人突变载脂蛋白E(apoE4,apoE7)转基因小鼠模型

为揭示人突变apoE基因表达在整体引起的异常反应并得到人类相关疾病的小鼠模型,用显微注射法制备了人apoE4与apoE7转基因小鼠,用Southern印迹杂交及ELISA证明人apoE基因在首建鼠及其子代体内整合与表达良好,具有遗传稳定性.血清脂质与行为测定表明,人apoE表达使小鼠患高脂血症并出现大脑学习与记忆功能衰退和寿命缩短,高脂食物对正常鼠与转基鼠均可诱发高血脂,但机制不同.同时还证明,氨基端与羧基端突变的apoE具有相同的致病作用.     

The role of hepatic Surf4 in lipoprotein metabolism and the development of atherosclerosis in apoE−/− mice

Elevated plasma levels of low-density lipoprotein-C (LDL-C) increase the risk of atherosclerotic cardiovascular disease. Circulating LDL is derived from very low-density lipoprotein (VLDL) metabolism and cleared by LDL receptor (LDLR). We have previously demonstrated that cargo receptor Surfeit 4 (Surf4) mediates VLDL secretion. Inhibition of hepatic Surf4 impairs VLDL secretion, significantly reduces plasma LDL-C levels, and markedly mitigates the development of atherosclerosis in LDLR knockout (Ldlr^?/?) mice. Here, we investigated the role of Surf4 in lipoprotein metabolism and the development of atherosclerosis in another commonly used mouse model of atherosclerosis, apolipoprotein E knockout (apoE^?/?) mice. Adeno-associated viral shRNA was used to silence Surf4 expression mainly in the liver of apoE^?/? mice. In apoE^?/? mice fed a regular chow diet, knockdown of Surf4 expression significantly reduced triglyceride secretion and plasma levels of non-HDL cholesterol and triglycerides without causing hepatic lipid accumulation or liver damage. When Surf4 was knocked down in apoE^?/? mice fed the Western-type diet, we observed a significant reduction in plasma levels of non-HDL cholesterol, but not triglycerides. Knockdown of Surf4 did not increase hepatic cholesterol and triglyceride levels or cause liver damage, but significantly diminished atherosclerosis lesions. Therefore, our findings indicate the potential of hepatic Surf4 inhibition as a novel therapeutic strategy to reduce the risk of atherosclerotic cardiovascular disease.     

Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice

The role of farnesoid X receptor (FXR) in the development of atherosclerosis has been unclear. Here, LDL receptor (LDLR^−/−) or apolipoprotein E (apoE^−/−) female or male mice were fed a Western diet and treated with a potent synthetic FXR agonist, WAY-362450. Activation of FXR blocked diet-induced hypertriglyceridemia and elevations of non-HDL cholesterol and produced a near complete inhibition of aortic lesion formation. WAY-362450 also induced small heterodimer partner (SHP) expression and repressed cholesterol 7α-hydroxylase (CYP7A1) and sterol 12 α-hydroxylase (CYP8B1) expression. To determine if SHP was essential for these protective activities, LDLR^−/−SHP^−/− and apoE^−/−SHP^−/− mice were similarly treated with WAY-362450. Surprisingly, a notable sex difference was observed in these mice. In male LDLR^−/−SHP^−/− or apoE^−/−SHP^−/− mice, WAY-362450 still repressed CYP7A1 and CYP8B1 expression by 10-fold and still strongly reduced non-HDL cholesterol levels and aortic lesion area. In contrast, in the female LDLR^−/−SHP^−/− or apoE^−/−SHP^−/− mice, WAY-362450 only slightly repressed CYP7A1 and CYP8B1 expression and did not reduce non-HDL cholesterol or aortic lesion size. WAY-362450 inhibition of hypertriglyceridemia remained intact in LDLR^−/− or apoE^−/− mice lacking SHP of both sexes. These results suggest that activation of FXR protects against atherosclerosis in the mouse, and this protective effect correlates with repression of bile acid synthetic genes, with mechanistic differences between male and female mice.     

