(jié)構(gòu)的改變;采用Real-time Q-PCR(Real-time quantitation PCR)方法檢測各組小鼠肝臟Egr-1、TNF-α及末端回腸組織中TNF-α、Occludin m RNA的表達情況;蛋白免疫印跡(Western Blot)法檢測肝臟Egr-1、TNF-α及末端回腸組織中Occludin、TNF-α的蛋白表達情況。結(jié)果:1實驗動物的一般情況正常對照組小鼠皮毛順滑、濃密、有光澤,精神狀態(tài)良好,活動靈活,反應靈敏,體重逐漸增加;模型組小鼠自造模第4周開始皮毛欠光滑,稍顯油膩,精神差,體重增長迅速,逐漸呈腹型肥胖,至24周,小鼠精神萎靡,喜臥懶動,反應遲緩,毛發(fā)無光澤,造模完成。給予布拉氏酵母菌灌胃干預后,TB組小鼠體重較前減輕,活動增多,情況好轉(zhuǎn)。實驗期間,TB組及TD組小鼠各死亡1只,剩余5只,其余組小鼠無死傷發(fā)生。2實驗動物體重、肝重指數(shù)的比較MG組較CG組小鼠體重及肝重指數(shù)顯著增高(P0.05);灌胃干預后,TB組較TD組體重及肝重指數(shù)顯著降低(P0.05),但仍明顯高于NG組(P0.05),差異有統(tǒng)計學意義。3實驗動物肝臟及末端回腸組織病理肝臟病理:CG組小鼠肝小葉結(jié)構(gòu)完整,肝細胞排列整齊,大小正常,可見圍繞中央靜脈呈放射狀排列的肝細胞索,細胞核大而飽滿,未見脂肪滴及炎性細胞浸潤;MG組肝細胞排列疏松、紊亂,脂肪滴呈彌漫性堆積,肝細胞呈氣球樣變,可見不同程度的炎性細胞浸潤,點狀壞死。經(jīng)灌胃后,TB組較TD組肝臟脂肪變性程度減輕,炎癥細胞浸潤減少,但與NG組相比其脂肪變及炎癥程度仍較明顯�；啬c病理:CG組小鼠末端回腸黏膜結(jié)構(gòu)完整,絨毛及腺體排列整齊,上皮細胞形態(tài)正常,呈高柱狀;MG組小鼠回腸絨毛變疏松,上皮細胞出現(xiàn)壞死和脫落,間隙增寬,可見上皮層與固有層分離。灌胃干預后,TB組較TD組黏膜損傷減輕,但仍比NG組損傷嚴重。4透射電鏡觀察回腸組織超微結(jié)構(gòu)改變CG組腸黏膜上皮細胞排列緊密,表面微絨毛豐富,排列規(guī)則,緊密連接比較長,連接縫隙小;MG組腸黏膜上皮排列較規(guī)則,部分上皮細胞表面微絨毛倒伏、稀疏,緊密連接結(jié)構(gòu)破壞。布拉氏酵母菌灌胃后,TB組較TD組上皮細胞表面微絨毛逐漸豐富,緊密連接結(jié)構(gòu)恢復,連接縫隙減小,但仍比NG組損傷嚴重。5血清生化指標比較MG組較CG組血清ALT、AST、TC及TG顯著升高(P0.05);灌胃干預后,與TD組相比,TB組血清ALT、AST、TC及TG顯著降低(P0.05),但仍高于NG組(P0.05),差異有統(tǒng)計學意義。6血清LPS水平比較MG組較CG組血清LPS顯著升高(P0.05);灌胃干預后,TB組血清LPS較TD組降低(P0.05),但仍高于NG組(P0.05),差異有統(tǒng)計學意義。7 Egr-1、TNF-α在動物肝臟組織的表達情況應用PCR及Western Blot技術(shù)從基因及蛋白水平檢測肝組織中Egr-1、TNF-α表達,結(jié)果均顯示,MG組Egr-1、TNF-α表達明顯高于CG組(P0.05);灌胃干預后,TB組表達量較TD組顯著降低(P0.05),但仍明顯高于NG組(P0.05),差異有統(tǒng)計學意義。8 Occludin、TNF-α在動物末端回腸的表達情況PCR及Western Blot結(jié)果均顯示:MG組回腸組織Occludin蛋白較CG組表達降低(P0.05);灌胃干預后,TB較TD組表達增加(P0.05),但仍低于NG組(P0.05),差異有統(tǒng)計學意義。MG組回腸組織中TNF-α較CG組表達增加(P0.05);灌胃干預后,TB較TD組表達降低(P0.05),但仍高于NG組(P0.05),差異有統(tǒng)計學意義。結(jié)論:1西方飲食可誘導NAFLD,NAFLD存在內(nèi)毒素血癥。2 NAFLD小鼠腸粘膜機械屏障受損,腸道通透性增加,可能加重了腸源性內(nèi)毒素血癥。3 NAFLD小鼠肝臟Egr-1表達增加,可能參與了NAFLD的發(fā)病過程。4布拉氏酵母菌可能通過改善腸粘膜機械屏障,減輕腸源性內(nèi)毒素血癥,降低了肝臟Egr-1的表達,從而改善了肝臟炎癥及脂肪變程度。
[Abstract]:Objective: to establish nonalcoholic fatty liver disease (non-alcoholic fatty liver disease in the Western diet, NAFLD) mouse model, observe the level of serum endotoxin and liver of early growth response factor -1 (early growth response-1, Egr-1), tumor necrosis factor alpha (tumor alpha factor- alpha necrosis, TNF-) expression and intestinal mucosal changes. And to explore the intervention effect of Saccharomyces boulardii, preliminary discuss the curative effect of drugs on NAFLD and related mechanism. Methods: 30 6-8 week old male C57BL/6 mice were fed for 1 weeks after application were randomly divided randomly into normal control group (CG, n=12) and model group (MG. N=18). Normal control group was given normal diet (AIN93M control diet + normal drinking water feeding), model group were given the Western diet (high fat diet + high glucose in drinking water) feeding. Feeding 24 weekends were killed Each group of 6 mice, take corresponding specimens, the liver and the HE staining, the pathological changes of NAFLD mouse model confirmed successful. Since the twenty-fifth week, the model group, the remaining 12 mice were randomly divided into Saccharomyces boulardii gavage group (TB, n=6) model and gavage control group (TD, n=6), to the Western diet the normal control group feeding. The remaining 6 mice into normal gavage control group (NG, n=6), continue to give normal diet.TB group was given boulardii (5.98 x 108 CFU/kg/day) by gavage, TD group and NG group also began to give the corresponding materials of Saccharomyces boulardii fructose (284mg/kg/day) + lactose (19mg/kg/day) solution by intragastric administration in distilled water gavage 1 times, a total of 6 weeks, all mice were killed after 6 weeks, the specimen of the corresponding detection. Observe the hair color, behavior, mental state, and body weight was recorded weekly. After the death of small rats, the general situation of the observation of liver and ileum, And complete resection of the liver, specimens from the distal ileum, called wet liver weight; the serum alanine aminotransferase automatic biochemical analyzer (alanine