【作者】 沈茜；

【导师】 徐虹； 查锡良； 张志刚；

【作者基本信息】 复旦大学 ， 儿科学， 2010， 博士

【摘要】 宫内生长迟缓(intrauterine growth restriction, IUGR)可通过“胎儿程序化”途径对机体各器官功能产生长期以至终生的影响,与成年期严重危害人类健康的许多疾病如高血压、冠心病、糖尿病、慢性肾脏病等均有一定的关系。有关IUGR与肾脏疾病关系的人类研究发现,IUGR胎儿肾脏体积较正常胎儿明显缩小同时肾小球数目明显减少,而生后的长期随访发现,IUGR组肾小球滤过功能较正常组明显降低而蛋白尿发生率明显增高。而我科以往动物实验的资料又表明,IUGR大鼠出生时肾小球数目明显减少,同时伴有皮质区变薄和生肾区增厚,且生后随访中蛋白尿和高血压的发生率明显增高。但目前有关IUGR引起出生时肾小球数目减少、肾脏发育异常以及生后肾脏疾病的确切发病机制仍未完全阐明。以往认为,生后早期的营养干预尤其是高蛋白饮食对IUGR患儿体格的快速增长具有一定的帮助。但近来有人提出了“预计的适应性改变”(predictive adaptive response, PAR)的理论：胎儿可根据发育阶段的环境而对生后环境进行预计,引发一系列的适应性改变。研究显示,宫内营养不良的仔鼠如果生后饮食越丰富,其寿命越短,而我科前期有关IUGR新生仔鼠生后高蛋白饮食干预的研究发现,高蛋白饮食不仅不能纠正生后肾小球数目的减少,反而加重了高血压和蛋白尿的严重程度,但目前有关IUGR生后高蛋白饮食干预对肾脏影响的可能机理仍未完全阐明。近年来,蛋白质组学技术在医学基础研究领域得到了广泛应用,它可以整体、定量、动态地研究组织或细胞中蛋白质谱的变化,以及与疾病发生发展不同阶段的关系,在疾病机制的研究和寻找新的药物治疗靶点中具有重要意义。因此,本课题通过蛋白质组学方法研究IUGR新生鼠与正常新生鼠肾脏蛋白质表达谱的差异、IUGR成年鼠与正常成年鼠肾脏蛋白质表达谱的差异以及生后高蛋白饮食干预对肾脏蛋白质表达谱的影响,并进一步进行验证,以探讨IUGR大鼠肾脏发育异常的可能机制、IUGR生后肾脏损害相关的关键蛋白分子及高蛋白饮食干预对肾脏影响的可能机理。为研究IUGR新生鼠与正常新生鼠肾脏蛋白质表达谱的差异,采用孕期全程低蛋白饮食法(6%低蛋白等热卡饲料)建立IUGR大鼠模型,选择新生鼠仔出生体重在正常对照组平均体重-2SD以下者为IUGR组。对照组以孕期常规饲料(含22%蛋白质)饲养至自然分娩,所生新生鼠仔作为正常对照组。8个IUGR组肾脏(平均肾重48mg±3mg,总肾重385mg)和6个正常对照组肾脏(平均肾重64mg±4mg,总肾重383mg)分别混合后抽提总蛋白,双向凝胶电泳后使用双胺银染法染色,凝胶图像分析后切取组间差异明显的蛋白质点通过质谱仪进行肽指纹谱和串级质谱鉴定。差异表达分析及质谱鉴定共筛选出仅在IUGR组表达的蛋白11个(包括非特异性二肽酶、甲基丙二酸半醛脱氢酶、转酮醇酶、尿嘧啶核苷磷酸合成酶、波形蛋白、载脂蛋白A-Ⅳ、细胞角蛋白10、26S蛋白酶调节亚基7、δ-1-吡咯啉-5-羧化脱氢酶、视黄醇脱氢酶1、Rho GDP解离抑制因子α等),仅在正常对照组表达的蛋白1个(即剪接因子精氨酸/丝氨酸丰富9);IUGR组较正常对照组表达大于5倍的蛋白7个(包括尿卟啉原脱羧酶、异质性胞核核糖核蛋白K、75kDa葡萄糖调节蛋白、227kDa纺锤体-着丝点相关蛋白、二硫化物异构酶A3、细胞分裂蛋白激酶2、α电子转运黄素蛋白等),正常对照组较IUGR组表达大于5倍的蛋白2个(即γ-肌动蛋白和串珠素)。这些差异蛋白质主要参与能量代谢、氧化还原、信号转导以及细胞增殖和凋亡等过程,其中结构分子有4个,包括波形蛋白、串珠素、γ-肌动蛋白和细胞角蛋白10。对波形蛋白和串珠素进一步通过western blot和免疫组化方法在肾脏组织中对其差异表达进行验证,结果显示波形蛋白主要表达于肾小球且IUGR组的表达明显高于正常对照组,而串珠素可同时表达于肾小球和肾小管且IUGR组的表达明显低于正常对照组,这一验证结果与蛋白质组的研究结果相一致。同时,为进一步探讨IUGR成年鼠与正常成年鼠肾脏蛋白质表达谱的差异以及生后高蛋白饮食干预对肾脏蛋白质表达谱的影响,对IUGR新生鼠生后分别给予常规饲料(含22%蛋白质,IUGR组)和高蛋白饲料(含30%蛋白质,高蛋白组)喂养直至12周龄(成年期),同时设立正常对照组(孕期常规饲料饲养至自然分娩,所生新生鼠仔生后给予常规饲料喂养直至生后12周龄)。12周龄时,每组各选取6个肾脏分别混合后抽提总蛋白,双向凝胶电泳后使用考马斯亮蓝法染色,凝胶图像分析后切取组间差异明显的蛋白质点通过质谱仪进行肽指纹谱和串级质谱鉴定。差异表达分析及质谱鉴定结果显示,与正常对照组相比,共有11个蛋白质在IUGR组和高蛋白组发生了趋势一致的表达改变,其中包括参与体内代谢的转酮醇酶、谷胱甘肽S转移酶α1、果糖-二磷酸醛缩酶A、乌头酸水合酶,参与氧化还原的羟酸氧化酶2、长链特异性乙酰辅酶A脱氢酶、视黄醇脱氢酶1和谷氨酸脱氢酶1及参与转录和凋亡调控的α烯醇酶、α晶体蛋白和核苷二磷酸激酶B,提示这些差异蛋白质可能参与了IUGR生后肾脏病变的发生。另有8个蛋白质仅在高蛋白营养干预后出现差异表达,其中包括参与氧化还原的异柠檬酸脱氢酶、二硫化物异构酶A3和细胞色素bc1复合物,参与凋亡调控的细胞内氯离子通道蛋白1及参与体内代谢的GM2神经节苷酯活化蛋白、天冬氨酸酰基转移酶2、酰化氨基酸水解酶1和5,10-次甲基四氢叶酸合成酶,提示这些差异蛋白质可能参与了高蛋白饮食干预对IUGR大鼠肾脏的影响。同时还发现有2个蛋白质即抗增殖蛋白和戴帽蛋白(肌动蛋白丝)出现了等级相关的表达差异(正常对照组/IUGR组大于5倍而高蛋白组无明显表达),提示这2个蛋白质可能同时参与了IUGR生后肾脏病变的发生及高蛋白饮食干预对肾脏的影响。对抗增殖蛋白进一步通过western blot和免疫组化方法在肾脏组织中对其差异表达进行验证,结果显示抗增殖蛋白主要表达于肾小管上皮细胞,IUGR组的表达明显低于正常对照组而高蛋白组的表达更弱,这一验证结果与蛋白质组的研究结果相一致。综上所述,能量代谢异常、氧化还原和凋亡的失衡以及信号转导和细胞增殖的异常可能参与了IUGR大鼠肾单位数目减少和肾脏发育的异常。一些关键蛋白分子如谷胱甘肽S转移酶α1、α烯醇酶、核苷二磷酸激酶B、α晶体蛋白等可能参与了IUGR生后肾脏病变的发生,另一些关键蛋白分子如异柠檬酸脱氢酶、细胞内氯离子通道蛋白1等可能参与了高蛋白饮食干预对IUGR者肾脏的影响,而抗增殖蛋白和戴帽蛋白(肌动蛋白丝)则可能同时参与了IUGR生后肾脏病变的发生及高蛋白饮食干预对肾脏的影响。蛋白质组学的方法为研究IUGR肾脏发育异常和生后肾脏损害的可能机制以及探讨生后高蛋白饮食干预对肾脏影响的发病机理提供了新的研究线索和思路,对上述差异蛋白质的深入研究,包括蛋白质表达的验证、蛋白质作用机制的研究和信号通路分析等将有助于今后进一步揭示IUGR肾脏发育异常和生后肾脏损害的可能机制以及IUGR生后高蛋白饮食干预对肾脏影响的发病机理。更多还原

