|摘要||目的 探讨肝脏过氧化物体增殖物激活受体γ (peroxisome proliferator-ativated receptorγ,PPARγ)基因启动子甲基化状态及其mRNA表达在胎儿生长受限(fetal growth restriction,FGR)大鼠胰岛素敏感性降低中的作用. 方法 20只雌鼠受孕后第1天随机分为对照组和低蛋白组,各10只.低蛋白组采用低蛋白(粗蛋白含量为8.00％)法建立FGR模型.测定对照组仔鼠和低蛋白组FGR仔鼠生后3、7、14、30、60及90 d(每组每个时间点取雄性仔鼠8只)空腹血浆血糖和空腹血清胰岛素,计算胰岛素抵抗指数及胰岛素敏感指数.采用甲基化特异性聚合酶链反应和逆转录-聚合酶链反应技术测定仔鼠生后7和90 d时肝脏组织PPARγ基因启动子甲基化水平及其mRNA表达情况.采用Pearson相关分析及秩和检验分析肝脏组织中PPARγ基因启动子甲基化及其mRNA表达改变与胰岛素敏感性变化间的关系. 结果 (1)低蛋白组新生仔鼠平均出生体重为(4.92±0.36)g,低于对照组的(6.43±0.59)g(t=14.73,P＜0.05).低蛋白组仔鼠中FGR发生率为88.2％(97/110),其中雄性仔鼠FGR发生率为94.1％(48/51).(2)生后90 d时FGR仔鼠空腹血浆血糖、血清胰岛素和胰岛素抵抗指数显著高于对照组[空腹血浆血糖:(8.95±1.83) mmol/L与(6.21±1.14) mmol/L,t=-3.291,P＜0.05;血清胰岛素:(59.57±9.89) mU/L与(36.10±7.32) mU/L,t=-4.916,P＜0.05；胰岛素抵抗指数:0.967±0.297与0.410±0.135,t=-4.472,P＜0.05)],而胰岛素敏感指数低于对照组仔鼠(-3.043±0.294与-2.172±0.354,t=4.774,P＜0.05).(3)生后7d时,对照组仔鼠与FGR仔鼠PPARγ基因启动子甲基化程度差异无统计学意义(0/8与2/8,Fisher精确概率法,P＞0.05),生后90 d时FGR仔鼠甲基化程度高于对照组仔鼠(8/8与2/8,Fisher精确概率法,P＜0.05).PPARγ基因完全甲基化仔鼠PPARγ基因mRNA相对表达水平最低(27.2±1.6),其次是甲基化与非甲基化共存组(47.3±33.0),完全非甲基化组最高(144.6±21.2),差异均有统计学意义(P均＜0.05).(4)FGR仔鼠生后90 d时PPARγ基因mRNA表达量分别与空腹血浆血糖、血清胰岛素和胰岛素抵抗指数呈负相关(r分别为-0.819、-0.906和-0.860,P均＜0.05),而与胰岛素敏感指数呈正相关(r=0.947,P＜0.05). 结论 PPARγ基因启动子区高甲基化可能抑制其基因转录,从而参与FGR大鼠胰岛素抵抗的发生.
Objective To explore the effect of methylation of peroxisome proliferator-activated receptor γ(PPARy) gene promoter in liver and its mRNA expression changes on decreasing of insulin sensitivity in fetal growth restriction (FGR) rats.Methods Twenty pregnant rats were randomly divided into two groups on their first day of pregnancy:normal-protein group (NP) and low-protein group (LP),ten in each.During pregnancy the LP group rats were fed with low-protein diet (8.00％ protein),while the NP group rats were fed with normal-protein diet (20.00％ protein).The offspring rats were fed with standard feed after 21 days of birth.Male offsprings in NP group were as control offsprings,and male FGR offsprings in LP group ware as FGR offsprings.At day 3,7,14,30,60 and 90,fasting blood of offsprings was collected to measure fasting plasma glucose (FPG) and fasting insulin(FINS).Then insulin resistance index of homeostasis model assessment (HOMA-IR) and insulin sensitivity index (ISI) were calculated to evaluate insulin sensitivity.At day 7 and 90,liver tissue of male offsprings was collected to extract DNA and total RNA.The methylation level of PPARγ gene promoter and its mRNA expression were detected by methylation specific-polymerase chain reaction (MS-PCR) and reverse transcription-RCR,respectively.The relationships between methylation of PPARγ gene promoter and mRNA expression and insulin sensitivity were analyzed by Pearson correlation and nonparametric test method.Results (1) The mean offspring birth-weight of LP group was (4.92±0.36) g,which was lower than that [(6.43±0.59) g] of control group (t=14.73,P＜0.05).In LP group,the incidence of FGR offspring was 88.2％ (97/110) and the FGR incidence of male ones was 94.1％ (48/51).(2) At day 90,compared with control offsprings,FPG [(8.95±1.83) mmol/L vs (6.21±1.14) mmol/L,t=-3.291,P＜0.05],FINS [(59.57±9.89) mU/Lvs (36.10±7.32) mU/L,t=-4.916,P＜0.05] and HOMA-IR (0.967±0.297 vs 0.410±0.135,t=-4.472,P＜0.05) of FGR offsprings were significantly higher; while ISI of FGR offspring was lower than that of control offsprings (-3.043±0.294 vs -2.172±0.354,t=4.774,P＜0.05).(3) There was no significant difference in methylation degree of PPARγ gene promoter in liver between FGR and control offsprings at day 7 (0/8 vs 2/8,Fisher exact test,P＞0.05).The methylation degree of PPARγ gene promoter in liver in FGR offsprings was significantly higher than that of control offsprings at day 90 (8/8 vs 2/8,Fisher exact test,P＜0.05).Compared with control offsprings,PPARγ gene mRNA expression level of FGR offsprings decreased significantly at day 90 (4.3.07±7.51 vs 146.72± 40.66,t=7.09,P＜0.05).mRNA expression of PPARγ gene was the lowest in exhaustive methylation offsprings (27.2± 1.6),and then in partial methylation ones (47.3±33.0),the highest in no methylation ones (144.6 ± 21.2) (P＜0.05).(4) The correlation analysis showed that PPARγ mRNA expression level negatively correlated to the level of FPG (r=-0.819),FINS (r=-0.906) and HOMA-IR (r=-0.860),P＜0.05 respectively; but positively correlated to ISI level (r=0.947,P＜0.05).Conclusions Hypermethylation in promoter region of PPARγ gene might inhibit gene transcription,and be involved in the occurrence of insulin resistance in FGR rats.|