干酪乳杆菌F0822调控大鼠胆固醇代谢的关键因子及途径分析

心脑血管疾病已成为中国居民的首要死因，而高胆固醇血症则是引发该疾病的重要因素之一。本项目在我们前期工作观察到干酪乳杆菌F0822能有效降低大鼠血清胆固醇水平并基于体外研究推测其胆盐水解酶和S层蛋白可能与其功能性密切相关的基础上，采用同原序列双交换技术分别构建胆盐水解酶基因、S-层蛋白基因以及两个基因均敲除的突变菌株，将野生菌株和基因敲除菌株分别灌胃高胆固醇进食大鼠，通过考察不同菌株在降胆固醇能力上的差异，揭示胆盐水解酶和S-层蛋白在干酪乳杆菌F0822调控大鼠胆固醇代谢过程中发挥的作用；基于生化和分子水平采用液相色谱-电喷雾串联质谱、酶比色、酶联免疫、实时荧光定量反转录PCR和蛋白印迹等手段对大鼠血清、肝脏、肠道和粪便相关指标进行检测，通过考察干酪乳杆菌F0822对大鼠胆汁酸的肠道吸收、肝脏合成及粪便排泄环节的影响，系统的阐明其调控大鼠胆固醇代谢的具体途径。

Cardio-cerebrovascular disease has become the leading cause of death in China, and hypercholesterolemia is one of important factors resulting in such desease. In our previous work, Lactobacillus casei F0822 isolated from human feces showed significantly greater hypocholesterolemic activity and we speculated that its bile salt hydrolase and S-layer protein involved with the hypocholesterolemic activity based on in vitro experiments. In the present study, we will construct bile salt hydrolase gene, S-layer protein gene and both gene knockout mutant strains by double-crossover homologous recombination. Rats fed cholesterol-enriched diets will be intragastricly administrated with the wild and mutant strains respectively, and role of bile salt hydrolase and S-layer protein of Lactobacillus casei F0822 in regulation of cholesterol metabolism in rats will be analyze by comparing difference between the wild and mutant strains in cholesterol-lowering ability. Hypocholesterolemic pathways of Lactobacillus casei F0822 in rats will be explain systematically through investigating its influences on intestinal absorption, hepatic synthesis and fecal excretion process of bile acids by using biological chemistry and molecular biology technologies such as liquid chromatography electrospray ionisation tandem mass spectrometry, enzymatic colorimetry, enzyme-linked immunosorbent assay, real time quantitative reverse transcription PCR and western blot to analyze corresponding parameters in serum, liver, fecal and intestinal samples.