【Abstract】 Intrauterine growth restriction (IUGR) has long-term effects on various organisms through fetal programming. IUGR causes hormone imbalance and metabolic disorders, affects growth and development in many organs and is associated with many diseases that can jeopardize human health such as hypertension, coronary heart disease, diabetes and chronic kidney disease. Human studies of the association between IUGR and renal diseases indicate that, in contrast to the normal fetal kidney, the IUGR fetal kidney has less volume, with a significant reduction in the number of glomeruli. In addition, long-term follow-up after birth shows significantly lower glomerular filtration and greater incidence of proteinuria in the IUGR group compared to the control group. Our previous animal studies have also shown a decrease in the number of glomeruli in IUGR newborn rats. At the same time, the cortex thickness in IUGR group was significantly thinner than that in control group, while the thickness of nephrogenic zone in IUGR group was significantly larger than that in control group, with an increased incidence of proteinuria and hypertension in the postnatal follow-up period. However, the mechanism underlying IUGR-induced abnormal nephrogenesis and the pathogenesis of IUGR-induced postnatal kidney diseases have not been fully clarified.Previous theories have suggested that a high protein diet for infants in whom IUGR is diagnosed, at an early postnatal stage may assist rapid postnatal growth, but the high protein diet may further exacerbate kidney injury. Our previous animal studies have shown that postnatal overnutrition following IUGR causes more severe hypertension and proteinuria than IUGR itself. Currently, there is no definite conclusion on the proper postnatal nutritional intervention for IUGR fetuses.In the past few years, proteomic technology has been used extensively in basic medical research. It has been used to study systemic and quantitative proteomic changes in tissues and cells at different disease progression stages. This is very important when studying disease pathogenesis and when searching for new drug targets. Therefore, we investigated the differences of proteomic profiles between normal and IUGR neonatal rats, the differences of proteomic profiles between normal and IUGR adult rats and the effects of postnatal high-protein diet on the proteomic profiles of kidney by a comparative proteomic method to identify the possible mechanism of nephrogenesis in IUGR rats, the key proteins that are associated with IUGR-induced kidney injury and the effects of high-protein diet intervention on the kidney.In order to explore the possible mechanism of abnormal nephrogenesis in IUGR rats, a low-protein isocaloric diet consisting of 6% protein was provided to the study group throughout the entire pregnancy period until natural labor. The resulting newborn rats with body weight 2 standard deviations below the average were assigned to the neonatal IUGR rats (IUGR group). The normal control group was supplied during the gestation period with conventional feed (22% protein) until natural labor. The