【作者】 王正根；

【导师】 卢放根；

【作者基本信息】 中南大学 ， 消化内科， 2010， 博士

【摘要】 目的从正常人肠道粪便中分离的潜在目的菌中筛选出有降胆固醇能力的菌株,并进行鉴定。方法通过改良的胆固醇MRS培养基作为筛选模型进行目的菌的筛选,并对降胆固醇能力较好的菌株进行初步鉴定。结果经过初筛和复筛,筛选出4株降胆固醇能力在30%以上的菌株,降解率分别为M7-544.7%,M2-134.2%,M10-2-8 32.5%,MM1-16为32.4%；将M7-5和M2-1进行鉴定,M7-5为发酵乳酸杆菌,M2-1为植物乳酸杆菌。结论菌株M7-5和M2-1在体外有较强的降胆固醇能力,经鉴定分别为发酵乳杆菌及植物乳杆菌,有待于将其应用于动物体内进行进一步研究。目的观察2株乳酸杆菌对高脂饮食喂养的SD大鼠胆固醇代谢的影响,评价其对高脂饮食喂养的SD大鼠降胆固醇作用的有效性和安全性,从而为筛选到有利于人体降胆醇的功能菌提供依据。方法40只SD大鼠随机分为4组,K组喂养普通饲料,E、F、I组喂养改良高脂饲料,同时以2株乳酸菌干预,E组是M2-1,F组是M7-5。I组不加益生菌,为高脂模型组,6周末处死大鼠,检测大鼠血脂指标,血清总胆叶酸、肝脂质、粪脂质,体重增加量,Lee指数,肝组织病理学检查及血清谷丙转氨酶等指标。结果l、高脂模型组血清TC、TG、LDL-C较正常对照组比均升高(P<0.05),差异有显著性,乳酸菌E组和F组比高脂模型组下降,以F组效果最好,差异有显著性。2、第4周和第6周,高脂模型组血胆汁酸与正常对照组比升高,但无显著性差异,益生菌E组较高脂模型组下隆,差异有统计学意义。3、粪胆汁酸和胆固醇高脂模型组升高,与正常对照组比,P<0.05,差异有显著性；乳酸菌E组的胆汁酸和F组的胆固醇均升高,分别与高脂模型组及正常对照组比较,P<0.05,差异有显著性。4、高脂模型组Lee’s指数比正常对照组高,P<0.05,差异有显著性；乳酸菌E组和F组均比高脂模型组低,比正常组高,尤其在第6周,F组与高脂模型组比差异有显著性(P<0.05)。5、高脂模型组及乳酸菌E组和F组,每周体重增加量较正常对照组高,有显著性差异；第6周乳酸菌F组体重增加量下降,比高脂模型组降低,差异有显著性。6、肝病理检查HE染色高脂模型组肝脂肪变性较明显,与正常对照组比有显著性差异而乳酸菌E组和F组不明显,与正常对照组无差异。7、高脂模型组血清谷丙转氨酶升高,但分别与其他各组比较无显著性差异。结论1、改良高脂饮食配方连续喂养SD大鼠6周,可以成功造出高脂固醇血症模型。2、对两株乳杆菌M7-5和M2-1对SD大鼠高脂血症模型有降胆固醇作用,以M7-5效果更好。3两株乳酸菌可能的降脂机制是减少了肝脂质沉积和增加了粪脂的排泄,它们的机制可能不完全相同,前者以增加粪胆固醇的排泄为主,而后者则以增加粪胆汁酸的排出为主。目的探讨两株乳酸菌影响SD大鼠饮食性高脂血症脂质代谢的可能机制方法实验动物、分组、建模和干预方法同第2章,第6周末处死大鼠,留取各组肝脏及回肠粘膜上皮标本,用实时荧光定量PCR技术检测肝脏孤核受体FXR及细胞表面受体ABCA1和ABCG5基因mRNA的表达,检测回肠粘膜上皮组织LXRa、ABCA1和ABCG5基因mRNA的表达。结果1、高脂模型组,肝组织FXR受体及LXRα受体,回肠粘膜上皮组织LXRα、ABCA1及ABCG5受体与正常对照组比较差异有统计学意义(P<0.05),而肝组织ABCG5表达无明显上调。2、乳酸菌E组(植物乳肝菌M2-1)肝组织FXR表达与高脂模型组相比显著下调,LXRα则显著上调,回肠粘肠上皮组织细胞表面受体ABCG5上调(P<0.05)。3、乳酸菌F组(发酵乳杆菌M7-5)肝组织孤核受体FXR与高脂模型组比表达下调,肝组织LXRα、ABCG5,回肠粘膜上皮组织LXRα、ABCA1、ABCG5与高脂膜型组相比均上调,有统计学意义(P<0.05)。结论1、发酵乳杆菌M7-5和植物乳杆菌M2-1可降低高脂饮食喂养的SD大鼠血清胆固醇。2、发酵乳杆菌M7-5和植物乳杆菌M2-1作用靶点可能涉及了肝组织和回肠粘膜的某些受体。前者抑制肝脏FXR的表达,可能上调肝、肠粘膜组织LXR2、ABCA1、ABCG5的表达,降胆固醇作用主要以增加体内胆固醇的排泄为主。后者则显著抑制肝组织FXR的表达,上调肝组织LXRa回肠粘膜上皮组织ABCG5的表达,其降胆固醇作用主要在于增加体内胆汁酸的合成,加速胆汁酸的排泄为主。更多还原

