【作者】 郭瑞；

【导师】 史莹华；

【作者基本信息】 河南农业大学 ， 动物营养与饲料科学， 2014， 硕士

【摘要】 近年来,随着人们生活水平的提高,高血脂、动脉粥样硬化、心肌梗死等心血管疾病已成为危害人类身体健康的头号杀手。血脂异常,特别是血液中过高的胆固醇含量是引发这些疾病的主要因素之一。因此,控制高脂血症的发生对于预防心脑血管疾病有着重要意义。苜蓿皂苷具有调节脂质代谢,降低机体胆固醇含量等作用,但是目前对其机理研究并不多,特别是细胞水平上的研究更少。为了研究苜蓿皂苷在细胞水平上对胆固醇代谢的作用,试验一以大鼠BRL肝细胞为研究对象研究苜蓿皂苷对细胞胆固醇代谢相关基因的影响；试验二构建胆固醇代谢关键基因-低密度脂蛋白受体(LDLR)的串联亲和纯化载体并在293T细胞中表达。具体情况如下：试验一苜蓿皂苷对大鼠BRL肝细胞基因表达的影响。1、将培养2d的BRL正常细胞随机分为4组,正常细胞对照A组、正常细胞+苜蓿皂苷50 mg/L B组、正常细胞+苜蓿皂苷100mg/LC组和正常细胞+苜蓿皂苷150mg/LD组。正常BRL细胞分组后培养24 h,加入不同浓度的苜蓿皂苷液,继续培养24 h后,收集细胞,运用qRT-PCR检测细胞内LDLR、LXR(肝X受体)和FXR(胆汁酸受体)mRNA的表达。2、采用50%胎牛血清诱导BRL肝细胞48 h建立细胞脂肪变性模型。随后将脂变BRL细胞随机分为4组,脂变细胞组、脂变细胞+苜蓿皂苷50 mg/L组、脂变细胞+苜蓿皂苷]00 mg/L组和脂变细胞+苜蓿皂苷150 mg/L组,并建立正常细胞对照组。各处理组在添加苜蓿皂苷液24 h后,收集细胞,运用qRT-PCR检测肝细胞内LDLR、LXR和FXR mRNA的表达。结果表明：(1)添加苜蓿皂苷后,正常细胞LDLR mRNA的表达量显著性上调,脂变细胞LDLR mRNA的表达量有增加的趋势,但差异不显著；(2)苜蓿皂苷可以降低正常细胞和脂变细胞的LXR mRNA的表达量；(3)苜蓿皂苷能显著下调正常细胞FXR mRNA的表达,极显著下调脂变细胞FXR mRNA的表达。结论：在细胞水平上,苜蓿皂苷可能通过促进LDLR mRNA的表达,抑制LXR和FXR mRNA的表达,从而改善脂变肝细胞胆固醇的代谢。试验二重组质粒pNTAP-LDLR的构建及表达。根据GenBank中LDLR的mRNA序列及串联亲和纯化载体pNTAP-B中的多克隆位点设计引物,然后提取大鼠肝脏总RNA,利用逆转录聚合酶链式反应(RT-PCR)方法扩增大鼠LDLR编码区,进行LDLR的克隆和载体构建。将重组载体在真核细胞中进行表达,通过Western blotting方法鉴定蛋白的表达情况。结果表明：扩增出大鼠肝脏LDLR编码区并成功构建了pNTAP-LDLR哺乳动物串联亲和纯化表达载体；Western blotting检测结果显示,转染重组质粒后的293T细胞LDLR蛋白有稳定的表达。结论：LDLR基因的克隆、表达载体的构建和蛋白表达,为进一步研究其功能及互作蛋白奠定了一定的技术基础。更多还原

【Abstract】 In recent years, with the improvement of people’s living standards, high cholesterol, atherosclerosis, myocardial infarction and other cardiovascular diseases have become the top harmful factor to human health. Dyslipidemia, especially high blood cholesterol level is one of the main factors causing these diseases. Therefore, the control of hyperlipidemia is of great significance for the prevention of cardiovascular disease. Alfalfa saponins have regulating lipid metabolism, cholesterol-lowering effect and other biological activity, but there is a paucity of information on the mechanism of its cholesterol-lowering effect, especially from the cellular level. In order to study the effect of alfalfa saponins on cholesterol metabolism at the cellular level, in trial 1, we used rat BRL liver cells to study the effect of alfalfa saponins on some genes related to cholesterol metabolism; in trial 2, we constructed a key gene involved in cholesterol metabolism-low density lipoprotein receptor (LDLR) tandem affinity purification expression vector and further expressed in the HEK-293T cell. The details are as follows:Exp.l Effects of alfalfa saponins on genes involved in cholesterol metabolism in rat BRL liver cells.The normal BRL cells cultured for 2 d were divided into four groups:normal cells, normal cells with 50 mg/L,100 mg/L,150 mg/L alfalfa saponins. After cultured for 24 h, four groups were treated with different levels of alfalfa saponins for 24 h. Then the mRNA expression of LDLR, liver X receptor (LXR) and bile acid receptor (FXR) in treated cells were detected by qRT-PCR.Hyperlipidemic BRL cell model was established after treated with 50% fetal bovine serum for 48 h. After detection, hyperlipidemic BRL cells were divided into four groups: hyperlipidemic cells, hyperlipidemic cells with 50 mg/L,100 mg/L,150 mg/L alfalfa saponins. After cultured for 24 h, cells were collected and dected the mRNA expression of LDLR, LXR and FXR by qRT-PCR.The results showed that:(1) alfalfa saponins upregulated the mRNA expression of LDLR in normal cells. (2) after treated with alfalfa saponins, the mRNA expression of LXR in normal cells and hyperlipidemic BRL cells were downregulated. (3) after treated with alfalfa saponins, the mRNA expression of FXR in normal cells was significantly reduced, and extremely significantly decreased in hyperlipidemic BRL cells.Conclusion:alfalfa saponins might ameliorate hepatic steatosis by regulation of some genes involved in cholesterol metabolism, including upregulation of LDLR as well as downregulation of LXR and FXR.Exp.2 Construction and expression of pNTAP-LDLR.A pair of primers were designed according to multiple clone sites in pNTAP-B and LDLR mRNA sequence in Genbank. The total RNA was extracted from SD rat liver, and the cDNA encoding LDLR was obtained by qRT-PCR, which was confirmed by sequencing and BLAST, then it was cloned into T vertor and subsequently inserted in the expression vertor pNTAP-B. The expressed plasmids were transfected into eukaryotic cells, the expression product of pNTAP-LDLR was identified by Western blotting. The results showed that the coding region of LDLR was successfully amplified, and pNTAP-LDLR was successfully constructed. Western blotting analysis showed that LDLR was successfully expressed in transfected 293T cells. Conclusion:in the present study, LDLR was cloned, pNTAP-LDLR was successfully construsted and expressed in 293T cells, which lay a foundation for studying the function of LDLR and its interacting proteins in the further.更多还原