【作者】 夏水秀；

【导师】 徐智策；

【作者基本信息】 苏州大学 ， 胚胎生理与围产基础医学， 2014， 博士

【摘要】 研究背景：高血压和肾疾病的发病率及死亡率日益增加，严重威胁人类健康。长期以来，认为遗传和生活方式在其发病中起重要作用。近年来，大量的流行病学研究表明宫内不良环境造成胎儿的发育程序化，导致其发生高血压和肾脏疾病的风险增加。出生体重低于2500克的婴儿均称为低出生体重（Low Birth Weight, LBW）儿，通常由早产（Preterm Birth, PB）或宫内生长迟缓（Intrauterine Growth Retardation,IUGR）所导致。据统计，LBW发生率约为7-15%。1989年David Barker教授首次提出心血管疾病与出生体重有关，进而发展成胚胎编程学说：低出生体重（Low BirthWeight, LBW）是心血管疾病发生的独立危险因素之一，对成年期的肾脏疾病和高血压可产生不同的影响。PB或IUGR导致的LBW伴发肾及肾单位发育不良，是日后肾疾病发生的危险因素。充足的氧供对胎儿的生长发育是必需的，缺氧会影响胎儿发育。孕期缺氧是胚胎发育中的常见现象，妊娠期母体的多种生理因素或病理因素诱发孕妇缺氧。研究表明胎儿孕期缺氧不仅会引起胎儿宫内生长受限，造成胎儿LBW，还会造成胎儿重要组织器官譬如肾的发育不良。孕期缺氧对胎儿肾发育造成怎样的病理改变，目前国内外报道甚少。大量研究显示：成熟肾脏在病理状态下高表达的分子，在肾脏发育过程中也高表达，而且细胞表型的改变类似于胚胎肾发育过程中后肾间充质细胞向上皮细胞转分化的逆过程。这些研究结果提示肾脏损伤后细胞的再生过程和肾脏胚胎发育过程有着极其密切的关系，因此，对肾发育的研究有助于理解后天性肾脏疾病发生的病理生理机制。自噬和凋亡在胚胎、器官的发育以及组织分化过程中起重要作用。自噬，又名II型细胞程序性死亡，是一个自体吞噬的过程，即：亚细胞膜结构发生动态的形态学改变，并通过溶酶体介导蛋白质和细胞器降解的过程，主要包括4个环节：底物诱导自噬前体（proautophagosome，PAS）的形成、自噬体（autophagosome）形成、自噬体与溶酶体融合和自噬体内容物被降解。LBW患者肾及肾单位发育落后的机制还不完全清楚。研究表明I型细胞程序性死亡即凋亡是LBW患者肾及肾单位发育落后的原因之一。自噬是否也参与了这一过程国内外尚未有报道。自噬激活过程，往往与凋亡共存或同时发生。一个突破性的研究表明凋亡抑制分子BCL-2可以通过与自噬的标记物之一Beclin-1相互作用，进而对自噬发挥作用。自噬在孕期缺氧大鼠肾单位发育的作用及其机制目前国内外都未见报道。因此，进一步论证自噬与肾单位发育的关系，深入分析其分子调控机制就显得十分必要。PI3K/Akt/mTOR和HIF-1α/BNIP3/Beclin-1是细胞低氧条件下发生自噬上调的二条重要信号通路。本课题对此二条信号通路是否参与了孕期缺氧诱导的肾发育障碍的肾细胞的损伤进行初步的研究。为探讨孕期缺氧对胎鼠肾脏发育落后的机制，本实验利用大鼠及大鼠肾细胞株（NRK-52E），分别从整体、组织器官、细胞、分子水平，就孕期缺氧诱导的自噬对胎鼠肾脏发育的影响及自噬信号通路进行了研究。第一部分孕期缺氧对胎鼠形态学、血气及肾脏发育和功能的影响目的：观察孕期缺氧对胎鼠形态学的影响，以及对肾脏发育和功能的影响。方法：孕鼠从妊娠第4天至第21天（Gestation Day，GD4-21）置缺氧箱（常压低氧，O2浓度为10.5%），建立LBW动物模型。缺氧期间每天称量孕鼠饮食量及体重，计算体重增长量。于GD21天，以剖宫术取胎鼠。以正常胎鼠做对照，测量胎鼠（GD21）的身长、尾长，称量胎鼠体重、脏器如心、肝、肺、肾和脑等重量；用血气分析仪检测胎鼠血气指标；用酶标法分析胎鼠血液中尿酸（UricAcid, UA）、血尿素氮（Blood UreaNitrogen, BUN）、肌酐（Creatinine, CRE）含量；分别对上述样本肾脏组织进行HE染色，光镜观察组织结构；电镜观察胎鼠肾脏的超微结构。结果：与对照组相比，孕期缺氧对孕鼠饮食量有一过性影响、对体重增长量无明显影响，但可显著降低胎鼠的体重、肾量、心重、肝重、肺重及肾重指数、肝重指数；明显降低胎鼠血液中pO2、SO2%水平。组织形态学结果显示孕期缺氧可使胎鼠肾间质增生、肾囊腔增宽、肾小球固缩；足细胞足突分支减少且变低平，线粒体肿胀变形，嵴固缩。结论：孕期缺氧导致胎鼠及肾脏发育落后。第二部分孕期缺氧诱导IUGR胎鼠肾脏自噬及凋亡的机制研究目的：探讨孕期缺氧对胎鼠肾脏自噬及凋亡程度的影响；研究孕期缺氧调控自噬及凋亡的信号通路。分析凋亡及自噬与孕期缺氧诱导的肾发育障碍的相关联系。方法：用放免法检测IUGR胎鼠肾脏组织中可溶性抗凋亡蛋白及自噬相关蛋白；用透射电镜法观察实质细胞中自噬结构的改变；用免疫印迹法检测肾脏组织中凋亡相关蛋白、自噬标记蛋白以及自噬通路、凋亡通路关键蛋白；采用real-time PCR法检测缺氧后肾脏组织LC3mRNA的表达水平。TUNEL染色法检测肾脏细胞发生凋亡的程度。结果：孕期缺氧可导致胎鼠肾脏组织APG5L含量增加，而sFAS无显著变化。孕期缺氧可使肾细胞中自噬小体及自噬溶酶体等自噬结构显著增加，自噬活性增加。与对照组相比，缺氧组自噬相关蛋白LC3-II表达明显增加，而LC3-I表达无明显变化，LC3-II/LC3-I显著增高；p62及BCL-2蛋白表达明显减少。缺氧胎鼠肾脏中的自噬通路关键蛋白Beclin-1, p-S6, HIF-1α表达增加，而AKT及p-AKT含量减少。TUNEL染色阳性细胞缺氧组明显多于对照组。结论：孕期缺氧引起胎鼠肾脏组织中自噬上调及自噬活性增加。激活自噬的机制可能是HIF-1α/BNIP3/Beclin-1通路的激活，而与PI3K/Akt/mTOR通路关系不大。细胞凋亡增加的可能机制是抑制BCL-2信号通路而激活了caspase-3信号途径，与sFas/FasL信号受体途径无关。第三部分低氧对NRK-52E细胞自噬的影响目的：观察低氧对NRK-52E细胞的生长以及自噬程度的影响；探讨自噬在低氧环境下对细胞存亡的影响。方法：首先常规培养NRK-52E细胞，通过倒置显微镜下观察细胞生长及细胞计数，绘制生长曲线图，探索NRK-52E细胞的增殖期，明确NRK-52E细胞进行药物干预的最佳时间；通过western-blot法检测不同浓度3-MA干预后对自噬标记蛋白表达水平影响程度，确定药物干预的最适药物浓度。在最适时机用最佳3-MA药物浓度干预后，用免疫印迹法检测LC3、Beclin1及p62的蛋白表达水平，推测低氧对细胞自噬及其活性的影响；通过MTT法及LDH法分别检测细胞存活率及乳酸脱氢酶漏出率的检测，分析低氧诱导的自噬对细胞存亡的影响。通过使用三气培养箱的使用建立NRK-52E细胞缺氧模型，应用自噬抑制剂，检测自噬表达被抑制时NRK-52E细胞的生长情况；分析在低氧环境中自噬对NRK-52E细胞的存活的作用。结果：在24小时内细胞呈指数形式生长，24小时后进入生长平台期，获得了低氧干预的最佳时间；低氧环境使NRK-52E细胞增殖速度减慢，细胞存活率下降而死亡率上升；同时细胞死亡比率也随缺氧时间的延长而递增；缺氧24h内，细胞数量仍可增加，但其后随缺氧时间延长，细胞数量逐步下降。对低氧的细胞给予3-MA处理后，能明显抑制低氧引起的LC3-II、Beclin1、激活型caspase-3（p20）上调，并维持p62的蛋白表达水平。结论：低氧可使NRK-52E细胞增殖减缓、存活率下降。抑制自噬可减少缺氧引起的细胞死亡（包括凋亡）。更多还原

