【作者】 邱冬妮；

【导师】 孙大裕； 钟良； 孙旭； 须国融；

【作者基本信息】 复旦大学 ， 内科学， 2008， 博士

【摘要】 第一部分高胆固醇摄入对胆汁酸经典合成途径中基因调节的影响[目的]检测短期(2天)和较长期(2周)高胆固醇摄入新西兰白兔血清胆固醇、胆汁酸水平及肝脏胆汁酸经典合成途径限速酶CYP7A1、核受体FXR和LXR的激活状态,揭示短期和较长期高胆固醇摄入对胆汁酸合成的不同影响,探讨核受体不同的激活状态对胆汁酸合成不同的基因调节机制。[方法]将36只新西兰兔随机分为4组:2天对照组、2天胆固醇组、2周对照组和2周胆固醇组,每组各9只。其中对照组分别喂饲普通兔粮2天及2周,高胆固醇组分别喂饲含2%胆固醇兔粮2天及2周,喂饲结束后测定血清总胆固醇、低密度脂蛋白和胆汁酸水平并测定肝脏CYP7A1活性及CYP7A1 mRNA、FXR的靶基因SHP mRNA、BSEP mRNA和LXR的靶基因ABCA1 mRNA、CETPmRNA。[结果]2天对照组和2周对照组各项指标无差异(P＞0.05)。高胆固醇组的血清总胆固醇、低密度脂蛋白水平均较对照组升高,且2周高胆固醇组高于2天高胆固醇组(P＜0.05);2天高胆固醇组血清胆汁酸水平与对照组相比无变化(P＞0.05),而2周高胆固醇组血清胆汁酸水平较对照组升高(P＜0.05);2天高胆固醇组肝脏CYP7A1活性及CYP7A1mRNA均较对照组升高(P＜0.05),而2周高胆固醇组肝脏CYP7A1活性及CYP7A1mRNA均较对照组降低(P＜0.05);2天高胆固醇组肝脏SHPmRNA、BSEP mRNA与对照组相比无变化(P＞0.05),而2周高胆固醇组肝脏SHPmRNA、BSEP mRNA均较对照组升高(P＜0.05);高胆固醇组肝脏ABCA1mRNA、CETP mRNA均较对照组升高,且2周高胆固醇组高于2天高胆固醇组(P＜0.05)。[结论]高胆固醇摄入使血清总胆固醇、低密度脂蛋白水平升高。短期高胆固醇摄入可激活肝脏LXR受体继而使CYP7A1活性增加,胆汁酸合成增加;此时体内胆汁酸水平尚未升高故肝脏FXR受体活性不变。当较长期的高胆固醇摄入后,体内胆汁酸水平升高从而激活FXR受体;尽管LXR仍被进一步激活,但FXR对CYP7A1的抑制作用强于LXR对CYP7A1激活作用,CYP7A1活性下降,胆汁酸合成减少。第二部分考来烯胺对胆汁酸经典合成途径的基因调节及其降低血清胆固醇的机制[目的]研究考来烯胺对胆汁酸经典合成途径的基因调节及其降低血清胆固醇的作用机制。[方法]20只新西兰兔随机分为考来烯胺组和对照组,每组10只。考来烯胺组每天予考来烯胺混悬液20ml灌胃共2周;对照组每天予20ml蒸馏水灌胃共2周。实验结束后抽血测定血清总胆固醇、低密度脂蛋白和胆汁酸水平并测定肝脏CYP7A1活性及CYP7A1 mRNA、SHP mRNA、BSEP mRNA和LDL-R mRNA。[结果]考来烯胺组血清总胆固醇水平和低密度脂蛋白水平较对照组下降(P＜0.05);考来烯胺组血清胆汁酸水平与对照组差异无统计学意义(P＞0.05);考来烯胺组CYP7A1活性及mRNA较对照组升高(P＜0.05);考来烯胺组SHPmRNA和BSEP mRNA均较对照组下降(P＜0.05);考来烯胺组LDL mRNA较对照组升高(P＜0.05)。[结论]考来烯胺使回流入肝的胆汁酸减少,FXR失活从而对CYP7A1的抑制减弱,CYP7A1活性增高,胆汁酸经典途径合成增加,弥补经肠道丢失的胆汁酸并消耗了肝内的LDL,肝脏摄取循环LDL增加,使血清LDL下降。第三部分考来烯胺对高胆固醇摄入新西兰兔胆汁酸-胆固醇代谢的影响[目的]研究考来烯胺对长期(4周)高胆固醇摄入新西兰兔经典途径胆汁酸合成及胆固醇代谢的影响。[方法]将30只新西兰兔随机分为对照组、高胆固醇血症模型组和考来烯胺组,每组10只。对照组给予普通兔粮,每天予20ml蒸馏水灌胃4周;模型组给予高胆固醇兔粮,每天予20ml蒸馏水灌胃4周;考来烯胺组给予高胆固醇兔粮,每天予考来烯胺混悬液20ml灌胃4周。4周后抽血测定血清总胆固醇、低密度脂蛋白、胆汁酸水平并测定肝脏HMGCoA还原酶活性、CYP7A1活性及CYP7A1mRNA、SHP mRNA、BSEP mRNA和LDL-R mRNA。[结果]模型组、考来烯胺组血清总胆固醇及低密度脂蛋白均较对照组上升(P＜0.01);考来烯胺组血清总胆固醇及低密度脂蛋白均较模型组下降(P＜0.01)。模型组、考来烯胺组血清胆汁酸水平较对照组上升(P＜0.01),模型组和考来烯胺组血清胆汁酸水平差异无统计学意义(P＜0.01)。模型组CYP7A1活性及mRNA较对照组降低(P＜0.01);考来烯胺组CYP7A1活性及mRNA组较模型组升高(P＜0.01)。模型组HMGCoA还原酶活性较对照组降低(P＜0.01);考来烯胺组HMGCoA还原酶活性较模型组升高(P＜0.01)但与对照组差异无统计学意义(P＞0.05)。模型组肝脏SHPmRNA和BSEPmRNA均较对照组升高(P＜0.01);考来烯胺组肝脏SHP mRNA和BSEP mRNA均较对照组升高(P＜0.01)且较模型组下降(P＜0.01)。模型组LDL mRNA较对照组下降(P＜0.01);考来烯胺组LDL mRNA较对照组和模型组升高(P＜0.01)。[结论]长期高胆固醇摄入新西兰兔肝脏FXR被激活,CYP7A1活性及其mRNA、HMGCoA还原酶活性、LDL mRNA下降,血清胆固醇、低密度脂蛋白和胆汁酸升高;同时应用考来烯胺可降低FXR活性,CYP7A1活性及其mRNA、HMGCoA还原酶活性、LDL mRNA升高,从而降低血清胆固醇、低密度脂蛋白而胆汁酸水平不变。更多还原

【Abstract】 PartⅠThe effect of high cholesterol intake on gene regulation of bile acids classic synthesis pathwayObjective To detect the different effect of short term(2 days) and relative long term(2 weeks) high cholesterol intake on serum cholesterol,bile acid,CYP7A1 and the activative status of FXR and LXR on New Zealand white rabbit.Methods Thirty-six New Zealand white rabbits were divided into four groups randomizely:2 days control group,2 days cholesterol group,2 weeks control group,2 weeks cholesterol group.Each group contain 9 rabbits.The control groups were fed regular forage for 2 