·综述·
目前，世界范围内2型糖尿病(type 2 diabetes mellitus，T2DM)发病率逐年递增。Barker等[1]在上世纪90年代初率先报道，低出生体重与成年T2DM关系密切，突破了糖尿病主要与成年肥胖有关的传统观点，从而将“成年疾病的发育起源”这一全新理论引入糖尿病的研究领域。宫内发育迟缓(intrauterine growth retardation，IUGR)主要指发育时期胎儿受到各种因素的影响，导致其正常生长态势受到阻滞，应有的生长潜能遭到削弱，从而造成胎儿在出生前、后出现各种不良生理和病理改变[2]。IUGR的病因除了先天遗传因素外，很大程度上是因为孕期不良宫内环境(包括母体和外源环境因素)所致。已有研究表明，IUGR是糖耐量异常和T2DM最重要的独立危险因素之一[3]。
胰腺是人体第二大分泌腺体，其胰岛β细胞主要通过分泌体内唯一能够降血糖的胰岛素调节机体血糖平衡。胚胎胰腺发育过程受到多因素调控，其中宫内环境作为胚胎胰腺发育的外环境，是胰腺正常发育、胰腺功能最终完成的重要保证[4]。本文将系统阐述各种不良宫内环境所致的IUGR子代发生胰腺发育和功能的编程改变及其可遗传性效应。
在啮齿类动物模型上，人们根据不同诱因分别建立了多种IUGR模型，主要包括：饮食限制、蛋白摄入限制、子宫动脉结扎、地塞米松暴露。
妊娠末期与出生后早期是胰岛β细胞生长发育的关键时期，该时期营养物质的数量会对胰腺结构及功能产生长期以至终生的影响。妊娠晚期对大鼠进行饮食限制，导致新生鼠IUGR及胰岛β细胞数量明显减少[5]。进一步发现，饮食限制所致的IUGR胎胰腺β细胞分化调控关键因子神经元素3(neurogenin 3，Ngn-3)和胰腺-十二指肠同源框基因(pancreatic and duodenal homeobox 1，Pdx-1)蛋白表达减少，表明饮食限制引起的β细胞数量降低可能与β细胞分化失衡有关[6]。
在保持能量摄入恒定的情况下，孕期选择性的蛋白限制大鼠(给予正常量的40%～50%)也能够导致IUGR以及β细胞数和胰岛面积的减少[7]，其原因可能与IUGR胎鼠胰岛细胞中血管表皮生长因子(vascular endothelial growth factor，VEGF)及其受体胎肝激酶-1(fetal liver kinase 1，Flk-1)的阳性细胞表达减少,导致胰岛血管化作用减弱，进而影响胰岛细胞的增殖有关[8]。除增殖受到抑制以外，蛋白限制所致IUGR的胰腺β细胞还存在胰岛素样生长因子2(insulin like growth factor 2，IGF-2)低表达介导的异常凋亡[9]。
子宫-胎盘功能不足对IUGR子代胰岛素分泌和糖耐量有着显著的影响，孕第18～19天双侧子宫动脉结扎能够导致大鼠IUGR，子代出生后存在β细胞数目减少、胰岛素含量下降现象[10]。子宫胎盘功能不足所致IUGR胎儿存在氧化应激[11-13]。由血供不足所引起的氧化还原状态的改变在某些敏感的胎儿组织会导致氧化应激的发生。与其他组织相比，胰腺组织对线粒体功能障碍与活性氧簇(reactive oxygen species,ROS)暴露更敏感。已证实，线粒体功能缺陷和氧化应激增加都将对β细胞功能产生严重的不良影响，如损伤葡萄糖刺激胰岛素的分泌，降低β细胞关键基因的表达以及诱导细胞死亡等[14-16]。
外源性糖皮质激素(glucocorticoids，GC)通常用于加速有早产风险产妇的胎儿肺成熟。但如果长时间采用这种疗法则会导致IUGR[17]。孕期最后一周给予大鼠地塞米松(一种外源性合成GC)暴露的IUGR子代3周龄时，胰岛内α和β细胞分布正常，但与对照组相比，β细胞内胰岛素表达降低[18]。地塞米松不仅难以被胎盘11-β羟基类固醇脱氢酶2(11β hydroxysteroid dehydrogenase type 2，11β-HSD-2)灭活，同时还能够抑制其活性。因此，地塞米松往往直接通过胎盘对胎儿胰腺发挥直接作用。另外，本实验室也发现，孕期外源物暴露(咖啡因、尼古丁和乙醇)也能够引起胎鼠β细胞数目减少，胰岛素合成功能受损。
