【作者】 高倩；

【导师】 盛建中；

【作者基本信息】 浙江大学 ， 病理学与病理生理学， 2014， 博士

【摘要】 1.研究目的：尽管已经有研究结果显示IVF子代会出现血管代谢紊乱、全身性和肺循环功能紊乱以及心血管结构的重塑,但是这方面的机制尚且缺乏研究。我们的课题首先针对中国3-13岁的IVF子代及其条件吻合的对照组子代进行了大样本的血压监测；然后借助脐静脉来模拟子代血管,试图从蛋白水平上发现差异功能蛋白；关于IVF子代后期生长发育中发生紊乱的原因,目前说法不一,有人认为是IVF技术本身导致,有人认为是不孕因素导致,而我们的研究最后试图通过临床和基础研究相结合的方式来发现这些蛋白差异表达的原因,从而挖掘出IVF子代血管功能紊乱的机制。2.材料和方法：(1)对在浙江大学医学院附属妇产科医院借助IVF技术和自然妊娠出生的3-13岁的子代进行随访,以新鲜胚胎移植出生的单胎为实验组,以同期相应的自然妊娠出生的单胎作为对照组,对其身高、体重、BMI、收缩压、舒张压和心率进行统计分析。所选的IVF均为管性不孕,另外两组均排除子代出生时血管功能缺陷、家族型血管疾病、妊娠高血压和妊娠高血糖。(2)从浙江大学附属妇产科医院分别收集IVF组和对照组脐血管14例以及45例IVF组和48例对照组脐血。入选标准为：产妇的年龄为25-35岁,孕周足月,单胎妊娠、出生体重为大于2500g小于4000g,剖腹产,并且排除各种妊娠并发症和子代出生缺陷。其中3例IVF组和3例对照组的脐静脉用来提取蛋白用于蛋白组学实验。其余用于后面的验证实验(荧光定量PCR和western blotting)和免疫组化实验。利用碘雌二醇放射免疫分析药盒测量了脐血中的雌激素浓度。(3)体外实验以人类脐静脉内皮细胞(HUVECs)为研究对象,通过不同浓度的雌激素处理,采用荧光定量PCR技术和western blotting技术检测lumican和vimentin的表达水平。(4)利用小RNA干扰技术将HUVECs的vimentin干扰后,利用免疫荧光、流式细胞术和transwell方法分别检测细胞的形态、细胞周期和细胞的迁移能力。3.实验结果：(1)大规模流行病学调查结果显示在3-6岁和6-13岁组中,IVF组的收缩压和舒张压均显著性高于对照组。(2)应用iTRAQ检测2组脐血管差异蛋白的表达,我们总共鉴定到723个蛋白,其中上调蛋白335个,下调蛋白388个。借助特征分析(Correlation-based Feature Selectim)技术,根据变化倍率至少为+/-1.2倍,最终筛选到了47个差异蛋白,其中20个上调,27个下调。对这47个差异蛋白进行IPA生物信息学分析发现这些差异蛋白与血管系统发育和代谢功能相关。(3)从蛋白组学结果中筛选了一个上调蛋白lumican和下调蛋白vimentin进行验证。荧光定量PCR和western blotting结果与蛋白组学结果是一致的。(4)IVF组的脐血雌激素浓度是显著高于对照组的。说明IVF子代在胚胎发育过程中受到了高雌激素的刺激,因此高雌激素有可能是引发这些蛋白差异表达的重要原因。(5)不同浓度的雌激素处理HUVECs后发现,高浓度的雌激素会上调lumican的表达,却抑制vimentin的表达。这一结果和蛋白组学的结果是吻合的。说明我们前面的假设“雌激素是引发IVF子代脐血管蛋白差异表达的重要因素”是正确的。(6)把HUVECs中的vimentin干扰后,细胞的形态发生了皱缩,细胞周期最阻滞于S期,细胞的迁移能力也受到了抑制,说明该蛋白的表达下调会影响到血管内皮行使正常的生理功能。4.结论：IVF子代脐静脉血管中存在着与血管系统发育和代谢相关的差异功能蛋白的表达,提示这些蛋白可能是IVF子代发生血管功能紊乱的一个重要原因,另外还发现IVF胚胎发育过程中的宫内高雌激素有可能是这些蛋白差异表达的重要影响因素。这些结果初步筛选了IVF相关脐静脉差异表达蛋白,为深入探讨IVF妊娠过程中的血管异常发育机制,进一步明确IVF与不良妊娠结局和子代健康之间的关系提供了可靠的资料。更多还原