aminotransferase, ALT), aspartate aminotransferase (aspartate, aminotransferase, AST), triglycerides (triglyceride, TG), total cholesterol (total, cholesterol, TC) level of serum; lipopolysaccharide using Limulus amebocyte lysate (lipopolysaccharide, LPS) level; observe the pathological changes of liver and terminal ileum mucosa HE staining; TEM ileal tissue changes; using Real-time Q-PCR (Real-time quantitation PCR) method to detect the mouse liver Egr-1, TNF- alpha TNF- alpha and terminal ileum tissue, the expression of Occludin m RNA; Western blot (Western Blot) method for the detection of liver Egr-1, TNF- alpha and terminal ileum tissue of Occludin, expression of TNF- protein. Results: 1 The general situation of the experimental animal control mice skin smooth, dense, shiny, good mental state, flexible, responsive, body weight increased gradually; the model mice model was made fourth weeks fur less smooth, slightly greasy, poor spirit, weight increase rapidly, gradually abdominal obesity, to 24 week, apathetic, lie lazy, sluggish, hair dull, modeling is completed. Given boulardii gavage intervention, group TB mice weight decreased, the activity increased, the situation improved. During the experiment, TB group and TD group were 1 rats died, the remaining 5, the remaining group no casualties occurred in mice.2 experimental animal body weight, liver weight and liver weight index in MG group compared with CG group of mice weight index increased significantly (P0.05); gavage intervention, TB group than in TD group significantly decreased body weight and liver weight index (P0.05), but still higher than that in group NG (P0.05), there are differences statistical significance The meaning of liver and.3 terminal ileum tissue pathology experimental animal liver pathology: in CG group the structure of hepatic lobule integrity, liver cells arranged in neat rows, normal size, visible around the central vein radially cords of liver cells, the nucleus is big and full, no fat droplets and inflammatory cell infiltration; MG group of liver cells arranged in loose and disorder that fat droplets diffuse accumulation, the liver cells showed ballooning, showed different degrees of inflammatory cell infiltration, spotty necrosis. After intragastric administration, TB group than in TD group reduced the degree of liver steatosis, inflammatory cells infiltration, but compared with the NG and the degree of fatty degeneration and inflammation of the ileum was still obvious. Pathology: CG group were terminal ileum mucosa structure integrity, villi and glands lined epithelial cells, normal, columnlike; MG group of mouse ileum villi became loose, epithelial cell necrosis and shedding, gap widened, visible epithelial layer and solid A layer of separation. Intragastric administration intervention, reduce the TB group than in TD group mucosal injury, but still higher than the NG group was seriously damaged.4 transmission electron microscope to observe the ultrastructural changes of ileal tissue in CG group intestinal epithelial cells arranged closely, microvilli on the surface of rich, arranged and closely connected to long connection gap of small intestinal mucosa in MG group; epithelial cells were regular, part of epithelial cell microvilli on the surface of lodging, sparse, tight junction damage. Boulardii after intragastric administration, TB group and TD group of epithelial cell surface microvilli gradually, tight junction recovery, gap junction decreased, but still higher than the NG group was seriously damaged.5 serum biochemical indexes in MG group than in CG group serum ALT, AST, TC and TG were significantly increased (P0.05); gavage intervention, compared with the TD group, TB group, serum ALT, AST, TC and TG were significantly reduced (P0.05), but still higher than that of NG group (P0.05), the difference had statistical significance between the serum level of LPS in.6 MG group than in CG 緇勮娓匧PS鏄捐憲鍗囬珮(P0.05);鐏岃儍騫查鍚，

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