重组人apoE基因表达活性和转基因小鼠研究

人ａｐｏＥ基因组ＤＮＡ,去除其自身妄动仓,代之以小鼠金属硫蛋白启动子,重组质粒经脂质体介异转入小鼠ＮＩＨ／３Ｔ３细胞后,以人ａｐｏＥ基因组ＤＮＡ／ＥｃｏＲⅠ片段为探针检测ｍＲＮＡ表达,可见ａｐｏＥ ｍＲＮＡ杂交信号很强,经重金属诱导后杂交信息更强,表明ＭＴ启动子功能良好,ｐＭＥ表达正常。     

重组人apoE基因表达活性和转基因小鼠研究

人apoE基因组DNA,去除其自身启动子,代之以小鼠金属硫蛋白启动子,重组质粒经脂质体介导转入小鼠NIH／3T3细胞后,以人apoE基因组DNA／EcoRⅠ片段为探针检测mRNA表达,可见apoEmRNA杂交信号很强,经重金属诱导后杂交信号更强,表明MT启动子功能良好,pME表达正常．将人apoE基因组DNA用显微注射法导入小鼠受精卵雄性原核,再将胚胎移植入假孕母鼠输卵管内,仔鼠分娩四周后,自鼠尾提取DNA,鉴定人apoEDNA在小鼠染色体上的整合,最终得到有人apoE基因整合的转基因首建鼠．     

马尾松树皮提取物预防apoE基因敲除小鼠肝脏脂肪变性

目的探究马尾松树皮提取物(Pinus massoniana bark extract, PMBE)对载脂蛋白E(ApoE)基因敲除小鼠肝脏脂肪变性的影响。方法给8只ApoE KO雄性小鼠每天口服PMBE(30 mg/kg)2周,然后喂养高胆固醇及高脂饮食8周后,与普通饮食(NC)组及高脂饮食(HCD)组进行对比,取各组小鼠肝脏组织进行油红染色及Real-time PCR试验,检测脂质代谢相关基因的表达。结果经PMBE治疗后的小鼠肝细胞中脂质沉积减少,肝脏脂质含量减低;随后检测脂质代谢相关基因,发现PMBE通过减少脂质合成和增加脂解作用显著抑制了肝脏脂肪的蓄积。结论 PMBE可以预防肝脏脂肪变性的发生和发展。     

The influence of angiotensin-(1–7) Mas receptor agonist (AVE 0991) on mitochondrial proteome in kidneys of apoE knockout mice

Excessive action of angiotensin II on mitochondria has been shown to play an important role in mitochondrial dysfunction, a common feature of atherogenesis and kidney injury. Angiotensin-(1–7)/Mas receptor axis constitutes a countermeasure to the detrimental effects of angiotensin II on AT1 receptors. The aim of the study was to assess the effects of angiotensin-(1–7) peptidomimetic AVE0991 on the kidney mitochondrial proteome in widely used animal model of atherosclerosis (apoE^?/? mice). Proteins changed in apoE^?/? mice belonged to the groups of antioxidant enzymes, apoptosis regulators, inflammatory factors and metabolic enzymes. Importantly, AVE0991 partially reversed atherosclerosis-related changes in apoE^?/? mice.     

脂代谢相关基因在apoE基因缺失幼龄小鼠肝脏中的表达

本文旨在分析脂代谢相关基因在载脂蛋白E(apolipoprotein E,apoE)基因缺失(apoE-/-)幼龄小鼠肝脏中的表达特征及其与血脂紊乱和动脉粥样硬化(atherosclerosis,AS)早期病变的关系.利用半定量RT-PCR和荧光实时定量RT-PCR技术,分析14 d龄、1、2和3月龄apoE-/-小鼠及同龄野生型(wild type,WT)小鼠肝脏脂代谢相关基因的表达,并进行血生化指标及主动脉病理形态学榆测.apo-/-小鼠肝脏中apoAI、apoAIV表达在14d龄时即发生显著变化(P<0.05);在1月龄时apoB10G表达较同龄WT小鼠明显上调(P＜0.05);apoA V表达则在2月龄时较同龄WT小鼠上调(P＜0.05),此时可观察到apoE-/-小鼠主动脉内膜出现AS早期病变;Faf/CD36和Angptl 3表达在3月龄时较同龄WT小鼠上调(P＜0.05);实验中检测的其它基因的mRNA表达与同龄WT小鼠相比无显著性差异.apoE-/-小鼠血清总胆同醇、甘油三酯、低密度脂蛋白胆固醇和高密度脂蛋白胆固醇含量均高于同龄WT小鼠,并随年龄增长而升高.apoE-/-小鼠和同龄WT小鼠血清中apoB100蛋白浓度在14 d龄到3月龄问变化趋势与其在肝脏中mRNA表达及血清中低密度脂蛋白胆崮醇含量变化趋势基本一致.上述部分脂代谢相关基因表达在幼龄小鼠即发生改变,与血脂紊乱以及主动脉AS病变发生发展过程呈正相关,说明其可能在幼龄小鼠脂质代谢紊乱发生过程中起重要作用,从而引起动脉内皮细胞功能改变乃至AS早期病变的发生.     