【Abstract】 Background: The morbidity and mortality of hypertension and kidney diseaseshowed a highly prevalent and increasing tendency in recent decades. It has long beenrecognized that genetic and lifestyle factors play an important role in these diseases.However, epidemiologic studies have demonstrated that the suboptimal intrauterineenvironment can induce fetal development programming, leading to increased risk ofhypertension and kidney disease. Low birth weight (LBW) infers.Infant birth weight lessthan2500grams are known as. Preterm birth (PB) or intrauterine growth retardation(IUGR) often leads to LBW. According to statistics, the incidence of LBW is about7-15%.In1989, a professor, named David Barker, firstly demonstrated that cardiovascular diseasewas associated with birth weight, and the viewpoint was put forth the provocativehypothesis of fetal programming or developmental plasticity of adult disease: LBW is oneof the independent risk factors for cardiovascular disease, and affects adulthood on renaldisease and high blood pressure. LBW infants, caused by PB or IUGR, are oftenaccompanied with kidney and nephron dysplasia, which is a risk factor for kidney diseasein the future. Adequate oxygen is necessary for the fetal growth and hypoxia can affectdevelopment of fetuses. Hypoxia is a common phenomenon in embryonic development.During pregnancy, a number of maternal physiologic or pathologic factors, includingplateau-hypoxia, gestational hypertension, heart failure, anaemia, preeclampsia, pathologicand umbilical cord factors, which induced maternal hypoxia and lead to fetal poorintrauterine fetal growth (PIFG) because of nutrient substance insufficent. Many studiesreported that prenatal hypoxia (PH) not only caused litter size reduction and small bodysize at birth, but also induced fetal programming of abnormality in many important organssuch as kidney. It is unknown how prenatal hypoxia affects the fetal renal development. Alarge amount of research shows that some molecules which are high expression in maturepathological kidney as well as in fetal kidney, and moreover, the changes of cellularphenotype are similar to the inverse process of cellular differentiation from metanephronMSCs to epithelial cell. These results indicate that cellular regeneration after renal damage closely relates to embryonic development of kidney. Therefore, the study on renaldevelopment helps to understand the pathophysiology of renal diseases. Sufficient oxygenis necessary for fetal development, and in utero hypoxia has been demonstrated adverseeffects on fetal development. In the study of possible mechanisms underlying the reducednumber of nephron by insults during pregnancy, previous work demonstrated the roles ofapoptosis which is known as type I programmed cell death. Apoptosis, which is known astype I programmed cell death (PCD), plays an important role in nephrogenesis. Recentstudies