宫内发育不良能够波及胰腺发育并永久性编程改变β细胞的结构与功能，由此诱发子代糖和胰岛素代谢功能异常以及T2DM风险[19]。临床研究表明，IUGR胎儿和新生儿胰腺β细胞的数量减少[20]，脐带血糖和胰岛素水平降低，血糖/胰岛素比率升高[21]。出生时虽胰岛素敏感性升高，但婴幼儿时期胰腺β细胞功能减低[22-23]。尽管IUGR子代存在着出生后胰岛素敏感性降低的风险[24]，但只有当β细胞数量和功能依赖的胰岛素分泌无法代偿机体胰岛素抵抗时，才能诱发糖尿病。大多数的研究提示，相对于胰岛素敏感性而言，IUGR的儿童和成年人群胰岛素的分泌实际上已经发生障碍且比胰岛素抵抗更早出现[25-27]。低出生体重的青年男性尽管胰岛素敏感性正常，但胰岛素分泌却降低了30%，即胰岛素分布指数(insulin disposition index,IDI)降低[28]，表明β细胞数量和功能不足所致胰岛素分泌反应性降低是编程IUGR成年个体糖代谢稳态功能异常中重要的始发现象。进一步，研究者通过动物实验发现可能有多种机制参与IUGR子代糖代谢功能的编程改变。
1.孕期糖皮质激素过暴露编程机制：已知糖皮质激素受体(glucocorticoid receptor,GR)在胚胎期第15天的胰芽中已经有表达。采用GC处理体外培养的胰芽能够导致很多胰腺特异性转录因子表达下调，包括Pdx-1、Nk6同源框-1(Nk6 homeobox 1,Nkx6.1)和配对框基因6(paired box,Pax6)。发育时期胰腺细胞GR阻断的转基因小鼠表现出相反的症状，出生时β细胞数目会显著性升高；α细胞数目则相对稳定[29]。同时，地塞米松在体外能够通过下调Pdx-1并诱导CCAAT增强子结合蛋白β(CCAAT/enhancer-binding protein β,C/EBPβ)表达的方式来抑制胰腺β细胞的胰岛素表达[19]。有报道认为，过高水平GC影响甚至中断转录因子的表达，如增加啮齿类动物和人类胰岛丝裂原诱导的抗增殖因子Mig6基因表达[30]，减少β细胞促增殖基因Ngn3表达[31]。Valtat等[32]不仅证明孕期GC暴露可通过抑制β细胞发育参与编程子代成年糖尿病易感，还进一步证明GC能刺激GR的辅助调节因子过氧化物酶体增殖物激活受体-γ共激活因子-1α(peroxisome proliferator-activated receptor-γ coactivator-1α,PGC-1α)的表达。过表达的PGC-1α通过形成GR/PGC-1α复合体结合到β细胞关键转录因子Pdx-1启动子区从而抑制Pdx-1表达，提示孕期GC过暴露往往能够通过抑制胰腺发育和分化调节关键因子的表达，从而影响子代胰腺发育。
2.表观遗传修饰异常机制：不良宫内环境所致IUGR存在着关键基因的表观遗传修饰改变，这种改变往往能够导致子代出生后代谢表型异常[4]。研究已证实，子宫胎盘功能不足所致大鼠IUGR早期阶段Pdx-1的下调与其启动子区表观遗传修饰有关[33]，即胰岛中Pdx-1近端启动子招募组蛋白去乙酰化酶1和共抑制因子Sin3A，而失去了与Pdx-1的结合，从而导致Pdx-1表达受到抑制。出生后，组蛋白H3K4去甲基化而H3K9甲基化，诱导新生儿期Pdx-1表达持续性下调。成年后，随着H3K9me2的累积，Dnmt3a募集至Pdx-1启动子，促进启动子区近端一个高度保守CpG位点的DNA甲基化，最终导致Pdx-1的永久沉默而罹患糖尿病。
越来越多的证据表明，miRNAs在胚胎发育及胰腺的生理功能发育中发挥着重要的作用，这些miRNAs包括miR-483-3p、miR-375和miR17-92等[34]。有研究[35]发现，IUGR的成人及早期营养不良暴露所致IUGR成年大鼠体内脂肪组织miR-483-3p的表达显著增加，从而抑制其下游靶基因生长/分化转录因子3(growth/differentiation factor-3,GDF-3)的表达，提示miR-483-3p参与IUGR大鼠成年期代谢性疾病易感如脂毒性、胰岛素抵抗的分子编程作用。另外，Dumortier等[36]研究表明，孕期营养不良引起子代大鼠内分泌胰腺miRNAs编程性改变，其中miR-375变化最明显，主要表现为miR-375高表达介导的子代β细胞增殖减少以及葡萄糖刺激胰岛素分泌功能受损。
