【作者】 杨芳；

【导师】 马美湖；

【作者基本信息】 华中农业大学 ， 食品科学， 2013， 博士

【摘要】 针对鸡蛋胆固醇其营养作用目前仍有争议的现状,本文以鸡蛋及其活性成分作为研究对象,开展了鸡蛋胆固醇营养动物实验、鸡蛋调控胆固醇活性成分的筛选及鸡蛋活性脂质调控胆固醇吸收代谢的机理研究等系列工作。主要的研究内容和主要研究成果摘要如下：本实验以健康Sprague Dawley大鼠为动物模型,摄入同等胆固醇含量的化学胆固醇(CLG)、蛋黄(EYG)、全蛋(WEG)及不含胆固醇的普通饲料(CCG)进行饮食干预90d,系统检测胆固醇及其代谢物在大鼠体内各脏器、血液及粪便中的含量,通过肝脏切片观察病变,运用RT-qPCR检测胆固醇相关代谢酶的转录水平表达。实验结果发现EYG组与WEG组大鼠的身体状况、血脂、肝脂等与CCG组相比无显著差异,但与CLG组相比较,其肝脏TG、TC及LDL-胆固醇,肝脏TG与TC,肝脏损伤程度,肝脏羟甲基戊二酸单酰辅酶A还原酶(HMG-CoAR)与酰基辅酶A：胆固醇酰基转移酶(ACAT)的mRNA表达量都显著性地降低；而肝脏HDL-胆固醇与TBA,粪便中性固醇与TBA,肝脏低密度脂蛋白受体(LDLr)、胆固醇-7α羟化酶(CYP7A1)与卵磷脂-胆固醇酰基转移酶(LCAT)的mRNA表达量均显著性地升高。实验结果表明鸡蛋对健康SD大鼠血脂无显著性影响,鸡蛋的摄入能显著抑制胆固醇体内从头合成及以胆固醇酯的形式在体内储存,促进了胆固醇向胆汁酸的转化,并随粪便排出。建立了体外Caco-2细胞单层吸收模型,并从形态学、酶学、跨膜电阻及通透率对其进行评估。实验结果发现经过21d培养的Caco-2细胞可以作为小肠吸收的体外模型,倒置显微镜与电镜检查,发现细胞单层模型的A侧分化形成微绒毛与细胞间形成了完整的紧密连接结构,细胞单层模型的跨膜电阻值均符合220Ω·cm2-600Ω·cm2(?)勺要求,细胞单层的标志性酶(碱性磷酸酶)活力增加表现出明显的极化,荧光黄A-B侧转运的渗透系数低于转运实验规定的1.0×10-6cm/s。上述结果表明建立的Caco-2细胞单层模型符合物质转运实验的条件,可以作为胆固醇吸收转运研究的体外模型。以Caco-2细胞单层为模型,研究了一系列浓度的胆固醇在不同pH值下的双向转运情况,以及NPC1L1蛋白抑制剂依折麦布(Eetimibe)对胆固醇细胞转运的影响。实验结果发现,胆固醇在Caco-2细胞单层模型中的转运表现为一定的浓度与pH值依赖性和饱和性,在胆固醇添加浓度为100μmol/L供液pH值为7.4条件下,胆固醇在Caco-2细胞单层模型中的转运渗透系数最高,其摄入比为4.73,胆固醇在Caco-2细胞单层吸收过程中受到载体转运蛋白的调节,而胆固醇外排是以被动扩散的方式转运。当加入Ezetimibe (10μmol/L)之后胆固醇摄入比显著降低。实验结果说明胆固醇在Caco-2细胞单层模型中的转运为载体蛋白NPC1L1介导的主动转运。以Caco-2细胞单层为模型,提取鸡蛋中脂质、蛋白质类组分,借助体外人工胃肠模拟消化系统,采用液体闪烁追踪3-H-胆固醇,研究各组分对胆固醇在Caco-2细胞单层模型中摄取与转运的影响。实验结果发现,鸡蛋中的蛋白质(蛋清总蛋白质、蛋黄总蛋白质及卵黏蛋白)、磷脂(卵磷脂与神经鞘磷脂)及n-3系列的多不饱和脂肪酸(EPA与DHA)能显著地抑制Caco-2细胞中胆固醇的摄取与转运,棕榈酸与溶血卵磷脂能够促进Caco-2细胞中胆固醇的摄取与转运,而单不饱和脂肪酸与脑磷脂对Caco-2细胞中胆固醇的摄取与转运无显著性影响。上述结果表明鸡蛋中含有调控胆固醇吸收转运的成分,为进一步揭示其对胆固醇代谢网络的调控及机制提供理论基础。考察鸡蛋各磷脂成分对胆固醇微胶束化学组成、牛磺胆酸钠结合能力、微胶束中胆固醇与牛磺胆酸盐的溶解能力及微胶束分子量变化的影响。实验结果发现,蛋黄卵磷脂与神经鞘磷脂的添加促进了胆固醇参与微胶束的形成,且随着添加量的增加使得牛磺胆酸钠的结合能力增强。当微胶束孵育24h后,蛋黄卵磷脂与神经鞘磷脂组的盐溶液中胆固醇溶解度显著地低于其他组,蛋黄神经鞘磷脂组的牛磺胆酸钠的溶解度显著性降低。此外,微胶束的分子量随着蛋黄卵磷脂与神经鞘磷脂添加浓度的增加而增加,表明微胶束释放的速度受到了这两种磷脂的约束,减慢了微胶束中胆固醇通过静止水层向肠上皮细胞转运前的释放过程。以上结果表明鸡蛋卵磷脂与神经鞘磷脂通过改变胆固醇混合微胶束的物理化学性质,在一定程度上抑制了微胶束向小肠上皮细胞的转运过程。通过RT-qPCR与Western-blot检测不同浓度鸡蛋卵磷脂与神经鞘磷脂对Caco-2细胞胆固醇吸收通路中关键蛋白转录与翻译水平表达的影响。实验结果发现鸡蛋卵磷脂与神经鞘磷脂均能显著性抑制小肠胆固醇吸收关键蛋白NPC1L1(尼曼-匹克C1型类似蛋白1)的转录与翻译,从而抑制胆固醇在Caco-2细胞中的摄入与转运。鸡蛋卵磷脂与神经鞘磷脂对于NPC11L基因与蛋白表达的抑制可能与转录因子SREBP(固醇调节元件结合蛋白)激活受到抑制相关。鸡蛋卵磷脂通过促进胆固醇合成HDL的方式移除Caco-2细胞中多余的胆固醇。此外,高浓度的蛋黄神经鞘磷脂有可能通过下调Caveolin1基因表达从而抑制胆固醇的吸收。通过RT-qPCR与Western-blot检测鸡蛋中六种脂肪酸(PA、OA、LA、AA、EPA、 DHA)对Caco-2细胞胆固醇吸收通路中关键蛋白转录与翻译水平表达的影响。实验结果发现EPA与DHA均能显著性抑制小肠胆固醇吸收关键蛋白NPC1L1的转录与翻译,从而抑制胆固醇在Caco-2细胞中的摄入与转运,而高浓度的PA与OA通过促进NPC1L1基因的转录表达使得胆固醇在Caco-2细胞中的摄入与转运增加,且NPCL1L基因的下调与转录因子SREBP受到抑制相关。高浓度的AA与EPA通过抑制ABCA1(三磷酸腺苷结合盒转运体A1)的活性,从而抑制Caco-2细胞中胆固醇以合成HDL的方式外排,而高浓度的PA与OA通过上调ACAT2的转录表达促进胆固醇转化为胆固醇酯最终参与乳糜微粒的形成。一定浓度的EPA与DHA抑制了Caveolin1mRNA的表达,表明EPA与DHA有可能通过下调Caveolin1基因表达抑制胆固醇的吸收。更多还原

【Abstract】 Hen egg, as a cholesterol-rich food, nutritional advantages and disadvantages of the current status quo is still controversial. So the object of this study were hen eggs and its active ingredients, and the study was carried out to evaluate the egg cholesterol nutritional pros and cons though animal experiment, screen the active ingredients from eggs with regulation role of cholesterol absorption, investigate cholesterol absorption and metabolism regulation mechanism of egg active lipids. The main research contents and results are as follows:In order to investigate the influence of egg-enriched diets and cholesterol diet on SD rats’plasma, hepatic and fecal lipid levels, and on gene expression levels of transporters, receptors and enzymes involved in cholesterol metabolism after90d dietary interventions. Sprague-Dawley rats fed an egg-enriched diet had lower plasma triglycerides, total