【作者】 刘芬；

【导师】 彭文兴；

【作者基本信息】 中南大学 ， 药剂学， 2014， 硕士

【摘要】 目的：研究la,25-(OH)2D3对HepG2细胞上低密度脂蛋白受体相关蛋白1(Low-density lipoprotein receptor-related protein1, LRP1)及维生素D受体(vitamin D receptor, VDR)表达及摄取p-淀粉样蛋白(β-amyloid,Aβ)的影响。方法：体外培养人肝癌细胞HepG2, MTT实验确定最适药物干预浓度和干预时间。Western Blot及Real-Time PCR技术检测LRP1及VDR表达。酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)分析HepG2细胞摄取Aβ情况。为了考察LRP1在HepG2细胞摄取Ap中的作用,我们使用LRP1底物竞争性拮抗剂——受体相关蛋白(receptor associated protein, RAP)。结果：1.根据MTT实验结果,结合文献报道,我们选择1nM、10nM、50nM、100nM1α,25-(OH)2D3作为实验干预浓度,6h、24h作为实验干预时间。2.不同浓度1α,25-(OH)2D3干预HepG2细胞6h后,50nM、100nM1α,25-(OH)2D3显著上调LRP1蛋白表达(p<0.01),1nM、10nM浓度组对LRP1蛋白表达无影响。10nM、50nM、100nM1α,25-(OH)2D3显著上调VDR蛋白表达(p<0.05),1nM浓度组对VDR蛋白表达无影响。不同浓度1α,25-(OH)2D3干预HepG2细胞24h后,10nM、50nM、100nM浓度组显著上调LRP1蛋白表达(p<0.05),1nM浓度组对LRP1蛋白表达虽有上调趋势,但无统计学意义。1nM、10nM浓度组显著上调VDR蛋白表达(p<0.05),50nM、100nM浓度组对VDR蛋白表达虽有上调趋势,但无统计学意义。3.不同浓度1α,25-(OH)2D3干预HepG2细胞6h后,10nM、50nM、100nM la,25-(OH)2D3显著上调LRP1mRNA (p<0.05),1nM1α,25-(OH)2D3对LRP1mRNA无影响。50nM、100nM la,25-(OH)2D3显著上调VDR mRNA(p<0.01),1nM、10nM la,25-(OH)2D3对VDRmRNA无影响。不同浓度1α,25-(OH)2D3干预HepG2细胞24h后,药物各浓度组均显著上调LRP1和VDR mRNA (p<0.05)。4.不同浓度1α,25-(OH)2D3干预HepG2细胞611后,10nM、50nM、100nM la,25-(OH)2D3显著提高HepG2细胞摄取Aβ1-40(p<0.05),1nM浓度组虽有上调趋势,但无统计学意义。RAP显著降低HepG2细胞摄取Aβ1-40(p<0.05),且阻断1α,25-(OH)2D3对HepG2细胞摄取Aβ1-40的上调作用(p<0.05)。结论：1α,25-(OH)2D3能够诱导LRP1和VDR蛋白及mRNA表达,显著上调LRP1介导的HepG2细胞摄取Aβ。图20幅,表2个,参考文献48篇更多还原

【Abstract】 Objective:The purpose of this research were to study the effect of la,25-(OH)2D3on Low-density lipoprotein receptor-related protein1(LRP1) and vitamin D receptor (VDR) expression and uptake of β-amyloid (Aβ) in HepG2cells.Methods:The HepG2cells were cultured in vitro, the optimal concentration and action time were determined by MTT assay. The expression of LRP1and VDR were determined by western blot technique and Real-Time PCR; The uptake of AP in HepG2cells were analyzed by enzyme linked immunosorbent assay (ELISA). The role of LRP1in mediating the uptake of Aβ in HepG2cells was assessed using receptor associated protein (RAP), a competitive inhibitor ofLRP1.Results:1. Combined the results of MTT assay with reported researches, we chose1nM,10nM,50nM,100nM as intervention concentrations,6h and24h as intervention time.2. HepG2cells were treated with1α,25-(OH)2D3for6h,50nM,100nM1α,25-(OH)2D3significantly increased the expression of LRP1protein (p<0.05),1nM、10nM1α,25-(OH)2D3had no effect;10nM,50nM,100nM la,25-(OH)2D3significantly increased the expression of VDR protein (p<0.05),1nM1α,25-(OH)2D3had no effect. HepG2cells were treated with1α,25-(OH)2D3for24h,10nM,50nM,100nM la,25-(OH)2D3significantly increased the expression of LRP1protein (p<0.05), the up-regulation trend of the expression of LRP1protein was observed in1nM, but there were not statistical differences.1nM,10nM la,25-(OH)2D3significantly increased the expression of VDR protein (p<0.05), the up-regulation trend of the expression of VDR protein were observed in50nM,100nM groups, but there were not statistical differences.3. HepG2cells were treated with1α,25-(OH)2D3for6h, significant up-regulation of the expression of LRP1mRNA were observed in10nM,50nM,100nM groups (p<0.05),1nM group had no effect. Significant up-regulation of the expression of VDR mRNA were observed in50nM,100nM groups (p<0.01),1nM,10nM groups had no effect. HepG2cell were incubated with la,25-(OH)2D3for24h, All1α,25-(OH)2D3groups significantly enhanced the expression of LRP1and VDR mRNA (p<0.05).4. HepG2cells were treated with1α,25-(OH)2D3for6h, significant up-regulation of uptake Aβ1-40were discovered in10nM,50nM,100nM groups(p<0.05) and no difference was discovered in1nM group. RAP significantly reduced the uptake Aβ1-40in HepG2cells (p<0.05), and abolished the up-regulation of1α,25-(OH)2D3on uptake AP1-40in HepG2cells (p<0.05).Conclusions:la,25-(OH)2D3could induce the expression of LRP1and VDR protein and mRNA in HepG2cells, up-regulate the uptake of Aβ mediated by LRP1.更多还原