【Abstract】 ObjectiveTo screen the strains with the capability to reduce cholesterol from the potential target bacteria isolated by stool samples of healthy adults and identify them.MethodsWe screen the potential target bacteria through modified cholesterol MRS medium as the screening model, and preliminary identify the strains with relative strong capability to cholesterol-reducingResultsScreened out four strains with the 30% or more capability to cholesterol-reducing, with degradation rates M7-5 44.7%, M2-1 34.2%, M10-2-8 32.5%, and MM1-16 32.4% respectively. Identify M7-5 and M2-1 and M7-5 is Lactobacillus fermentum, M2-1 is Lactobacillus plantarum.ConclusionM7-5 and M2-1 had strong capability to cholesterol-reducing in vitro, The result of identification shows that M7-5 and M2-1 were Lactobacillus fermentum, and Lactobacillus plantarum., respectively andwe need Further Study of them to apply them to the animals in vivo. ObjectiveTo observe the effect of two strains of lactobacilli on the Cholesterol metabolism of the male Sprague-Dawley rats fed with high-fat diet and to evaluate their efficacy and safety on lowing cholesterol levels, so as to provide support for screening bacteria that could reduce cholesterol.Method40 SD rats were randomly divided into 4 groups, K, E, F and I group. K group was normal group fed with the normal diet, E group was interfered with high fat diet and lactobacillus M2-1, F group was interfered with high fat diet and lactobacillus M7-5.Ⅰgroup was model group, interfered with high fat diet only. All rats were sacrificed in the 6th week, then the levels of serum lipids, serum total bile acids, total cholesterol, liver lipids, fecal lipids, weight gain, Lee’s index, liver histopathology, serum glutamic-pyruvic transaminase and other index were detected.Results1. Interfered with lactobacilli, levels of serum TC, TG and LDL-C in the E and F group were lower compared with the model group, and higher compared with the normal group. The difference was significant. Effect of the M7-5 was the best, the difference was significant. 2. In the 4th and 6th week, levels of serum bile acid in the E group was higher compared with the normal group (P>0.05), and was lower compared with the model group(P<0.05).3. Levels of fecal bile acids and cholesterol in the model group were higher compared with the normal group(P<0.05), the difference was significant. Bile acid Level in the E group was higher compared with the model group. Cholesterol Level was lower compared with the normal group. The difference was significant.4. Lee’s index of model group was higher compared with the normal group (P<0.05). It was lower in the model group and higher in the normal group compared with Lactobacillus E and F group. Especially in the 6th week, difference was significant between the F and I group (P <0.05).5. weight gain daily in the model and Lactobacillus groups was higher compared with the normal group, difference was significant; From the 6th week, weight gain in the F group decreased. It was lower compared with the model group (P<0.05). Difference was significant.6. Adipose degeneration was very obvious in the model group and very slight in the Lactobacillus E and F groups with the method of HE staining of fatty liver7. Serum bile acid in the model group were increased, but there was no difference between the model and other groups. Conclusion1. Feeding the SD rats continuously with modified improvement compounding of high-fat diet for 6 weeks could successfully induce model of hyperlipoidemia, especially hypercholesterolemia.2. Two strains of Lactobacillus M7-5 and M2-1 could low the level of hyperlipemia in the SD model.M7-5 had a better effect.3. The possible mechanism of the two strains of Lactobacillus lowerng the hyperlipoidemia might be decreasing the deposition of liver lipid and increasing the excretion of fecal fat. Their mechanism might not be exactly the same. The former might be mostly increasing the excretion of the fecal cholesterol and the latter increasing the excretion of the fecal bile acid. ObjectiveInvestigate possible affection mechanism of SD rats alimentary hyperlipoidemia by two lactobacillusMethodsTest animals、group、group and intervention are same as the 2nd chapter, rats were executed after the 6th week, specimen of liver and ileal mucous membrane mucosae were kept to test expression of liver nuclear receptor FXR and cell surface receptor ABCA1 and ABCG5 mRNA, and expression of ileal mucous membrane mucosae LXRa,ABCA1 and ABCG5.Results1. Liver receptor FXR and LXRa, ileal mucous membrane mucosae LXRa, ABCA1 and ABCG5 in hyperlipoidemia group were obvious different with normal group (P<0.05), but liver ABCG5 were not obviously up-regulated.2. In E group of lactobacillus, liver receptor FXR were obviously down-regulated and LXRa were obviously up-regulated, ileal mucous membrane mucosae surface receptor ABCG5 were up-regulated in liver tissue.3.In F group of lactobacillus, liver nuclear receptor FXR were obviously down-regulated and LXRα、ABCG5、ileal mucous membrane mucosae surface receptor LXRα、ABCA1 and ABCG5 were obviously up-regulated with statistics significance compared to hyperlipoidemia group (P<0.05)Conclusion1. Lactobacillus fermentum M7-5 and Lactobacillus plantarum M2-1 can degrade serum cholesterol in high fat diet SD rats.2.Affection target of Lactobacillus fermenti M7-5 and Lactobacillus fermenti M2-1 possible related with hepatic tissue and receptors in ileal mucous. the fronter depressed FXR expression of liver and up-regulated expression of LXRα、ABCA1 and ABCG5 in mucous tissue in liver and ileal which leads to the enhancing of cholesterol excretion. the later obviously depressed FXR expression of liver and up-regulated expression of LXRa in liver and ABCG5 in ileal mucous membrane which enhance the synthesis of bile acid and cholesterol excretion to down-regulate cholesterol更多还原