resulting neonatal rats were assigned to the control group. Eight neonatal IUGR kidneys (average weight 48±4 mg/kidney, total weight 385 mg) and six neonatal normal kidneys (average weight 64±4 mg/kidney, total weight 383 mg) were mixed for total protein extraction. A series methods including two-dimensional gel electrophoresis, silver staining, mass spectrometry and database searching were used. The differential expression and mass spectrometry analysis found that eleven protein spots were expressed only in the IUGR group (incuding non-specific dipeptidase, methylmalonate semialdehyde dehydrogenase, transketolase, uridine monophosphate synthetase, vimentin, apolipoprotein A-IV, cytokeratin 10,26S protease regulatory subunit-7, delta-1-pyrroline-5-carboxylate dehydrogenase, retinal dehydrogenase-1 and Rho GDP dissociation inhibitor alpha) and one spot only in the control group, which was splicing factor (arginine/serine-rich 9). Seven protein spots were up-regulated more than fivefold (including uroporphyrinogen decarboxylase,75kDa glucose regulated protein, heterogeneous nuclear ribonucleoprotein K,227kDa spindle and centromere associated protein, disulfide-isomerase A3, cell division protein kinase-2 and alpha-electron transfer flavoprotein) and two spots were down-regulated more than fivefold (including gamma-actin and perlecan) in the IUGR group compared with those in the control group. These proteins are involved primarily in energy metabolism, oxidation and reduction, signal transduction, cell proliferation and apoptosis. Meanwhile, there were four structural molecules which were vimentin, perlecan, gamma-actin and cytokeratin 10. Confirmation of vimentin and perlecan expression by western blot and immunohistochemistry showed that vimentin was expressed primarily in glomeruli, and its expression was significantly increased in the IUGR group compared to the control group, while perlecan was expressed both in the glomeruli and the tubules, and the level of perlecan was significantly reduced in the IUGR group compared to the control group. These data were consistent with the findings using the proteomic approach.In order to identify the key proteins that are associated with IUGR-induced kidney injury and the effects of high-protein diet intervention on the kidney, IUGR pups were divided into two groups, fed with either a conventional diet (containing 22% protein, IUGR group) or a high-protein diet (containing 30% protein, HP group) until 12 weeks after birth. The normal control group was fed with a conventional pregnancy diet (22% protein) until natural delivery, and the newborn rats were fed with a conventional diet (22% protein, control group) until 12 weeks after birth. At 12 weeks of age,6 kidneys were selected from each group and were mixed for total protein extraction. A series methods including two-dimensional gel electrophoresis, coomassie brilliant blue staining, mass spectrometry and database searching were used. The differential expression and mass spectrometry analysis found that, compared with control