【作者】 苏红；

【导师】 周波；

【作者基本信息】 重庆医科大学 ， 内科学， 2013， 硕士

【摘要】 目的：以人系膜细胞（HMCs）作为体外模拟代谢记忆的研究对象，观察转录共激活子p300及组蛋白乙酰化H3（Ac-H3）、Ac-H4蛋白表达规律，并探讨p300在其中的潜在汇集点作用。方法：将培养的HMCs按以下分组处理：①高糖诱导代谢记忆模型，分为正糖组（NG，5.5mmol/L D-葡萄糖×2d）、渗透压组（LG，NG+19.5mmol/L L-葡萄糖×2d）、高糖组（HG，25mmol/L D-葡萄糖×2d）、记忆组（M1、M2、M3组，25mmol/L D-葡萄糖×2d+5.5mmol/L D-葡萄糖×3、6、9d）、持续正糖组（NC，5.5mmol/L D-葡萄糖×9d）；②采用H₂O₂模拟氧化应激记忆模型，分为正糖组（NG，5.5mmol/L D-葡萄糖×30min）；正糖+H₂O₂组（H₂O₂，5.5mmol/L D-葡萄糖+100μmol/LH₂O₂×30min）；H₂O₂记忆组（H₂O₂-M，5.5mmol/L D-葡萄糖+100μmol/LH₂O₂×30min+5.5mmol/L D-葡萄糖×3d）；正糖对照组（NG3，5.5mmol/LD-葡萄糖×3d）。③蛋白激酶C（PKC）β2激活记忆模型，分为正糖组（NG，5.5mmol/L D-葡萄糖×2d）；高糖组（HG，25mmol/L D-葡萄糖×2d）；记忆组（M，25mmol/L D-葡萄糖×2d+5.5mmol/L D-葡萄糖×3d）；空载体记忆组（HN，25mmol/L D-葡萄糖+Ad5-null×2d+5.5mmol/L D-葡萄糖×3d）； PKCβ2激活记忆组（PO，25mmol/L D-葡萄糖+Ad5-PKCβ2×2d+5.5mmol/L D-葡萄糖×3d）；PKCβ2抑制剂记忆组（PI，25mmol/L D-葡萄糖×2d+10μmol/L CGP53353+5.5mmol/L D-葡萄糖×3d）。④糖化终产物记忆模型,分为正糖组（NG，5.5mmol/L D-葡萄糖×2d）；正糖+AGEs组（AGEs，5.5mmol/L D-葡萄糖+250μg/ml AGEs×2d）；AGEs记忆组（AGEs-M，5.5mmol/L D-葡萄糖+250μg/mlAGEs×2d+5.5mmol/L D-葡萄糖×3d）；BSA组（NG+BSA，给予同浓度BSA对照）。二氢二氯荧光素（DCFDA）及酶标仪检测细胞内活性氧（ROS）的表达。Western blotting检测各组细胞p300、Ac-H3和Ac-H4及PKCβ2蛋白表达水平。结果：①HG组p300、Ac-H3和Ac-H4蛋白表达增加，分别较NG组增加了1.15倍、0.93倍和0.87倍（P<0.05），同时伴有PKCβ2蛋白及胞内ROS水平上调；M1、M2、M3组p300、Ac-H3、Ac-H4、PKCβ2蛋白水平及ROS表达水平较NG组仍显著为高，即使M3组亦较NG组分别增加75%、49%、47%、98%和48%（P<0.05）。②H₂O₂组p300、Ac-H3和Ac-H4蛋白表达均升高，较对照组分别升高了1.03倍、0.85倍和0.79倍（P<0.05），而H₂O₂-M组各蛋白表达较对照组差异无统计学意义。③与M组比较，PO组p300、Ac-H3和Ac-H4蛋白表达进一步上调，分别为M组的1.25倍、1.06倍和1.10倍（P<0.05），选择性PKCβ2抑制剂CGP53353可以显著降低上述蛋白表达。④AGEs组p300、Ac-H3和Ac-H4蛋白表达上调，较对照组分别升高了1.73倍、1.08倍和1.05倍（P<0.05），AGE-M组各蛋白较对照组分别增加了1.47倍、0.95倍和1.03倍（P<0.05）。结论：HMCs存在转录共激活子p300及表观修饰持续活化的记忆效应，p300可能系高糖致生化代谢及表观遗传记忆刺激的汇聚点。更多还原