【Abstract】 1. ObjectiveAlthough there have been studies showed that IVF offspring will appear vascular metabolic disorders, systemic and pulmonary circulation dysfunctions, and, cardiovascular structures remodeling, the mechanisms remained little known. In our study, we first tested the blood pressure of IVF children and NC children aged3-13year old; using the umbilical veins from IVF children and NC children, we tried to find the differentially expressed proteins in IVF children; someone thought that the IVF treatment or infertility itself led to vascular dysfunctions in IVF children, however there exited no agreement on such subject; but in this study, we attempted to study the clinical and basic research through a combination of to discover the reasons for the differential expression of these proteins, thus digging out the mechanisms of IVF offspring vascular dysfunction2Materials and methods(1) A following up study on the3-13years old children, born in the Women’s Hospital, School of Medicine, Zhejiang University, China, after IVF treatment or NC was performed. The standard IVF singletons were the test group, and the NC singletons were control group. The height, weight, BMI, diastolic blood pressure and systolic blood pressure was analyzed. All the IVF children were tube infertility, and offspring with birth defects, familial vascular disease, pregnancy-induced hypertension or gestational high blood sugar were excluded in both groups.(2)14umbilical vessels from IVF and NC group, and cord blood samples from45IVF and48NC babies were collected after caesarean delivery in Women’s Hospital, School of Medicine, Zhejiang University, China. The inclusion criteria used for mother and children was as follows:maternal mean age between25to35years; full-term delivery; singleton pregnancy; child birth weight between2500to4000g; no indication of pregnancy complications and no birth defect.3umbilical veins from IVF and NC group were used for the proteomic experiment, and the remaining umbilical veins were used for the verification experiment (q-PCR and western blotting). The serum E2level in cord blood was examined according to the protocol of Iodine [125I] Estradiol Radioimmunoassay Kit.(3) The in vitro experiment was performed in human umbilical vein endothelial cells (HUVECs). Treatment of HUVECs with E2at different concentrations, the expression of lumican and vimentin on mRNA and protein level was detected by quantitative PCR and western blotting.(4) After the treatment of vimentin special RNA interference in HUVECs, the cell morphology, cell cycle and migration was detected by immunofluorescence, flow cytometry and transwell, respectively.3. Results(1) The results of epidemiological investigations revealed that both in3-6years group and6-13years group the SBP and DBP were higher in IVF group.(2) Using iTRAQ, we identified723proteins in umbilical veins between the two group, including355up-regulated proteins and388down-regulated proteins. The Correlation-based Feature Selection (CFS) was employed for the protein selection from the MS intensity values of C (NC) and T (IVF) samples. The selected proteins were further inspected to retain the ones with differential expression ratio of at least over+/-1.2. Compared with the NC babies,47DEPs in umbilical veins of IVF babies were found,20proteins were up-regulated while27proteins were down-regulated. IPA software was used for bioinformatics analysis, and the results showed that these DEPs were related to cardiovascular development and metabolism.(3) To confirm the proteomic results, lumican, an up-regulated protein, and vimentin, a down-regulated protein, in IVF group umbilical veins were verified. The results of q-PCR and western blotting showed that the expression level of lumican was up- regulated and the expression level of vimentin was down-regulated in IVF group. And these results are in line with those of proteomics experiment.(4) The concentration of E2in IVF group was significantly higher than the NC group, indicating the fetal development might be stimulated by high concentration of E2. Therefore, the high level E2might be an important cause for the abnormal expression of these proteins.(5) Treatment of HUVECs with E2dose-dependently increased expression levels of lumican while reduced expression levels of vimentin, which was in line with the proteomic results. All these results revealed the high concentration of E2at pregnancy might be an important cause for these disorders.(6) After the treatment of vimentin special RNA interference in HUVECs, the cell morphology was changed, cell cycle was blocked in S phase and the ability of migration was inhibited, suggesting that the reduced expression of vimentin would affect the normal physiological function of vascular endothelial.4. ConclusionDifferentially expressed proteins which were related with the cardiovascular system development and metabolism were found in IVF offspring, suggesting that these proteins may be an important cause of IVF offspring vascular dysfunction. Besides, high intrauterine estrogen during IVF embryogenesis may be an important factor of these proteins differentially expressed. These results are preliminary screening of IVF-related differences in protein expression in human umbilical veins, providing information for further research on IVF vascular abnormal developmental mechanisms, and, for clarify the relationship between IVF and adverse pregnancy outcomes and the offspring health.更多还原