cDNA微阵列分析人apoE4转基因鼠肾脏基因表达谱的变化

目的研究人apoE4转基因鼠肾脏的基因表达谱变化.方法分别提取人apoE4转基因鼠和正常C57BL/6J小鼠的肾脏总RNA,经逆转录合成cDNA探针后分别与鼠cDNA表达点阵杂交,再用ESTblot软件进行分析,并用Northern印迹证明基因表达的改变.结果人apoE4转基因鼠肾脏中有38个基因的mRNA表达升高,22个基因的mRNA表达降低.其中血浆谷胱甘肽过氧化物酶前体、视黄酸γ受体和白介素5受体等基因的表达明显增加.B-raf原癌基因、促红细胞生成素受体、整联蛋白α4的基因表达显著降低.Northern杂交证明转基因鼠肾脏的c-Jun基因表达升高.结论人apoE4转基因鼠肾脏的c-Jun、血浆谷胱甘肽过氧化物酶前体、白介素5受体等基因的表达增加;促红细胞生成素受体、整联蛋白α4等基因的表达减少.     

A fish oil-rich diet reduces vascular oxidative stress in apoE–/– mice

Abstract

Oxidative stress contributes to lipid peroxidation and decreases nitric oxide (NO) bioavailability in atherosclerosis. While long-chain (n-3) polyunsaturated fatty acids (PUFA) are easily oxidized in vitro, they improve endothelial function. Hence, this study postulates that long-chain (n-3) PUFA decrease atherogenic oxidative stress in vivo. To test this, apoE^–/– mice were fed a corn oil- or a fish oil (FO)-rich diet for 8, 14 or 20 weeks and parameters related to NO and superoxide (O₂^.–) plus markers of lipid peroxidation and protein oxidative damage in the aortic root were evaluated. The FO-rich diet increased NO production and endothelial NO synthase (NOS) expression and lowered inducible NOS, p22^phox expression and O₂^.–production after 14 and 20 weeks of diet. Protein lipoxidative damage (including 4-hydroxynonenal) was decreased after a long-term FO-diet. This supports the hypothesis that a FO-rich diet could counteract atherogenic oxidative stress, showing beneficial effects of long-chain (n-3) PUFA.     

低氧性肺动脉高压小鼠肺组织apoE蛋白表达的变化及其意义

目的：观察低氧性肺动脉高压小鼠肺组织中载脂蛋白E（apoE）蛋白表达的变化,以探讨低氧性肺动脉高压形成过程中apoE蛋白表达的变化及可能的意义。方法：SPF级雄性野生型（WT）C57BL/6小鼠和雄性apoE基因敲除（apoE-KO）小鼠各20只,各随机再分为2组（n=10）：常氧组和低氧组,共4组。常压连续低氧3周（9%~11% O₂,23 h/d）复制慢性低氧性肺动脉高压模型,采用右心导管法测定小鼠右心室压（RVSP）,计算右心室与左心室加室间隔重量比RV/（LV+S）,ELISA法检测血浆中高密度脂蛋白（HDL）、低密度脂蛋白（LDL）和总胆固醇（TC）的含量;Western blot法检测肺组织中apoE和过氧化物酶体增殖物激活受体γ（PPARγ）蛋白的表达。结果：①低氧组WT小鼠RVSP、RV/（LV+S）分别较常氧组高68%和59%（P均<0.05）,血浆中HDL含量及HDL/LDL比值分别较常氧组低17%和40%（P均<0.05）,同时肺、肝组织中apoE及肺组织中PPARγ的蛋白表达分别较常氧组下调48%、52%和37%（P均<0.05）,RVSP与apoE及PPARγ蛋白表达均呈显著负相关（P均<0.01）;②低氧组apoE-KO小鼠RVSP、RV/（LV+S）较常氧组分别高96%和86%（P均<0.05）,低氧组apoE-KO小鼠RVSP和RV/（LV+S）较低氧组WT小鼠分别高29%和24%（P均<0.05）。结论：小鼠低氧性肺动脉高压的形成与肺组织中apoE蛋白表达下调有关。     