days or 2 weeks.The cholesterol groups were fed 2%cholesterol forage.Serum cholesterol,LDL,bile acid and hepatic CYP7A1 activity,CYP7A1 mRNA,FXR target gene SHP mRNA and BSEP mRNA,LXR target gene ABCA1 mRNA and CETP mRNA were measured.Results There’s no statistically difference between two control groups (P＞0.05).Total cholesterol and LDL were higher in cholesterol groups compared with those in controls,and 2 weeks were higher than those in 2 days(P＜0.05 ).There’s no statistically difference in bile acid between 2 days cholesterol group and control (P＞0.05).Bile acid in 2 weeks group was higher than that in control(P＜0.05). Hepatic CYP7A1 activity and mRNA in 2 day group elevated compared with control group(P＜0.05);2 weeks group were decreased compared with those in control group(P＜0.05).There’s no statistically difference in SHPmRNA,BSEP mRNA between 2 days cholesterol group and control(P＞0.05 ).SHPmRNA,BSEP mRNA were elevated compared with those in control(P＜0.05).ABCA1mRNA,CETP mRNA were elevated in cholesterol groups compared with those in controls(P＜0.05 ) and were higher in 2 weeks cholesterol group than those in 2 days cholesterol group (P＜0.05).Conclusions High cholesterol intake elevated serum total cholesterol and LDL level. Short term cholesterol intake activated hepatic LXR and CYP7A1,then bile acid synthesis increased.As bile acid level was not yet elevated,the activity of FXR remained unchanged.After relative long term cholesterol intake,bile acid level elevated and FXR was activated.Though LXR was further activated,the inhibition effect of FXR on CYP7A1 override the activation effect of LXR,CYP7A1 activity decreased and bile acid systhesis decreased.PartⅡThe gene regulation of cholestyramine on bile acid classic synthesis pathway and its mechanisms of lowering serum cholesterolObjective To investigate gene regulation mechanisms of cholestyramine on bile acid classic synthesis pathway and its mechanisms of lowering serum cholesterol.Methods Twenty New Zealand white rabbits were randomizely divided into cholestyramine group and control group,10 rabbits each group.The cholestyramine group was administrated 20ml cholestyramine suspension by gavage for 2 weeks.The control group was administrated 20ml distilled water by gavage for 2 weeks.Serum total cholesterol,LDL,bile acid and hepatic CYP7A1 activity,CYP7A1 mRNA,SHP mRNA and LDL mRNA were measured.Results Total cholesterol and LDL were decreased in cholestyramine group compared with those in control group(P＜0.05).There’s no statistically difference in bile acid between two groups(P＞0.05).CYP7A1 activity and mRNA were elevated in cholestyramine group compared with those in control group(P＜0.05).SHP mRNA and BSEP mRNA were declined in cholestyramine group compared with those in control group(P＜0.05 ).LDL mRNA was elevated in cholestyramine group compared with that in control group(P＜0.05).Conclusions Cholestyramine disturbed the reflux of bile acid from intestinal to liver, which inactivated FXR and the