脂代谢相关三基因突变小鼠模型动脉粥样硬化机理研究—蛋白质组学分析

【作者】 金晓蕾；

【导师】 潘杰；

【作者基本信息】 浙江大学 ， 生化与分子生物学， 2005， 硕士

【摘要】 动脉粥样硬化（atherosclerosis,AS）是由多种因素和作用环节介导的慢性病理变化过程,其发生发展与多基因异常以及遗传因素与环境因素的相互作用有关。深入研究AS关键基因、AS易感基因等重要遗传因素之间的相互作用机理以及环境因素对AS的影响,有助于进一步阐明AS的发生机制,为AS的预防和治疗提供有益的理论依据与研究平台。 目前对AS发病机制的研究重点已从单基因、单因素向多基因、多因素方向发展。本实验室首次成功建立了三基因突变(apoE⁺/LDLR⁺/Lepr^db/db小鼠模型,并从遗传因素和环境因素（高脂高胆固醇饮食）入手,对该小鼠的生物学特征、发生AS的病理生理特点以及AS发生机制进行了分子生物学和分子病理学研究。在前期研究结果的基础上,本论文进一步研究了三基因突变时基因与基因以及基因与环境间的相互作用对血脂、血糖及血浆中主要脂蛋白成分含量的变化、脂代谢重要器官肝脏的病理形态学改变的影响,探讨了肝脏中重要差异蛋白表达的变化规律,并且分析了上述变化与AS发生发展的关系,现得出以下结果: 1.血浆TC、TG、LDL—C水平的变化:5w龄普通饮食组ALO小鼠的TC水平为21.26+4.38.mM,略高于其他基因型小鼠;高脂高胆固醇饮食组ALO小鼠的。TC水平为110.51士12.99 mM,显著高于其他基因型小鼠。7w龄起,两种饮食喂养的ALO小鼠的TC水平均显著高于其他基因型小鼠。两种饮食喂养的ALO小鼠各年龄段的TG水平都略高于其他基因突变小鼠。ALO小鼠的LDL-C水平与TC的变化相似,且差异显著性更明显。血浆GLU水平的变化:两种饮食喂养的DB、ALO小鼠与WT小鼠相比有明显增加,A、L、AL小鼠与WT小鼠相比无差异。饮食性因素对小鼠GLU水平的影响不明显。血浆HDL-C水平的变化:普通饮食喂养时,DB、ALO小鼠的HDL-C水平与WT小鼠相比有明显增加,A、L、AL小鼠与WT小鼠相比无差异:高脂高胆固醇饮食喂养时,A、L、AL、DB、ALO小鼠的HDI-C水平都较WT小鼠有明显升高,ALO小鼠升高得尤为明显。 高脂高胆固醇饮食促进了高脂血症和AS的发生。各年龄时期高脂高胆固醇饮食组的ALO小鼠各项血浆生化检测指标均高于普通饮食组。且高脂高胆固醇饮食组的TC、HDL-C、LDL-C水平均在5w龄时达到最高,TG水平保持上升趋势,GLU水平略有升高,但不显著。更多还原

