The Characteristics Of Cholesterol Metabolism And Its Mechanism In Mfat-1Mice

ObjectiveTo observe the change of serum total cholesterol and efficiency of the synthesisand absorption of cholesterol in the mfat-1mice,which carrying the gene of ω-3fattyacid desaturase (fat-1) gene that has a proper ratio of ω-3/ω-6polyunsaturated fattyacids after high fat diet, and to explore the relative mechanism.Methods1.Let the mfat-1mice which given by professor Zhao Zijian and professor Dai Yifanfrom Nanjing Medical University,Interbreed with C57BL/6mice to get experimentalsubjects. identified the gene of fat-1in offspring expressed by semi-quantitative PCR.2. Select12mfat-1and12C57BL/6male mice with8-weeks-old randomly as theexperimental group and control group separately,giving high-fat diet after two weeksadaptability fed,take blood from the heart and clip the liver four weeks later,bothsaved in liquid nitrogen.3.Analyzed the serum by Gas Chromatography， quantitative detect the serumcholesterol levels with a single point external standard,cholesterol synthesis markers(squalene,desmosterol),absorption markers(campesterol, β-sitosterol,stigmasterol)with a single point internal standard.4.Extract the lipids through Chloroform-methanol from the mouse liver，analyzedthe percentage of eicosapentaenoic acid（EPA）、docosahexenoic acid（DHA）、 docosapentaenoic acid(DPA）、α-linolenic acid（ALA）、linoleic acid（LA）、arachidonic acid（AA）by Gas Chromatography ofter esterified.5.Analyse the expression levels of3-Hydroxy-3-methylglutaryl-coenzyme Areductase（HMG-COAR)，which is rate-limiting synthase of cholesterol synthesis inmouse liver，by ELISA.Result1.To identify the gene of ω-3fatty acid desaturase (fat-1) in the offspring mice wassuccessfully expressed by semi-quantitative PCR.2.The percentage of total ω-3polyunsaturated fatty acids in the liver of experimentalmice was8.06±0.76,andω-6PUFAs was13.71±0.98,and its ratio of totalω-6/ω-3PUFAs was significantly lower than control group (1.70vs.6.04p <0.01).3.The levels of serum total cholesterol in the experimental group are significantlylower than those in the control group（0.91±0.07vs.1.16±0.85,p＜0.01,mg/mL）. Thelevels of serum cholesterol synthesis markers (squalene,desmosterol) in theexperimental group are lower than those in the control group(0.63±0.17vs.1.24±0.36，3.89±0.54vs.6.57±1.06,p<0.05,ug/mL),and absorption markers(campesterol，β-sitosterol, stigmasterol)are lower than the control group too (16.10±2.28to21.24±2.96,1.07±0.18to1.76±0.43,28.78±5.60to42.30±5.81, p＜0.01,ug/mL).4.The levels of3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-COAR) inliver in the experimental group are significantly lower than those in the controlgroup(379.76±22.37to443.98±27.99, P <0.01,U/L).Conclusion:1.The gene of ω-3fatty acids desaturase,which can make the ratio of ω-6/ω-3polyunsaturated fatty acids tend to balance,is successfully expressed in the mfat-1 mice.2.The levels of plasma total cholesterol in the experimental group are significantlylower than those in the control group,which through inhibit the cholesterol synthesisand absorption to reduce the total cholesterol levels.3.One of the way to inhibit cholesterol synthesis in mfat-1mice is inhibitable3-Hydroxy-3-methylglutaryl-coenzyme A reductase expression.