【作者】 王昊；

【导师】 彭道泉；

【作者基本信息】 中南大学 ， 内科学， 2012， 博士

【摘要】 背景代谢综合征(metabolic syndrome, MS)患者体内高密度脂蛋白胆固醇(HDL-C)降低总是与甘油三酯(TG)升高相伴而行,而小鼠代谢综合征模型中无类似人体HDL-C降低的改变,这些现象仍无明确解释。我们观察到脂肪细胞与肝细胞共培养可影响肝细胞固醇调节元件结合蛋白(SREBP-1c)及ATP结合盒转运体A1(ABCA1)表达,结合最新发现SREBP基因内含子中隐藏了调节ABCA1的microRNA-33,我们推测代谢综合征的相关因素可调节SREBP1-miR33b,导致TG升高与HDL-C同步降低,而小鼠SREBP1基因中缺乏miR-33b是人鼠代谢综合征血脂异常差异的因素之一。目的探讨代谢综合征相关因素对肝细胞SREBP1/miR-33b的影响及其在调节肝细胞HDL代谢中的作用。方法分别以100nM胰岛素和200ng/ml TNF-α干预人原代肝脏细胞,以不加干预的细胞为对照组,干预24小时后提取各组细胞的总RNA及蛋白,通过实时荧光定量PCR (Real-time polymerase chain reaction)比较各组中miR-33b, SREBP-1c, SREBP-2, ABCA1, ABCG1, APOA1基因的相对表达量。通过免疫印迹(Western Blot)方法检测各组中ABCA1及ABCG1的蛋白表达量。通过闪烁计数法检测肝细胞胆固醇流出率。利用转染试剂LipofectamineTM2000分别以1miR-33b模拟物(mimic50nM)和miR-33b抑制物(inhibitor100nM)转染人原代肝细胞,6小时后再以100nM胰岛素干预,24小时后PCR检测miR-33b, SREBP-1c及ABCA1基因的相对表达量。结果1.与对照组相比,胰岛素干预人原代肝细胞24小时后,可见miR-33b及SREBP-1c表达明显上调,ABCA1, ABCG1和APOA1基因表达下调,其中ABCA1和ABCG1蛋白表达水平也出现相应下降,apoA-I介导的胆固醇流出效率下降,其变化均有统计学差异(P<0.05)。而胰岛素干预后SREBP-2基因无明显变化(P>0.05)2.与对照组相比,TNF-α干预人原代肝细胞24小时后,可见miR-33b及SREBP-1c表达明显下调,同时ABCA1和APOA1基因表达也出现下降,而ABCG1表达升高,其变化均有统计学差异(P<0.05)。TNF-α干预后SREBP-2基因无明显变化(P>0.05)3.过表达miR-33b可以下调人原代肝细胞中ABCA1表达水平,沉默miR-33b可以上调ABCA1表达水平：过表达miR-33b后,胰岛素干预可以进一步抑制ABCA1,而沉默miR-33b后,胰岛素对ABCA1的抑制作用消失(均P<0.05)4.无论过表达或沉默miR-33b后,SREBP-1c表达均无明显变化,P>0.05。而同时加入胰岛素后,SREBP-1c表达均明显上升,P<0.05。结论在人原代肝细胞中,高浓度胰岛素可以通过上调SREBP-1c和miR-33b基因的表达,从而抑制ABCA1, ABCG1mRNA和蛋白的表达,以及apoA-I介导的胆固醇流出。更多还原

【Abstract】 BackgroundMetabolic syndrome (MS) in humans is always associated with the reduction of high-density lipoprotein cholesterol (HDL-C) and the elevation of triglyceride (TG). However, the reduction of HDL-C would not happen in the mouse model of MS. Until now, there is no clear explanation for these. In our previous experiments, we observed that the gene expression of sterol-regulatory element binding protein-lc (SREBP-1c) and ATP-binding cassette transporter A1(ABCA1) in hepatocytes were changed when we co-cultured the hepatocytes with adipocytes. Combined with the fact that in the intron of SREBP-lc, a microRNA called miR-33could regulate the expression of ABCA1gene, we speculate that the relevant factors of MS might affect SREBP1c-miR33b, resulting in HDL-C decrease, accompanied by TG increase. In addition, there is a lack of miR-33b in the sequence of mouse SREBP1gene, and this may be a reason for differences of dyslipidemia between mice and men.ObjectiveExplore the effects of MS relevant factors on SREBP1/miR-33b in hepatocytes, and its role in regulating hepatocyte HDL metabolism.Methods Primary human hepatocytes were treated with100nM insulin and200ng/ml TNF-alpha, respectively. The untreated cells were used as control group. After24hours, the total RNA and protein were extracted. The relative gene expression of miR-33b, SREBP-1c, SREBP-2, ABCAl, ABCG1and APOA1were measured by real-time polymerase chain reaction (real-time PCR). The relative protein expression of ABCA1and ABCG1were measured by Western Blot. The efflux rates of3H-cholesterol in hepatocytes were detected by means of liquid scintillation counter. Primary human hepatocytes were transfected with50nM miR-33b mimic or100nM miR-33b inhibitor by liposome LipofectamineTM2000. After6hours, those cells were treated with100nM insulin and after24hours, the gene expression of miR-33b, SREBP-lc and ABCAl were detected by real-time PCR.Results1. Compared with the control group, the gene expressions of miR-33b and SREBP-1c in primary human hepatocytes of insulin intervention group were significantly up-regulated, while ABCA1, ABCG1and APOA1genes were down-regulated. In consistent with that, the expressions of ABCA1and ABCG1protein levels were also decline, while the cholesterol efflux rates form hepatocytes were decreased. All the changes were statistically significant (P<0.05). However, there is no significant change in SREBP-2gene expression (P>0.05). 2. Compared with the control group, the gene expressions of miR-33b and SREBP-1c in primary human hepatocytes of TNF-alpha group were significantly down-regulated; meanwhile, the gene expressions of ABCA1and APOA1were also down-regulated, while the expression of ABCG1gene was increased. All the changes were statistically significant (P<0.05). However, there is no significant change in SREBP-2gene expression (P>0.05).3. Overexpression of miR-33b could down-regulate ABCA1in primary human hepatocytes and inhibition of miR-33b could up-regulate ABCA1; The stimulation of insulin would further repress ABCA1when we overexpressed miR-33b, however, the depressant effect of insulin on ABCA1disappeared when we slience miR-33b (all P<0.05).4. Neither overexpression nor inhibition of miR-33b could affect the gene expression of SREBP-1c in primary human hepatocytes,P>0.05.ConclusionsHigh level of Insulin can increase the gene expressions of SREBP-1c and miR-33b in primary human hepatocytes simultaneously, thus inhibiting ABCA1and ABCG1mRNA and protein expressions. As a result, the cholesterol efflux mediated by apoA-Ⅰ is repressed.更多还原