干酪乳杆菌延缓衰老作用初步研究

发布时间：2018-02-11 06:27

本文关键词： 干酪乳杆菌衰老抗氧化肠黏膜屏障功能肠道菌群　出处：《青岛大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的建立小鼠衰老模型,研究干酪乳杆菌是否具有抗氧化、保护肠黏膜及调节肠道菌群的作用,探索干酪乳杆菌与衰老之间的关系。方法1.动物模型建立及分组:昆明种SPF级雄性小鼠,共75只,体重(20±2)g,按体重随机分为空白组(15只)和模型组(60只)。模型组颈背部皮下注射D-半乳糖,剂量为400 mg/kg/d,持续6周,建立衰老模型。然后模型组剪尾取血,并根据硫代巴比妥酸法(TBA法)所测红细胞中丙二醛(MDA)水平,将衰老模型组重新分组,随机分为模型组(10 m L/kg/d双蒸水灌胃)、干酪乳杆菌干预剂量1、2组(5、10m L/kg/d干酪乳杆菌灌胃),维生素E阳性对照组(80 mg/kg/d维生素E灌胃);正常对照组以10 m L/kg/d双蒸水灌胃。除正常对照组外其余各组继续颈背部皮下注射D-半乳糖,持续30天。最后一次灌胃后,用代谢笼收集小鼠粪便,用乙醚麻醉小鼠,采用摘眼球法取血,同时摘取肝脏和小肠。2.抗氧化指标检测:采用彗星电泳实验(单细胞凝胶电泳实验)检测小鼠外周血中淋巴细胞DNA损伤水平,利用TBA法检测小鼠红细胞中MDA含量,DNPH比色法(2,4-二硝基苯肼法)检测小鼠肝组织中蛋白质羰基含量,DTNB法(二硫代二硝基苯甲酸法)检测小鼠肝组织中GSH(还原型谷胱甘肽)含量及GSH-PX(谷胱甘肽过氧化物酶)活性。3.肠道屏障功能检测:利用透射电镜观察小鼠小肠黏膜组织超微结构的变化,采用ELISA法检测小鼠血清中D-LA(D-乳酸)含量、DAO(二胺氧化酶)活性及FABP2(肠脂肪酸结合蛋白)含量。4.肠道菌群含量检测:采用荧光实时定量PCR法检测各组小鼠粪便中双歧杆菌、乳酸杆菌、大肠杆菌和肠球菌的含量。结果1.与正常对照组相比,模型组淋巴细胞DNA损伤程度明显升高(P0.05);与模型组比较,干预剂量1、2组及维生素E组DNA损伤率明显下降(P0.05);与维生素E组比较,干预剂量1、2组DN A损伤情况无明显差异(P0.05)。2.与正常对照组相比,模型组MDA及蛋白质羰基含量均明显升高(P0.05);与模型组比较,干预剂量1、2组及维生素E组MDA及蛋白质羰基含量均明显下降(P0.05);与维生素E组比较,干预剂量1、2组蛋白质羰基含量降低(P0.05),MDA含量无明显差异(P0.05)。3.与正常对照组相比,模型组GSH和GSH-PX含量均显著降低(P0.05);与模型组比较,干预剂量1、2组GSH含量显著升高,干预剂量2组GSH-PX活性组显著升高(P0.05);与维生素E组比较,干预剂量1、2组GSH和GSH-PX含量均无明显差异(P0.05)。4.透射电镜观察显示,正常对照组小肠黏膜结构清晰完整,绒毛排列整齐紧密,细胞连接紧密;模型组小鼠小肠绒毛排列紊乱,部分倒伏脱落,细胞连接肿胀,桥粒数量减少;补充干酪乳杆菌及维生素E后,小肠绒毛及细胞连接状况均较模型组明显改善。5.与正常对照组相比,模型组D-LA、DAO及FABP2含量均显著增高(P0.05);与模型组比较,干预剂量1、2组及维生素E组D-乳酸含量、DAO活性及FABP2含量均显著降低(P0.05);与维生素E组比较,干预剂量1、2组D-乳酸、DAO及FABP2含量均无明显差别(P0.05)。6.与空白对照组比较,模型组双歧杆菌、乳酸杆菌及大肠杆菌含量均显著降低(P0.05);与模型组比较,干预剂量2组双歧杆菌、乳酸杆菌含量升高,肠球菌含量降低;干预剂量1组乳酸杆菌、大肠杆菌含量升高,肠球菌含量降低(P0.05)。结论1.干酪乳杆菌可以调节肠道菌群,优化菌群结构,提高衰老小鼠抗氧化能力,减轻氧化损伤水平,改善小肠黏膜屏障,降低小肠黏膜通透性,从而起到延缓衰老的作用。2.干酪乳杆菌作为一种有益的益生菌补充剂,对促进人体健康有极大意义,但是其如何通过调节肠道菌群等发挥延缓衰老作用的具体分子机制还需进一步研究。
[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.

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R151

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