showed that apoptosis was adversely involved in renal development. Autophagyand apoptosis play an important role in the development of embryo and the differentiationof organ and tissue. Whether autophagy, type II PCD, also contributes to impairment ofnephrogenesis remains unknown. Furthermore, there is no information on whetherintrauterine hypoxia may affect renal autophagy in the fetal kidney. Thus, the present studyinvestigated whether and how autophagy occurred in the fetal kidney following hypoxia.Autophagy is an evolutionarily conserved pathway which involves in degradation of theaging and damaged organelles or macromolecular proteins and maintains cellarhomeostasis when cells undergoing stress. In autophagy, double-membraneautophagosomes envelop and sequester intracellular components and then fuse withlysosomes to form autolysosomes which degrade their contents to regenerate nutrients.Autophagy often coexist with apoptosis. The relationship between autophagy andapoptosis is complicated but interesting. A groundbreaking research shows that apoptosiscan affect autophagy by BNIP3competeing with Beclin-1for binding to BCL2andthereby increasing the levels of free Beclin-1, which triggers autophagy. Whether and howhypoxia during pregnancy impacts on fetal renal development as well as renal functions isstill unclear. Thus, the present study investigated whether and how autophagy occurred inthe fetal kidney following hypoxia.There are two important signaling pathways in the regulation of autophagy induced byhypoxia. One is HIF-1α/BNIP3/Beclin-1pathway, the other PI3K/Akt/mTOR. The presentstudy examined the key elements in both signaling pathways associated with renaldevelopmental problems. Together, the present study tested the hypothesis that chronichypoxia during pregnancy may adversely affect renal development related to renalautophagy in the rat fetus. In order to clarify the function of autophahy on the fetal kidneywith hypoxia, we employ the glomerular endothelial cells of rats to analyze the harmful orbeneficial effect on cells by active autophagy which was induced by hypoxia. The presentstudy aimed at:1) to determine what fetal pathophysiological changes in vivo in the kidney of term fetal rats following hypoxia; and2) to investigate whether prenatal chronic hypoxiainfluence apoptosis or autophagy in the kidney of the fetus; and3) to explore the possiblemechanisms or pathways of autophagy in the fetal kidney. The data gained provided novelinformation on hypoxia-induced renal developmental risks and the related mechanisms forkidney health problems in fetal origins.Part1The effects of intrauterine hypoxia on morphology, bloodgas indexs, and renal function of fetusObjective: To determine the effects of the intrauterine hypoxia on morphology, bloodgas indexes, and renal function of fetus. Methods: Pregnant rats exposed to hypoxia (O210.5±0.5%) from day4to day21of pregnancy (GD4-21). The fetus (GD21) were studied.Physiological and histopathological examinations (with light microscope and withtransmission electron