本文關鍵詞： 干酪乳桿菌 衰老 抗氧化 腸黏膜屏障功能 腸道菌群　出處：《青島大學》2017年碩士論文　論文類型：學位論文

【摘要】：目的建立小鼠衰老模型,研究干酪乳桿菌是否具有抗氧化、保護腸黏膜及調節(jié)腸道菌群的作用,探索干酪乳桿菌與衰老之間的關系。方法1.動物模型建立及分組:昆明種SPF級雄性小鼠,共75只,體重(20±2)g,按體重隨機分為空白組(15只)和模型組(60只)。模型組頸背部皮下注射D-半乳糖,劑量為400 mg/kg/d,持續(xù)6周,建立衰老模型。然后模型組剪尾取血,并根據(jù)硫代巴比妥酸法(TBA法)所測紅細胞中丙二醛(MDA)水平,將衰老模型組重新分組,隨機分為模型組(10 m L/kg/d雙蒸水灌胃)、干酪乳桿菌干預劑量1、2組(5、10m L/kg/d干酪乳桿菌灌胃),維生素E陽性對照組(80 mg/kg/d維生素E灌胃);正常對照組以10 m L/kg/d雙蒸水灌胃。除正常對照組外其余各組繼續(xù)頸背部皮下注射D-半乳糖,持續(xù)30天。最后一次灌胃后,用代謝籠收集小鼠糞便,用乙醚麻醉小鼠,采用摘眼球法取血,同時摘取肝臟和小腸。2.抗氧化指標檢測:采用彗星電泳實驗(單細胞凝膠電泳實驗)檢測小鼠外周血中淋巴細胞DNA損傷水平,利用TBA法檢測小鼠紅細胞中MDA含量,DNPH比色法(2,4-二硝基苯肼法)檢測小鼠肝組織中蛋白質羰基含量,DTNB法(二硫代二硝基苯甲酸法)檢測小鼠肝組織中GSH(還原型谷胱甘肽)含量及GSH-PX(谷胱甘肽過氧化物酶)活性。3.腸道屏障功能檢測:利用透射電鏡觀察小鼠小腸黏膜組織超微結構的變化,采用ELISA法檢測小鼠血清中D-LA(D-乳酸)含量、DAO(二胺氧化酶)活性及FABP2(腸脂肪酸結合蛋白)含量。4.腸道菌群含量檢測:采用熒光實時定量PCR法檢測各組小鼠糞便中雙歧桿菌、乳酸桿菌、大腸桿菌和腸球菌的含量。結果1.與正常對照組相比,模型組淋巴細胞DNA損傷程度明顯升高(P0.05);與模型組比較,干預劑量1、2組及維生素E組DNA損傷率明顯下降(P0.05);與維生素E組比較,干預劑量1、2組DN A損傷情況無明顯差異(P0.05)。2.與正常對照組相比,模型組MDA及蛋白質羰基含量均明顯升高(P0.05);與模型組比較,干預劑量1、2組及維生素E組MDA及蛋白質羰基含量均明顯下降(P0.05);與維生素E組比較,干預劑量1、2組蛋白質羰基含量降低(P0.05),MDA含量無明顯差異(P0.05)。3.與正常對照組相比,模型組GSH和GSH-PX含量均顯著降低(P0.05);與模型組比較,干預劑量1、2組GSH含量顯著升高,干預劑量2組GSH-PX活性組顯著升高(P0.05);與維生素E組比較,干預劑量1、2組GSH和GSH-PX含量均無明顯差異(P0.05)。4.透射電鏡觀察顯示,正常對照組小腸黏膜結構清晰完整,絨毛排列整齊緊密,細胞連接緊密;模型組小鼠小腸絨毛排列紊亂,部分倒伏脫落,細胞連接腫脹,橋粒數(shù)量減少;補充干酪乳桿菌及維生素E后,小腸絨毛及細胞連接狀況均較模型組明顯改善。5.與正常對照組相比,模型組D-LA、DAO及FABP2含量均顯著增高(P0.05);與模型組比較,干預劑量1、2組及維生素E組D-乳酸含量、DAO活性及FABP2含量均顯著降低(P0.05);與維生素E組比較,干預劑量1、2組D-乳酸、DAO及FABP2含量均無明顯差別(P0.05)。6.與空白對照組比較,模型組雙歧桿菌、乳酸桿菌及大腸桿菌含量均顯著降低(P0.05);與模型組比較,干預劑量2組雙歧桿菌、乳酸桿菌含量升高,腸球菌含量降低;干預劑量1組乳酸桿菌、大腸桿菌含量升高,腸球菌含量降低(P0.05)。結論1.干酪乳桿菌可以調節(jié)腸道菌群,優(yōu)化菌群結構,提高衰老小鼠抗氧化能力,減輕氧化損傷水平,改善小腸黏膜屏障,降低小腸黏膜通透性,從而起到延緩衰老的作用。2.干酪乳桿菌作為一種有益的益生菌補充劑,對促進人體健康有極大意義,但是其如何通過調節(jié)腸道菌群等發(fā)揮延緩衰老作用的具體分子機制還需進一步研究。
[Abstract]:Objective to establish a model of aging mice, to study whether Lactobacillus casei has antioxidant, protect the intestinal mucosa and intestinal flora regulation function, explore the relationship between Lactobacillus casei and aging. Methods 1. animal models and grouping: Kunming male mice of SPF grade, a total of 75, weight (20 + 2) g, according to were randomly divided into blank group (15 rats) and model group (60 rats). The model group subcutaneous injection of D- galactose, a dose of 400 mg/kg/d for 6 weeks. Then, a model of aging model group cut the tail blood, and according to the thiobarbituric acid method (TBA method) were measured by red blood cells in (MDA) level, the aging model group to re group, were randomly divided into model group (10 m L/kg/d double distilled water), Lactobacillus casei intervention dose 1,2 group (5,10m L/kg/d, Lactobacillus casei gavage), positive control group (vitamin E 80 mg/kg/d vitamin E orally) in the normal control group; 10 m L/kg/d double distilled Water gavage. Groups except the normal control group to subcutaneous injection of D- galactose for 30 days. The last time after intragastric administration, collected mice feces using metabolic cages, with ether anesthesia in mice, using the method of eyeball blood detection and the removal of.2. in liver and intestine antioxidant index: using comet assay (single cell gel electrophoresis) were detected in peripheral blood DNA lymphocyte in the injury level, detection of MDA content in mice red blood cells by TBA assay, DNPH colorimetric method (2,4- method two 4-dinitrophenylhydrazine) protein carbonyl content in liver tissue of mice were detected in DTNB (two - two nitrobenzo method) were detected in liver tissue GSH (glutathione) content and GSH-PX (glutathione peroxidase) barrier