inhibition effect of FXR on CYP7A1 was weakened. The activity of CYP7A1 elevated and the bile acid classic synthesis increased,which compensated for the loss of bile acid from intestine and consumpted hepatic LDL.As a result,the liver uptook more LDL from circulation so that serum LDL was declined.PartⅢThe effect of cholestyramine on bile acid and cholesterol metabolism in cholesterol fed New Zealand white rabbit Objective To investigate the effect of cholestyramine on bile acid classic synthesis pathway and metabolism of cholesterol in cholesterol fed New Zealand white rabbits.Methods Thirty New Zealand white rabbits were randomizely divided into 3 groups: control group,model group and cholestyramine group,10 rabbits each group.The control group was fed regular forage and administrated 20ml distilled water by garage for 4 weeks.The model group was fed 1%cholesterol forage and administrated 20ml distilled water by gavage for 4 weeks.The cholesyramine group was fed 1% cholesterol forage and administrated 20ml cholestyramine suspension by gavage for 4 weeks.Serum total cholesterol,LDL,bile acid and hepatic HMGCoA reductase activity,CYP7A1 activity,CYP7A1 mRNA,SHP mRNA,BSEP mRNA and LDL mRNA were measured.Results Total cholesterol and LDL level elevated in model group and cholestyramine group compared with those in control group(P＜0.01).Total cholesterol and LDL decreased in cholestyramine group compared with those in model group(P＜0.01). Bile acid level elevated in model group and cholestyramine group compared with that in control group(P＜0.01).There’s no statistically difference in bile acid between cholestyramine group and model group(P＞0.05).CYP7A1 activity and mRNA decreased in model group compared with those in control group(P＜0.01).CYP7A1 activity and mRNA elevated in cholestyramine group compared with those in model group(P＜0.01 ).HMGCoA reductase activity decreased in model group compared with that in control group(P＜0.01) HMGCoA reductase activity elevated in cholestyramine group compared with that in model group(P＜0.01),but there’s no statistically difference in HMGCoA reductase activity between cholestyramine group and model group(P＞0.05).SHP mRNA and BSEP mRNA were elevated in model group and cholestyramine group compared with those in control(P＜0.01 ).SHP mRNA and BSEP mRNA decreased in cholestyramine group compared with those in model group(P＜0.01).LDL mRNA was decreased in model group compared with that in control group(P＜0.01 ).LDL mRNA was elevated in cholestyramine group compared with that in control group and model group(P＜0.01 ).Conclusions Long term cholesterol intake activated hepatic FXR,so that CYP7A1 activity and mRNA decreased;HMGCoA reductase activity and LDL mRNA decreased;serum cholesterol,LDL and bile acid level elevated.Cholestyramine intervention lowered FXR activity,CYP7A1 activity and mRNA elevated,HMGCoA reductase activity and LDL mRNA increased,then serum cholesterol,LDL level decreased while bile acid level remained unchanged.更多还原