microscope) of the fetal kidney were evaluated. Food intake andbody weight of the pregnant rats were measured during gestation period; Fetal body weight,heart weight, kidney weight, brain weight, lung weight, liver weight, stem length and taillength were measured at gestation21day; Blood gases and electrolytes were determinedwith a Nova analyzer. Blood samples were centrifuged and serum was used formeasurements of albumin (ALB), uric acid (UA), creatinine (CRE), and urea nitrogen(BUN) using the enzyme colorimetric method. For histological analysis, the slices of fetalkidney were stained with HE, and then analyzed with microscope. The ultrastructure wasobserved under electron microscopy. Results: Hypoxia during gestation briefly influencethe food intake during GD4-7, and during other times there was no influence on the foodintake of pregnant. Compared with the control, body weight gain of the pregnant ratremained unchanged. In hypoxia group, the survival rate of fetal rat decreased, and theincidence of IUGR increased significantly. Except brain, hypoxia significantly decreasedweight of the fetal body, heart, kidney, lung and liver, accompanied with increased BUN.Intrauterine hypoxia reduced fetal stem and tail lengths, also decreased plasma pO2andSO2%level of fetus, others blood gas index (pH、Na+、K+、Osm、pCO2、Hb、Hct、Glu and Lac) did not changed. Intrauterine hypoxia significantly decreased viscera indexessuch as kidney, liver, and lung index, while the brain/body weight ratio apparentlyincreased, and the heart/body weight ratio remained unchanged. There was no difference infetal ALB, UA and CRE between the normoxia and hypoxia groups. Meanwhile, fetal serum BUN was higher in the hypoxia group than that of the control. The notablehistological changes included that the interstitium was much wider, and cells in theinterstitium increased in the hypoxia fetal kidney. In the glomeruli, the Bowman’s spacewas obviously enlarged, while the sizes of the solid parts in the glomeruli were reduced inthe fetuses exposed to hypoxia. Podocyte foot process was flat and decreased in theglomeruli of the hypoxic fetuses. Conclusion: Intrauterine hypoxia adversely affected thedevelopment of fetus and fetal heart, liver, lung, and kidney. Chronic prenatal hypoxia hada negative influence on the fetal renal structure and renal function. Part2The mechanism of autophagy and apoptosis induced byintrauterine hypoxia in fetal rat kidney with IUGRObjective: To study the autophagy and apoptosis induced by intrauterine hypoxia infetal rat kidney with IUGR; To exploit the mechanism of autophagy and apoptosis inducedby intrauterine hypoxia in fetal rat kidney with IUGR. Methods: Pregnant rats exposed tohypoxia (O210.5±0.5%) from day4to day21of pregnancy (GD4-21). The fetus (GD21)with IUGR were studied. Analyzed sFAS and APG5L of the fetal kidneys. Theultrastructure was observed under transmitted electron microscopy (TEM). The proteinswere analyzed by Western-blotting. The TUNEL technique was used to discriminateapoptotic nuclei. Real-time PCR was used to detect