function of intestinal.3. activity assay: To observe the ultrastructural changes of intestinal mucosa by TEM, serum were detected by ELISA D-LA (D- acid) content, DAO (two amine oxidase) activity and FABP2 (intestinal fatty acid binding protein) to examine the content of.4. content of intestinal flora: bifidobacteria, mice feces were detected by real-time fluorescence quantitative PCR method in the content of lactic acid bacteria, Escherichia coli and enterococci. Results 1. compared with normal control group the model group DNA damage of lymphocytes was significantly elevated (P0.05); compared with the model group, the intervention dose 1,2 group and vitamin E group DNA injury rate decreased significantly (P0.05); compared with vitamin E group, 1,2 group and DN intervention dose A injury had no significant difference (P0.05.2.) compared with the normal control group, model group MDA and protein carbonyl content were significantly increased (P0.05); compared with the model group, the intervention dose 1,2 group and vitamin E group MDA and protein carbonyl content were significantly decreased (P0.05); compared with vitamin E group, intervention dose 1,2 group decreased the protein carbonyl content (P0 .05), no significant difference between the content of MDA (P0.05).3. compared with normal control group, model group, GSH and GSH-PX were significantly lower (P0.05); compared with the model group, the intervention dose 1,2 group GSH content increased significantly, the intervention dose group 2 GSH-PX activity group was significantly increased (P0.05); compared with vitamin E group. There were no significant difference between the intervention group GSH dose of 1,2 and the content of GSH-PX (P0.05).4. transmission electron microscope showed that the normal structure of small intestinal mucosa villi was clear and complete, closely aligned cells connected tightly; mice model of intestinal villi arranged in disorder, partial lodging loss, cell junction swelling, reduce the number of desmosomes; Lactobacillus casei and vitamin E, intestinal villi and cell connection status were significantly improved compared with the model group.5. compared with normal control group, D-LA model group, DAO and FABP2 were significantly increased (P0.05); compared with the model group, 1,2 group and vitamin intervention dose E group D- lactic acid content, DAO activity and FABP2 content were significantly decreased (P0.05); compared with vitamin E group, intervention dose 1,2 group D- lactic acid, DAO and FABP2 were no significant difference (P0.05.6.) compared with control group, model group, Bifidobacterium, Lactobacillus and Escherichia coli were significantly reduce (P0.05); compared with the model group, the intervention group increased 2 doses of Bifidobacterium, lactic acid content, Enterococcus decreased; intervention dose group 1 Lactobacillus increased, Escherichia coli, Enterococcus decreased (P0.05). Conclusion: 1. Lactobacillus casei can regulate the intestinal flora, flora structure optimization, improve antioxidant capacity the aging mice, reduce oxidative damage, improve intestinal mucosal barrier, reduce the intestinal permeability, which play a role in delaying aging of.2. casei as a useful supplement of probiotics, promote human health is very careless But it needs further study on how to play a specific molecular mechanism to postpone aging by regulating intestinal microflora.

【學位授予單位】：青島大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R151

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