【Abstract】 Atherosclerosis is a kind of complex multifactorial disease. Many complicated factors interaction and interrelated biological processes contribute to atherogenesis. Study of its mechanisms can provide available preventive measures and clinical therapy strategies. At present study, apoE^-/-/LDLR^-/-/Lepr^db/db treble-gene mutant mice were established and used for study the mechanisms of polygenetic disfunction in dyslipidemia as well as atherogenesis. The relationship between treble-genetic mutation and hyperlipidemia/ hyperlipoprotein, following atherosclerotic pathological changes in the mutant livers were investigated. The influence of high fat/cholesterol diet induction on the development of hyperlipidemia and atherosclerosis was studied. I also discussed the differential expression of protein profiles in the livers of young treble-gene mutants and double-gene mutants (apoE^-/-/LDLR^-/-) compared with that of wild type mice to find the key proteins related to atherosclerosis and disfunction of lipid metabolism.In the first experiment the results showed that with normal chow and high fat/cholesterol diet, plasma TC, TG, LDL-C levels of all mutant mice significantly elevated compared with that of wild type mice at all stages of age, except that of DB mice had only slight elevations. Plasma TC level of 5-week-old treble-gene mutants was 21.26±4.38 mM, only slightly higher than that of double-gene mutants, while after fed with high fat/cholesterol diet, it was 110.51±12.99 mM, dramatically elevated than that of any other mice. After 7w of age, TC levels of treble-gene mutants were both significantly higher than that of the other mutants at the condition of both normal chow and high fat /cholesterol diet. The TG level of treble-gene mutants increased with age and was a little higher compared with that of other mutant mice when fed with two diets. Plasma LDL-C level almost had the same trend but more significant change when compared with TC level. Plasma GLU levels of treble-gene mutants and DB mice elevated compared with that of the other type mice at all stages of age fed with two diets. A, L, AL mice had no difference with wild type mice. When fed with normal chow plasma HDL-C levels of treble-gene mutants and DB mice were higher than the other mice. HDL-C levels of single-gene, double-gene mutants had no significant difference with wild type mice. But after fed with high fat/cholesterol diet theHDL-C levels of all mutant mice were higher than that of wild type mice.High fat/cholesterol diet promoted the development of hyperlipidemia/ hyperlipoprotein. Concentrations of TC and LDL-C of treble-gene mutants persistently elevated with age when fed normal chow diet, while when fed high fat/cholesterol diet the levels went up sharply at 5w of age and then slightly went down later till 15w of age. TG levels of treble-gene mutants steadily increased with age fed with both diets. GLU levels of all mutant mice were not significantly influenced by the different diets. HDL-C levels of all mutants elevated after fed with high fat /cholesterol diet and that of treble-gene mutants had the most significant change.In the second experiment treble-gene mutant mice and DB mice appeared a sharp increase in body weight at 3 weeks old fed with both two diets. And the weight increased smoothly with the age after 7w of age. The high fat/cholesterol diet affected this alteration slightly. The other mutants and wild type mice had a stable increasing process of body weight and the diet had no effect on their weight alteration.Treble-gene mutant mice had atherogenetic lesions accompany with fatty changes of hepatic cells and the accretion of cells volume even at 5w of age, and appeared more severe lesions later. Till 15w of age the extent of lipid accumulation in hepatic cells of treble-genetic mutants was more significant than that of the other mutants. DB mice and other mutants all had different degrees of liver pathological alteration. High fat /cholesterol diet accelerated the process of liver fatty change.In the third proteomic experiment two-dimensional gel electrophoresis and mass spectrometry were used for analyse differential displayed proteomics of 5-week-old treble-gene mutants, double-gene mutants and wild type mice after fed a regular chow for 2 weeks. Approximately 841±57, 928±15 and 1017±50 spots were detected in these three kinds of mouse livers （n=3）. When one of the wild type mouse liver gels acted as the reference gel, the average matched spots of wild type mice, double-gene mutants, treble-gene mutants were 846±22 （n=2）, 773±16 （n=3） and 706±29 （n=3） and the average matched ratio were 83.2%, 78.7% and 71.9%, respectively. Compared with wild type mice, 108 spots were not matched in that of apoE^VLDLR"’’ mice and 140 spots were not matched in that of apoE"/7LDLR’/7Leprdb/db mice. Ten over expression spots （>5 fold） and 45 lowerexpression spots （>5 fold） were noted in the former and 7 over expression spots （>5 fold） and 39 lower expression spots （>5 fold） were noted in the latter. Six significantly differentially expressed proteins in gels were identified by LTQ-ESI, including endoplasmin precursor, acidic leucin-rich nuclear phosphoprotein 32 family member A, serotransferrin precursor, elongation factor-2 homolog, fructose bisphosphatase 1, fibronectin precursor, complement C3 precursor, Complement C4 precursor, fibrinogen B beta polypeptide, fibrinogen, gamma polypeptide, which were related to lipid and carbohydrate metabolism, aortic intima lesions, immune reactions and inflammation. The protein profiles of multiple-gene mutant livers exhibited obviously difference compared with that of wild type mice. The results implied that lipid metabolism relative polygenetie mutation contributed to the alteration of protein expression profiles, especially which lipid metabolism related and participated in dysfunction in lipid metabolism during atherogenesis.Take all results together, which suggested that treble-genetic mutation, especially the lipid metabolism related genes dysfunction contributed to hyperlipidemia/ hyperlipoprotein and atherogenetic lesions. Atherogenic diet stimulation could promote hyperlipidemia, aggravated pathological changes of liver and even developed atherosclerosis in the treble-gene mutants.更多还原