the amount of mRNA.Results: Fetal renal BCL-2was decreased accompanied with higher positive TUNELstaining in the hypoxia group. Prenatal hypoxia increased apoptosis related renal APG5Lwithout change of sFAS. Hypoxia also increased autophagic structures, includingautophagosome and autolysosome, in the fetal kidney. There was a significant increase inLC3-II, Beclin-1, p-S6, HIF-1α, ratio of LC3-II/LC3-I, and a decrease in P62, AKT, andp-AKT, while LC3-I unchanged, in the kidney of the hypoxia group. The account of LC3mRNA apparently increase in hypoxia group compared with normoxia group. Conclusion:Intrauterine hypoxia adversely affect renal development in the rat fetus, up-regulated therenal autophagy and the possible mechanism involved may include the Beclin-1pathway.Intrauterine hypoxia also up-regulated apoptosis by inhibiting the BCL-2signaling wayand activating the caspase-3pathway. Part3The impact of hypoxia on autophagy in NRK-52E cellsObjective: To investigate the effects of hypoxia on the growth and cell autophagy inNRK-52E cells; and to investigate the roles of autophagy in cells under hypoxia survival.Methods: Firstly, NRK-52E cells were cultured conventionally, and cell growthtogether with cell counts were observed under an inverted microscope. And then wedrawed growth chart, explored NRK-52E proliferative cells, and found the best time ofNRK-52E cells which drug intervention. Using western-blot assay, we explored the effectsof the concentration of3-MA intervention on the expression levels of marker autophagyproteins, to determine the optimal drug concentration. After finding the optimal timing ofthe best3-MA concentration of drug intervention, the protein levels of LC3, Beclin1andp62wered detected by Western blot, to study the impact of hypoxia on autophagy and itsactivity. Moreover, cell viability and lactate dehydrogenase leakage rate were detected byMTT and LDH assay, to analyze the impact of hypoxia-induced autophagy on cell survival.Using three gas incubators to establish NRK-52E hypoxia model, and using autophagyinhibitor, to study cell growth of NRK-52E cells when autophagy expression wassuppressed. Additionally, we analysised the effects of autophagy on NRK-52E cellsurvival in hypoxic environment.Results: the NRK-52E cells growed exponentially within24hours, and then enteredthe growth plateau after24hours. So that the best time to hypoxia intervention wasabtained. Cell proliferation of NRK-52E cells slowed, cell viability decreased andmortality increased in hypoxic environment. Meanwhile, the rate of cell death alsoincreased with hypoxia time prolonged. The number of cells is still increasing within24h,but the subsequent hypoxia time prolonged, the number of cells gradually declined.Treatment with3-MA could inhibit hypoxia-induced LC3II, Beclin1, activated caspase-3(p20) upregulation and maintained the levels of p62protein expression.Conclusion: Hypoxia could slow NRK-52E cell proliferation and decrease survival rate.Autophagy inhibition may reduce hypoxia-induced cell death (including apoptosis).更多还原