Nur77 Decreases Atherosclerosis Progression in apoE?/? Mice Fed a High-Fat/High-Cholesterol Diet

Rationale

It is clear that lipid disorder and inflammation are associated with cardiovascular diseases and underlying atherosclerosis. Nur77 has been shown to be involved in inflammatory response and lipid metabolism.

Objective

Here, we explored the role of Nur77 in atherosclerotic plaque progression in apoE^−/− mice fed a high-fat/high cholesterol diet.

Methods and Results

The Nur77 gene, a nuclear hormone receptor, was highly induced by treatment with Cytosporone B (Csn-B, specific Nur77 agonist), recombinant plasmid over-expressing Nur77 (pcDNA-Nur77), while inhibited by treatment with siRNAs against Nur77 (si-Nur77) in THP-1 macrophage-derived foam cells, HepG2 cells and Caco-2 cells, respectively. In addition, the expression of Nur77 was highly induced by Nur77 agonist Csn-B, lentivirus encoding Nur77 (LV-Nur77), while silenced by lentivirus encoding siRNA against Nur77 (si-Nur77) in apoE^−/− mice fed a high-fat/high cholesterol diet, respectively. We found that increased expression of Nur77 reduced macrophage-derived foam cells formation and hepatic lipid deposition, downregulated gene levels of inflammatory molecules, adhesion molecules and intestinal lipid absorption, and decreases atherosclerotic plaque formation.

Conclusion

These observations provide direct evidence that Nur77 is an important nuclear hormone receptor in regulation of atherosclerotic plaque formation and thus represents a promising target for the treatment of atherosclerosis.     

apoE/LDLR双基因缺失幼龄小鼠主动脉中动脉粥样硬化相关基因的表达

利用RT-PCR以及实时定量RT-PCR检测11个动脉粥样硬化(atherosclerosis,AS)相关基因在1、2和3月龄的载脂蛋白E(apolipoproteinE,aopE)/低密度脂蛋白受体(low-density lipoprotein receptor,LDLR)双基因缺失(apoE-/-/LDLR-/-)小鼠主动脉中的表达变化,同时应用血生化指标和病理形态学观察AS早期病变特点,探讨apoE和LDLR基因联合缺失引发的血脂代谢紊乱和血管炎症损伤的关系以及AS的炎症反应机制.结果显示,apoE-/-/LDLR-/-小鼠IL-18、TLR2、MCP-1、ICAM-1、VCAM-1、GM-CSF、CD36和ET-1表达在1月龄时较同龄野生型(wild type,WT)小鼠显著上调(P<0.05,P<0.01),PDGF-α和TNF-α表达在2月龄时较同龄WT小鼠显著上调,除ET-1表达在2月龄时以及了LR2、VCAM-1和ICAM-1表达在3月龄时降至WT小鼠水平以外,其余各基因表达随年龄增长继续升高(P<0.05,P<0.01),其中MCP-1表达在2月龄时达到峰值.NF-kB在各年龄段apoE-/-/LDLR-/-小鼠中的表达与同龄WT小鼠相比均无显著差异.各年龄段apoE-/-/LDLR-/-/小鼠血清了C、TG、LDL、HDL、TNF-α、IL-1β和ox-LDL含量均显著高于同龄WT小鼠(P<0.05,P<0.01),并随年龄增长逐渐升高.apoE-/-/LDLR-/-小鼠1月龄时主动脉内膜出现少量的散在的脂质沉积,随着年龄增长病变区域增多,脂质沉积增厚.上述结果提示:apoE和LDLR双基因缺失形成的高脂血症可能通过刺激主动脉中炎症基因时序表达,起始并扩大病变部位的炎症反应,共同促进AS的发生发展.     

