【作者】 曾德星；

【导师】 易光辉；

【作者基本信息】 南华大学 ， 病理学与病理生理学， 2007， 硕士

【摘要】 大量的流行病学调查及基础研究、临床观察均已显示血浆高密度脂蛋白胆固醇(High density lipoprotein-cholesterol,HDL-C)水平与动脉粥样硬化的发病率呈负相关。B类I型清道夫受体(Scavenger receptor class B type I, SR-BI)是第一个在分子水平上得到证实的高密度脂蛋白(High density lipoprotein, HDL)受体,其主要功能是介导高密度脂蛋白胆固醇酯(High density lipoprotein-cholesterol ester,HDL-CE)的选择性摄取和细胞内未酯化胆固醇的流出。载脂蛋白E(Apolipoprotein E,apoE)是富含三酰甘油脂蛋白的蛋白组分之一,作为乳糜微粒(Chylomicron,CM)残粒受体和低密度脂蛋白(Low density lipoprotein,LDL)受体的配体在脂蛋白残粒的代谢中起重要作用。尽管当前已有大量关于SR-BI和apoE的研究报道,但对于apoE和SR-BI这两个脂质代谢关键基因之间的关联关系目前并不完全清楚。为此,本实验检测了ApoE-/-小鼠腹膜巨噬细胞SR-BI表达及其介导的胆固醇转运的变化,为进一步阐明apoE和SR-BI之间的关联关系提供依据。第一部分:ApoE-/-小鼠腹膜巨噬细胞SR-BI表达及胆固醇转运的变化目的:探讨ApoE-/-小鼠腹膜巨噬细胞SR-BI表达及胆固醇转运的变化。方法:以ApoE-/-小鼠腹膜巨噬细胞为实验组,具有相同遗传背景的C57BL/6J小鼠腹膜巨噬细胞为对照组, RT-PCR、Western blotting检测SR-BI mRNA转录和蛋白表达水平变化,高效液相色谱检测细胞内脂质蓄积情况,液体闪烁计数法检测胆固醇流出率。结果:RT-PCR、Western blotting检测ApoE-/-小鼠和C57BL/6J小鼠腹膜巨噬细胞SR-BI mRNA转录和蛋白表达水平变化,结果显示,与对照组相比,ApoE-/-小鼠腹膜巨噬细胞SR-BI mRNA转录及蛋白表达下调(P<0.05);高效液相色谱检测表明,ApoE-/-小鼠腹膜巨噬细胞的总胆固醇含量、游离胆固醇含量、胆固醇酯含量以及所占比例(分别为116.9±9.3mg/g蛋白、84.2±4.1 mg/g蛋白、32.7±3.7%mg/g蛋白、28.0±2.5%)明显高于对照组(分别为75.3±6.8mg/g蛋白、67.8±3.6 mg/g蛋白、7.5±1.8%mg/g蛋白、9.9±1.2%),差异具有显著性意义(P<0.05);液体闪烁计数法检测也发现,ApoE-/-小鼠腹膜巨噬细胞胆固醇流出率(35.18±4.85%)明显低于对照组(56.39±6.63%),差异具有显著性意义(P<0.05)。小结:1、ApoE-/-小鼠腹膜巨噬细胞SR-BI表达下调。2、ApoE-/-小鼠腹膜巨噬细胞总胆固醇和胆固醇酯含量增高,胆固醇酯所占比例上升。3、ApoE-/-小鼠腹膜巨噬细胞的胆固醇流出率下降。第二部分:脂质转运阻断剂BLT4对小鼠腹膜巨噬细胞SR-BI表达及其介导的胆固醇转运的影响目的:观察脂质转运阻断剂BLT4对小鼠腹膜巨噬细胞SR-BI表达及其介导的胆固醇转运的影响。方法:以ApoE-/-小鼠腹膜巨噬细胞+DMSO溶媒对照或+100μmol/L BLT4为I组、II组,具有相同遗传背景的C57BL/6J小鼠腹膜巨噬细胞+DMSO溶媒对照或+100μmol/L BLT4为III组、IV组。RT-PCR、Western blotting检测SR-BI mRNA转录和蛋白表达水平变化,高效液相色谱检测细胞内脂质蓄积情况,液体闪烁计数法检测胆固醇流出率。结果RT-PCR、Western blotting检测ApoE-/-小鼠和C57BL/6J小鼠腹膜巨噬细胞实验组和对照组SR-BI mRNA转录和蛋白表达水平变化,结果显示,分别与各自的对照组相比,ApoE-/-小鼠和C57BL/6J小鼠腹膜巨噬细胞BLT4组SR-BI mRNA转录和蛋白表达无明显变化;高效液相色谱检测表明,ApoE-/-小鼠和C57BL/6J小鼠腹膜巨噬细胞BLT4组的总胆固醇含量、游离胆固醇含量、胆固醇酯含量以及所占比例(分别为173±10.4mg/g蛋白、110.6±9.8 mg/g蛋白、62.4±6.7%mg/g蛋白、36.0±3.8%与100.5±7.8mg/g蛋白、75.4±4.1 mg/g蛋白、25.1±3.2%mg/g蛋白、25.0±2.2%)均明显高于各自的对照组(分别为123.2±10.3mg/g蛋白、89.8±3.9 mg/g蛋白、33.4±3.5%mg/g蛋白、27.3±2.5%与77.6±5.2mg/g蛋白、69.1±3.6 mg/g蛋白、8.5±1.7%mg/g蛋白、10.9±1.4%),差异具有显著性意义(P<0.05);液体闪烁计数法也发现,ApoE-/-小鼠和C57BL/6J小鼠腹膜巨噬细胞BLT4组的胆固醇流出率(分别为25.38±4.12%与32.66±4.97%)明显低于各自的对照组(37.45±5.12%与54.39±7.64%),差异具有显著性意义(P<0.05)。小结:SR-BI脂质转运功能受BLT4抑制后小鼠腹膜巨噬细胞胆固醇流出率下降,总胆固醇和胆固醇酯蓄积增多。更多还原

