妊娠期糖尿病孕妇胎盘中哺乳动物雷帕霉素靶蛋白信号通路的表达情况

Activation of mammalian target of rapamycin signaling pathway in placental tissues of gravidas with gestational diabetes mellitus

目的探讨哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信号通路相关分子在妊娠期糖尿病(gestational diabetes mellitus, GDM)孕妇胎盘组织及高浓度葡萄糖处理后的JEG3细胞中的表达情况。 方法选择2016年12月至2017年6月在北京大学第一医院行择期剖宫产的足月单胎妊娠的60例孕妇,其中GDM孕妇30例为GDM组,正常糖耐量(normal glucose tolerance, NGT)30例孕妇为NGT组。采用实时荧光定量聚合酶链反应、蛋白质印迹法和免疫组织化学法检测2组胎盘组织中mTOR、Rictor、Raptor、S6激酶(S6 kinase, S6K)及磷酸化S6K(phosphorylated-S6K, p-S6K)等mTOR信号通路相关分子的表达水平。采用蛋白质印迹法检测不同浓度葡萄糖(5.5、10.0、15.0、25.0、50.0 mmol/L)的培养基处理JEG3细胞24 h后的mTOR、S6K及p-S6K的表达情况。采用两独立样本t检验和单因素方差分析对数据进行统计学分析。应用Spearman相关分析不同浓度葡萄糖与mTOR信号通路分子表达水平的相关性。 结果GDM组孕妇的孕前体重指数、新生儿出生体重,以及空腹、服糖后1及2 h血糖值高于NGT组[分别为(23.4±3.5)与(21.6±2.7) kg/m2,t=-2.192;(3 618.3± 580.9)与(3 337.5±347.7) g,t=-2.727;(5.2±0.8)与(4.6±0.4) mmol/L,t=-3.947;(9.6±1.9)与(7.4±1.2) mmol/L,t=-5.332;(8.1±1.5)与(6.3±1.0) mmol/L,t=-5.314;P值均< 0.05]。GDM组孕妇胎盘组织中mTOR、Rictor及Raptor的mRNA表达水平高于NGT组(0.051±0.015与0.031±0.011,t=-5.635;0.038±0.017与0.026±0.010,t=-3.485;0.036± 0.012与0.025±0.011,t=-3.312;P值均<0.05);mTOR、Rictor、Raptor及p-S6K的蛋白表达水平亦高于NGT组(0.834±0.432与0.386±0.361,t=-2.249;0.589±0.236与0.262± 0.075,t=-3.726;0.767±0.345与0.323±0.109,t=-3.472;1.847±1.025与0.834±0.432,t=-2.575;P值均<0.05);p-S6K的蛋白表达强度亦高于NGT组(0.29±0.09与0.18±0.08,t=-3.122,P=0.004)。不同浓度的葡萄糖处理后的mTOR(分别为0.190±0.085、0.301±0.089、0.419±0.065、0.562±0.108、0.412±0.058,F=18.351)和p-S6K蛋白表达(分别为0.753±0.150、1.146±0.289、2.148±0.188、2.248±0.115、2.134±0.214,F=66.242)差异均存在统计学意义(P值均<0.05);且随着葡萄糖浓度的增加,mTOR和p-S6K蛋白表达存在线性增加的趋势(r=0.314,P=0.035;r=0.457,P=0.002)。 结论GDM孕妇的胎盘组织及高浓度葡萄糖刺激的JEG3细胞中存在mTOR信号通路相关分子的表达水平上调,说明mTOR信号通路与GDM存在一定的相关性,但具体机制有待深入研究。

ObjectiveTo investigate the activation of mammalian target of rapamycin (mTOR) signaling pathway in placental tissues of gestational diabetes mellitus (GDM) women and in JEG3 cells after high glucose treatment. MethodsPlacental tissues were collected from 60 singleton pregnant women at term, who underwent elective cesarean section in Peking University First Hospital from December 2016 to June 2017, including 30 GDM women (GDM group) and 30 normal glucose tolerance (NGT) women (NGT group). Expressions of mTOR, Rictor, Raptor, S6 kinase (S6K) and phosphorylated-S6K (p-S6K), which were mTOR signaling pathway-related molecules in those placental tissues were detected by real-time fluorescence quantitative polymerase chain reaction, Western blotting and immunohistochemistry. At the same time, JEG3 cells were treated with glucose of different concentrations (5.5, 10.0, 15.0, 25.0 and 50.0 mmol/L) for 24 h, and the expressions of mTOR, S6K and p-S6K were detected by Western blotting. Two independent samples t-test, one-way analysis of variance test and Spearman correlation analysis were used for statistical analysis. ResultsCompared with the NGT group, the pre-pregnancy body mass index(BMI), neonatal birth weight, fasting blood glucose(FBG), and 1 h and 2 h post-load blood glucose in oral glucose tolerance test (OGTT) of the GDM group were significantly increased [pre-pregnant BMI:(21.6±2.7) vs (23.4±3.5) kg/m2, t=-2.192;neonatal birth weight:(3 337.5±347.7) vs (3 618.3±580.9) g, t=-2.727;FBG:(4.6±0.4) vs (5.2±0.8) mmol/L, t=-3.947;1 h glucose level:(7.4±1.2) vs (9.6±1.9) mmol/L, t=-5.332;2 h glucose level:(6.3±1.0) vs (8.1±1.5) mmol/L, t=-5.314;all P<0.05]. The mRNA expressions of mTOR, Rictor and Raptor were significantly higher in the GDM group than in the NGT group (0.051±0.015 vs 0.031±0.011, t=-5.635;0.038±0.017 vs 0.026±0.010, t=-3.485;0.036±0.012 vs 0.025±0.011, t=-3.312;all P<0.05). The expression of mTOR, Rictor, Raptor and p-S6K protein in the GDM group were enhanced as compared with those in the NGT group (0.834±0.432 vs 0.386±0.361, t=-2.249;0.589±0.236 vs 0.262±0.075, t=-3.726;0.767±0.345 vs 0.323±0.109, t=-3.472;1.847±1.025 vs 0.834±0.432, t=-2.575;all P<0.05). The results of immunohistochemistry also showed that the p-S6K in the placental tissues of the GDM group was higher than that of the NGT group (0.29±0.09 vs 0.18±0.08, t=-3.122, P=0.004). The expressions of mTOR protein (0.190±0.085, 0.301±0.089, 0.419±0.065, 0.562±0.108, 0.412±0.058, F=18.351, P<0.05) and p-S6K protein (0.753±0.150, 1.146±0.289, 2.148±0.188, 2.248±0.115, 2.134±0.214, F=66.242, P<0.05) in JEG3 cells treated with different concentrations of glucose (5.5, 10.0, 15.0, 25.0 and 50.0 mmol/L) were significantly different and increased with increasing concentrations of glucose (r=0.314, P=0.035;r=0.457, P=0.002). ConclusionsThe up-regulated expressions of mTOR signaling pathway-related molecules in GDM placenta and high glucose-treated trophoblast cells (JEG3 cells) indicate a possible correlation between mTOR signaling pathway and GDM. However, the specific mechanisms need further study.