3.线粒体氧化损伤机制：作为体内胰岛素合成和分泌的主要场所，胰腺β细胞有着较高的氧化性能量需求。然而，胰岛本身抗氧化酶水平却非常低[37]，与其他组织相比，胰腺组织对线粒体功能障碍与ROS暴露更敏感。因此，任何线粒体功能缺陷和氧化应激增加都将对β细胞功能产生严重的不良影响，如损伤葡萄糖刺激胰岛素的分泌，降低β细胞关键基因的表达及诱导细胞死亡等[38]。Maechler等[39]研究发现，β细胞短期暴露于H2O2中，激活氧化应激可以使β细胞胰岛素mRNA减少，胞质和线粒体内钙离子流减少。Simmons等[40]在子宫动脉结扎所致IUGR大鼠模型上发现，胎儿β细胞线粒体存在功能障碍，ROS水平升高以及线粒体DNA损伤。这三者构成的恶性循环随年龄增长不断增强进而引起β细胞功能逐渐丧失，最终导致成年后糖尿病的发生。一方面，氧化应激产物增加可能导致胰岛细胞磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase，PI3K)活性降低，胰岛素信号通路受阻，进而导致胰岛细胞分泌功能障碍。另一方面，解耦联蛋白2(uncoupling protein 2,UCP2)在线粒体内膜上广泛存在，通过使线粒体的氧化磷酸化过程解耦联，导致ATP产生减少并影响胰岛素分泌[41]。此外，PGC-1α是线粒体生物合成的强诱导剂，Besseiche等[42]发现体内外β细胞PGC-1α特异性过表达可诱导氧化应激，进而激活AMPK通路，最终导致β细胞分泌功能受损。
众多动物实验表明，IUGR的代谢编程现象可以持续至第二代[43]。无论是饮食限制、蛋白限制还是缺血模型上观察到F1代糖耐量减弱以及高血糖发生的同时，F2代也呈现糖尿病发生的迹象[44-46]。哺乳期蛋白限制可以导致雄性F2代出现胰岛素抵抗，但如果低蛋白饮食发生在孕期，则受影响的是雌性F2代，表明性别差异来源于F1代胎鼠不良环境暴露时间窗[47]。在一项产前乙醇暴露研究中，糖代谢功能紊乱可以通过母系遗传至F2代[48]，提示母体代谢紊乱可能是一个重要的影响因素。
由于表观遗传可以受到环境的刺激而发生变化，而改变的表观遗传特性可在细胞分裂中得以保持并存在。因此，表观遗传很可能是不良宫内环境所致IUGR子代糖代谢功能编程改变的传代效应现象的重要分子基础[29]。现有的研究也证实，由不良宫内环境所致的IUGR子代糖代谢异常的遗传现象往往不依赖于基因序列的变化，而与表观遗传性状的改变密切相关[49]。尽管表观遗传机制包括DNA甲基化、组蛋白修饰和小RNA分子，但研究者目前主要关注的是营养或其他环境刺激所诱发的DNA甲基化模式改变[50-51]。DNA甲基化主要指甲基基团与CpG岛上半胱氨酸残基结合。甲基化模式在哺乳动物早期发育过程中会发生两次重置，首先是在着床前发育时来自于父方和母方的等位基因先后被移除[52]。新的甲基化模式产生于着床后发育的早期过程。在胚胎时期的原始生殖细胞中，DNA甲基化模式同样会被替换[53]。已证实，对雌鼠饮食进行干预能引起可遗传的表观遗传修饰改变，提示饮食中甲基供体的摄入量能够改变机体早期甲基化模式[54]。
综上所述，各种孕期不良环境所致IUGR可影响胰腺的结构发育、细胞分化、增殖和凋亡等多个方面，并最终导致β细胞功能的缺陷，其机制可能与GC过暴露、表观遗传修饰、线粒体氧化损伤等多个因素有关。胰腺发育编程的异常往往与代谢综合征、T2DM等代谢性疾病的发生发展紧密相连，而这种编程改变存在着以DNA甲基化修饰为基础的可遗传效应。开展IUGR胰腺发育编程和功能改变的相关机制研究，不仅对于揭示IUGR与成年期T2DM的内在联系具有重要的指导意义，同时也为未来减少IUGR发生及相关成年疾病治疗性干预措施的建立提供了美好的蓝图。
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