【Abstract】 Lots of epidemiological investigations, basic researches and clinical observations have manifested that high density lipoprotein-cholesterol is inverse correlated to the incidence of atherosclerosis. Scavenger receptor class B type I (SR-BI) was the first molecularly well-defined cell surface receptor for high density lipoprotein (HDL). The primary function of SR-BI is to mediate the selective intake of high density lipoprotein-cholesterol ester (HDL-CE) and the effusion of cellular unesterified cholesterol. Apolipoprotein E (ApoE) is one of the protein components of triglyceride-rich lipoprotein, plays an important part in the metabolism of lipoprotein remnant as the ligand of the receptors for the chylomicron remnants and low density lipoprotein. Although many researches related to SR-BI and apoE have been reported, the relation of the genes is not demonstrated completely. In these studies, we compared the differences of cholesterol trafficking and SR-BI expression in peritoneal macrophages between ApoE-/- and C57BL/6J mice. To illustrate the role of SR-BI in cholesterol trafficking, we subsequently explored the effects of block lipid transport 4 (BLT4), a relatively specific blocker to SR-BI, on cellular cholesterol transport in macrophages derived from ApoE-/- and C57BL/6J mice.Part I: Comparisons of cholesterol trafficking and SR-BI expression in peritoneal macrophages from ApoE-/- mice with that from C57BL/6J miceAIM: To study differences of SR-BI expression and cholesterol transport in peritoneal macrophages derived from ApoE-/- and C57BL/6J mice. METHODS: The peritoneal macrophages were prepared from ApoE-/- mice and its genetic background mice, C57BL/6J mice, respectively. RT-PCR, Western blotting were applied to detect the SR-BI mRNA and protein expression levels, respectively. Cellular lipid accumulation was determined and analyzed by high performance liquid chromatography. Cholesterol efflux rate was quantified by liquid scintillator.RESULTS: The results of RT-PCR and Western blotting showed that SR-BI mRNA transcription and protein expression level in peritoneal macrophages derived from ApoE-/- mice were decreased significantly, compared to that of C57BL/6J mice. High performance liquid chromatography analysis demonstrated that the cellular total cholesterol (TC), cholesterl ester (CE) and CE/TC ratio were increased in macrophages from ApoE-/- mice. Liquid scintillator evaluation demonstrated that the cholesterol efflux was lowered in macrophages from ApoE-/- mice.CONCLUSION: SR-BI expresstion of peritoneal macrophages from ApoE-/- mice is downregulated, which may be involved in the increase of cellular cholesterol accumulation and the decrease of cellular cholesterol efflux.Part II: The effect of BLT4 on cholesterol transport mediated by SR-BI in mouse peritoneal macrophagesAIM: To study the effect of BLT4 on cholesterol transport mediated by SR-BI in mouse peritoneal macrophages.METHODS: The peritoneal macrophages from ApoE-/- and C57 BL/6J mice were treated with 100umol/L BLT4 and without BLT4 (only solvent DMSO), respectively. RT-PCR, Western blotting were applied to detect the SR-BI mRNA and protein expression levels, respectively. Cellular lipid accumulation was determined and analyzed by high performance liquid chromatography. Cholesterol efflux rate was quantified by liquid scintillator. RESULTS: The results showed that no significant decrease of SR-BI expression level was observed in macrophages treated with BLT4. But, the accumulation of cholesterol in macrophges treated with BLT4 was illustrated by analysis of high performance liquid chromatography, and the decrease of cellular cholesterol efflux in macrophages treated with BLT4 was significant.CONCLUSIONS: SR-BI mediates the cellular cholesterol trafficking in peritoneal macrophages derived from both ApoE-/- mice and C57 BL/6J mice.更多还原