肝X受体激动剂通过上调NPC1基因的表达减轻apoE基因敲除小鼠动脉粥样硬化病变程度

本文研究肝X受体（LXR）激动剂对apoE基因敲除小鼠NPC1表达的影响．52只雄性apoE基因敲除小鼠被随机分成4组：（a）基础组（baseline group,n=10）;（b）对照组（control group,n=14）;（c）LXR激动剂治疗组（treatment group,n=14）;（d）LXR激动剂预防组（prevention group,n=14）．各组小鼠均被给予高脂／高胆固醇饲料喂养;基础组小鼠被赋形剂灌胃处理8周;对照组小鼠被赋形剂灌胃处理14周;LXR激动剂治疗组小鼠在前8周被赋形剂灌胃处理,后6周被T0901317灌胃处理;LXR激动剂预防组小鼠被T0901317灌胃处理14周．采用实时定量PCR,Western blot和免疫组织化学方法分别检测组织中NPC1 mRNA和蛋白质的表达;采用酶法测定血清中总胆固醇（TC）、甘油三酯（TG）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）、载脂蛋白A—I（ApoA—I）和载脂蛋白B（ApoB）含量．另外,我们采用RNA干扰技术沉默人THP-1巨噬细胞NPC1基因表达,并将细胞诱导成泡沫细胞,检测T0901317对该细胞胆固醇流出的影响．结果显示,LXR激动剂治疗组和预防组小鼠的血浆TC,TG,HDL-C和ApoA．I水平都较对照组明显增高（P〈0．05）;LXR激动剂治疗组和预防组小鼠的主动脉粥样硬化斑块和脂质条纹明显少于对照组（P〈0．05）,其中治疗组减少了58.3％,预防组减少了64．2％;LXR激动剂治疗组和预防组小鼠的小肠组织、肝脏组织和主动脉NPC1表达上调（P〈0.05）;和正常THP-1巨噬细胞源性泡沫细胞比较,RNA干扰组胆固醇流出明显减少,T0901317处理组胆固醇流出明显增加,总之,LXR激动剂T0901317能减轻高脂高胆固醇饲料喂养的apoE基因敲除小鼠动脉粥样硬化病变程度并上调肝脏组织、小肠组织和主动脉NPC1的表达,NPC1基因沉默能使细胞胆固醇流出减少。     

Developmental Exposure to Second-Hand Smoke Increases Adult Atherogenesis and Alters Mitochondrial DNA Copy Number and Deletions in apoE?/? Mice

Cardiovascular disease is a major cause of morbidity and mortality in the United States. While many studies have focused upon the effects of adult second-hand smoke exposure on cardiovascular disease development, disease development occurs over decades and is likely influenced by childhood exposure. The impacts of in utero versus neonatal second-hand smoke exposure on adult atherosclerotic disease development are not known. The objective of the current study was to determine the effects of in utero versus neonatal exposure to a low dose (1 mg/m³ total suspended particulate) of second-hand smoke on adult atherosclerotic lesion development using the apolipoprotein E null mouse model. Consequently, apolipoprotein E null mice were exposed to either filtered air or second-hand smoke: (i) in utero from gestation days 1–19, or (ii) from birth until 3 weeks of age (neonatal). Subsequently, all animals were exposed to filtered air and sacrificed at 12–14 weeks of age. Oil red-O staining of whole aortas, measures of mitochondrial damage, and oxidative stress were performed. Results show that both in utero and neonatal second-hand smoke exposure significantly increased adult atherogenesis in mice compared to filtered air controls. These changes were associated with changes in aconitase and mitochondrial superoxide dismutase activities consistent with increased oxidative stress in the aorta, changes in mitochondrial DNA copy number and deletion levels. These studies show that in utero or neonatal exposure to second-hand smoke significantly influences adult atherosclerotic lesion development and results in significant alterations to the mitochondrion and its genome that may contribute to atherogenesis.