【Abstract】 Objective：To investigate the proteins expression of transcriptionalcoactivator p300and acetylated histone H3（Ac-H3） as well as Ac-H4inhuman renal mesangial cell （HMCs）, imitating “metabolic memory” invitro and the potential role of convergence point.Methods：HMCs were divided into the following groups: The HMCswere divided into the following groups:①High glucose metabolic memorymodel: normal glucose （NG,5.5mmol/L D-glucose×2days） group, osmoticcontrol （LG,5.5mmol/L D-glucose+19.5mmol/L L-glucose×2days）group,high glucose （HG,25mmol/L D-glucose×2days） group, memory （M1, M2,M3,25mmol/L D-glucose×2days+5.5mmol/L D-glucose×3days,6days or9days） groups, continue normal glucose （NG,5.5mmol/L D-glucose×9days） group.②H₂O₂was used to simulate oxidative stress memorymodel:normal glucose（NG,5.5mmol/L D-glucose×2days）group, NG+H₂O₂（H₂O₂,5.5mmol/L D-glucose+100μmol/L H₂O₂×30min）group; H₂O₂memory [（5.5mmol/L D-glucose+100μmol/L H₂O₂×30min）+5.5mmol/LD-glucose×3days] group；normal glucose control（NG3，5.5mmol/L D-glucose×3d）group.③Transfected with PKCβ2memory model: normal glucose （NG,5.5mmol/L D-glucose×2days） group；high glucose（HG，25mmol/L D-glucose×2d）group；memory（M，25mmol/L D-glucose×2d+5.5mmol/L D-glucose×3d）group；Ad5-null memory（HN，25mmol/L D-glucose+Ad5-null×2d+5.5mmol/L D-glucose×3d）group；PKCβ2memory（PO，25mmol/L D-glucose+Ad5-PKCβ2×2d+5.5mmol/L D-glucose×3d）group；inhibitor of PKCβ2memory（PI，25mmol/L D-glucose×2d+10μmol/L CGP53353+5.5mmol/L D-glucose×3d）group.④Advanced glycation end products memory model: normal glucose （NG,5.5mmol/L D-glucose×2days） group, NG+AGEs （AGEs，5.5mmol/L D-glucose+250μg/ml AGEs×2d）group；AGEs memory（AGEs-M,5.5mmol/LD-glucose+250μg/ml AGEs×2days+5.5mmol/L D-glucose×3days）group； BSA control （NG+BSA，5.5mmol/L D-glucose+250μg/mlBSA×2d）group. ROS level was tested by fluorescence microscope andfluorescence microplate reader. The expression levels of p300, Ac-H3,Ac-H4as well as PKCβ2proteins were determined by Western blotting.Results：①p300, Ac-H3and Ac-H4protein expression levels in HGgroup increased, being2.15,1.93and1.87folds of those in group NG（P<0.05）, accompany with the upregulation expression of PKCβ2proteinlevels as well as ROS levels in group HG. More importantly, p300, Ac-H3,Ac-H4, PKCβ2protein expression and ROS levels in M1, M2, M3increased, even being1.75,1.49,1.47,1.98and1.48folds of those ingroup NG in M3.②The protein expression levels of p300, Ac-H3and Ac-H4in H₂O₂group increased significantly, increasing by1.03,0.85and0.79folds of those in control group （P<0.05）. However, no significantlywere observed between the indexes in H₂O₂-M and control groups.③Ascompared with group M, p300, Ac-H3and Ac-H4protein expression levelsin PO group increased more obviously, increasing1.25,1.06and1.10foldsof those in group M （P<0.05）. However, CGP53353could decrease thoseindexes significantly.④AS compared with the control group, the proteinexpression of p300, Ac-H3and Ac-H4in AGEs and AGEs-M groupsignificantly increased （1.73,1.47）,（1.08,0.95） and （1.05,1.03） folds ofthose in group BSA respectively （P<0.05）.Conclusion：Persistent activation of transcriptional co-activator p300and epigenetic modification exists after glucose levels are normalized inHMCs. p300may be the convergent point of glucose-induced metabolic“memory” stimulations.更多还原