cholesterol, low density lipoprotein (LDL)-cholesterol, hepatic triglyceride, and cholesterol concentrations, and greater plasma high-density lipoprotein cholesterol concentration, fecal neutral sterol and bile acid concentrations than those fed a plain cholesterol diet. Hen egg yolk had no effect on sterol12a-hydroxylase (CYP8B1) and sterol27α-hydroxylase (CYP27A1); but upregulated mRNA levels of hepatic LDL-receptor, cholesterol7α-hydroxylase (CYP7A1) and lecithin cholesterol acyltransferase, and downregulated hepatic hydroxymethylglutaryl-(HMG)-CoA reductase and acyl-CoA:cholesterol acyltransferase (ACAT) after90days. The present study has demonstrated that an egg-enriched diet has no significant impact on the plasma lipid levels of healthy SD rats, could restrain de novo cholesterol synthesis and deposits in the form of cholesterol esters, and activate bile acid synthesis and excretion in feces and deposition.Caco-2monolayers model was established and assessed with morphology, enzymology, transepithelial electrical resistance (TEER) and the apparent permeation rate (Papp)-The transmission electron micrograph showed well-differentiated cells with microvilli and a tight junction between the cells, the alkaline phosphatase activity in the apical side was significantly increased and higher than that in the basolateral side with the time, the transepithelial electrical resistance reached a relatively stable value within the scope of220Ω·cm2-600Ω·cm2, and the Lucifer Yellow apparent permeability coefficient was lower than1.0×10-6cm/s. Therefore, the Caco-2cell monolayers were appropriate for use in the transport study, and the best in vitro models to study cholesterol absorption and transport study.Caco-2monolayers model was used to study bidirectional transport of cholesterol in a series of concentrations with different pH values, and investigated impact of ezetimibe on cellular transport of cholesterol. The permeation rate of cholesterol across Caco-2cell monolayers was examined in both direction of A-B and B-A with micellar solutions (100μmol/L of cholesterol) in donor media at pH7.4and at pH7.4in receiving media, which were the best conditions according the preliminary experiments. The uptake ratio value was calculated as4.73, which show that the mechanism of cholesterol permeation was suggested as active uptake in absorptive transport with carrier proteins mediated-transport. In the presence of ezetimibe (10μmol/L), a significant decrease of the uptake ratio was observed compared to the data in the absence of the NPC1L1-inhibitor. It was found that the absorptive transport was competitively inhibited by ezetimibe.The lipids and proteins were extracted from hen eggs and digested with the help of in vitro artificial gastrointestinal simulation system. Then these functional factors were studied to compare the impact of cholesterol uptake and transport in Caco-2monolayers model by using liquid scintillation counter. It was found that egg proteins (egg white protein, egg yolk protein and egg ovomucin), phospholipids (phosphatidylcholine and sphingomyelin) and n-3series polyunsaturated fatty acids (EPA and DHA) significantly inhibited cholesterol uptake and transport of cholesterol in the Caco-2monolayers model, palmitic acid and lysophosphatidylcholine significantly promoted cholesterol uptake and transport of cholesterol in the Caco-2monolayers model. However, the effect of mono-unsaturated fatty acids and phosphatidyl ethanolamine on cholesterol uptake and transport of cholesterol in the Caco-2monolayers