group, a total of eleven proteins showed the same trend of expression changes in IUGR group and HP group. These eleven proteins included glutathione-S-transferase alpha-1, transketolase, fructose-bisphosphate aldolase A and aconitate hydratase that participate in body metabolism; long-chain specific acyl-CoA dehydrogenase, hydroxyacid oxidase-2, retinal dehydrogenase-1 and glutamate dehydrogenase-1 that participate in oxidation-reduction; alpha-enolase, alpha-crystallin and nucleoside diphosphate kinase B that participate in transcriptional and apoptosis regulation. These differentially expressed proteins probably participate in postnatal kidney disease in IUGR rats. Other eight proteins showed differential expression only after high-protein nutritional intervention. These included chloride intracellular channel-1 that participates in apoptosis regulation; isocitrate dehydrogenase, disulfide-isomerase A3 and cytochrome b-c1 complex that participate in oxidation-reduction; GM2 ganglioside activator protein, aspartoacylase-2, aminoacylase-1 and 5,10-methenyltetrahydrofolate synthetase that participate in body metabolism. These differential expression proteins may be related to the effects of high-protein diet intervention on the kidneys of IUGR rats. Meanwhile, it was found that compared with control group, two proteins which were capping protein (actin filament) and prohibitin had consecutive changes among three groups (the ratio of control group to IUGR group>5 and no obvious expression in HP group). These two proteins may be involved both in postnatal kidney disease in IUGR and the effects of high-protein diet intervention on the kidneys of IUGR. Confirmation of prohibitin expression by western blot and immunohistochemistry showed that prohibitin was primarily expressed in renal tubular epithelial cells. Its expression in IUGR group was lower than control group and the expression in HP group was even lower. This data was consistent with our findings using the proteomic approach.In conclusion, data from this study may provide, at least partly, evidence that abnormality of metabolism, imbalance of redox and apoptosis, and disorder of cellular signal and cell proliferation may be the major mechanisms responsible for abnormal nephrogenesis in IUGR. Some key proteins including glutathione-S-transferase alpha-1, alpha-enolase, alpha-crystallin and nucleoside diphosphate kinase B probably are associated with IUGR-induced kidney injury and some other key proteins including chloride intracellular channel-1, isocitrate dehydrogenase are involved in the effects of high-protein diet intervention on the kidney. Meanwhile, capping protein (actin filament) and prohibitin may be involved both in postnatal kidney disease in IUGR and the effects of high-protein diet intervention on the kidneys of IUGR. The comparative proteomic approach has provided new avenues for future research to explore the pathogenesis of abnormal nephrogenesis in IUGR, IUGR-induced kidney injury and the effects of high-protein diet intervention on the kidney.更多还原