model were no significant impact. These results indicate that egg contain the regulation of cholesterol absorption transport ingredients, which provide a theoretical basis for further study on the regulation of cholesterol metabolism network and mechanism.Determination of physicochemical characteristic of cholesterol micelles with egg-yolk phospholipids were evaluated by detecting the binding capacity with taurocholate, micellar solubility of cholesterol and taurocholate, and molecular weight changes of micelles after adding phospholipids. The present study indicated that egg-yolk SM and PC interfered with sodium taurocholate micelles, decreased the micellar solubility of cholesterol and taurocholate, and increased the bile acid-binding capacity and size of the taurocholatelecithin-cholesterol micelles. Since cholesterol has a higher affinity for bile salt micelles containing SM and PC, release from the micelles is reduced as a monomer and less absorbed to brush border membranes. Consequently, it is plausible that excess phospholipids especially SM could suppress Caco-2monolayers absorption of cholesterol by modulating the physicochemical properties of mixed micelles.Cholesterol micellar solutions with egg-yolk PC and SM in cultural medium were added in Caco-2cell, total RNA and protein were isolated from the cells and detected expression of transcriptional and protein levels of key proteins in the pathway of cholesterol absorption in Caco-2cells by RT-qPCR and Western-blot. Egg-yolk sphingomyelin and phosphatidylcholine could significantly inhibit Niemann-Pick Cl-like1protein (NPC1L1) levels and mRNA levels, which suggest that the cholesterol-inhibitory effect be mediated in part by their action at the NPC1L1gene and protein level, while NPC1L1gene might be relative with Sterol regulatory element binding protein (SREBP) activation was inhibited. High concentrations of egg-yolk PC increased ATP-binding cassette transporter (ABCA1) mRNA levels, suggesting that PC promoted ABCA1activity and accelerated the removal of cholesterol from Caco-2cell by reverse cholesterol transport process. Egg-yolk SM might inhibit cholesterol absorption by down-regulating mRNA expression of caveolin1.Cholesterol micellar solutions with egg-yolk fatty acids (PA, OA, LA, AA, EPA, DHA) cultural medium were added in Caco-2cell, total RNA and protein were isolated from the cells and detected expression of transcriptional and protein levels of key proteins in the pathway of cholesterol absorption in Caco-2cells by RT-qPCR and Western-blot. EPA and DHA could significantly inhibit NPC1L1levels and mRNA levels, which suggest that the cholesterol-inhibitory effect be mediated in part by their action at the NPC1L1gene and protein level, while NPC1L1gene might be relative with SREBP activation was inhibited. However, high concentrations of PA and OA could increase NPC1L1levels and mRNA levels, which were consistent with the increase of cholesterol uptake and transport in Caco-2monolayers model. High concentrations of EPA and DHA decreased ABCA1mRNA levels, suggesting that PC inhibited ABCA1activity and weakened the removal of cholesterol from Caco-2cell by reverse cholesterol transport process, while high concentrations of PA and OA increased acyl-CoA:cholesterol acyltransifying enzyme2(ACAT2) levels and mRNA levels to promote the conversion of cholesterol ester and eventually involved in the formation of chylomicrons. EPA and DHA might inhibit cholesterol absorption by down-regulating mRNA expression of caveolin1.更多还原