单绒毛膜双胎选择性胎儿生长受限胎盘差异蛋白相关研究
摘要
研究背景
随着辅助生殖技术的广泛开展及女性生育年龄的延后,近年多胎妊娠发生率正逐年上升。我国的多胎妊娠率虽缺少文献报道,但其发生呈上升趋势是无需置疑的事实。多胎妊娠尤其是最常见的双胎妊娠属于高危妊娠,可并发多种妊娠常见并发症,以及一些双胎特有的复杂并发症,例如：双胎输血综合征(twin-to-twin transfusion syndrome, TTTs)、选择性胎儿生长受限(selective intrauterine growth restriction, sIUGR)、双胎贫血-红细胞增多序列征(twin anemia-polycythemia sequence, TAPS)、单羊膜囊双胎、不一致生长双胎等复杂性双胎。目前对复杂性双胎的发病机制、理想的治疗方案、规范化监护、科学的咨询等多方面仍存在很多研究空间,引发世界范围内临床专业医生的关注。
双胎妊娠可分别依据受精卵性质或绒毛膜性进行分类,其中绒毛膜性较受精卵性质更能影响临床结局。绒毛膜性质的判断在双胎妊娠的产前监护中意义重大,对于产前咨询及临床干预有重要的指导意义。2011年加拿大妇产科医师协会(Society of Obstetricians and Gynaecologists of Canada, SOGC)指南指出,应对所有怀疑双胎妊娠孕妇在孕早期进行超声检查,重点确定孕周、胎儿是否成活、是否合并先天畸形及双胎绒毛膜性及羊膜性。推荐所有双胎在孕10-14周期间进行超声检查判定其绒毛膜性质,主要依靠胎儿胎盘数及胎盘λ或T征来明确绒毛膜性质。
选择性胎儿生长受限(sIUGR)是双胎妊娠的一个较为常见的并发症,不仅仅影响胎儿围生期结局,还对新生儿乃至成人后的心血管系统、内分泌代谢系统的功能有长远的影响,但其病因及科学规范的治疗原则尚未明确,是目前复杂性双胎亟待解决的热点问题。目前选择性胎儿生长受限概念应用比较广泛的是：双胎之一胎儿的超声检查估计体重(estimated fetal weight, EFW)小于相应孕周第十百分位。sIUGR临床预后不良,特别是其中单绒毛膜双胎因共用一个胎盘,99%拥有吻合血管支,生长受限胎儿发生宫内死亡之后,发育正常的胎儿有发生严重神经系统后遗症、甚至继发胎死宫内的风险；即便双胎均存活,仍有发生生长受限胎儿在出现短暂心动过缓或血压过低时,双胎间通过动脉-动脉(A-A)吻合支发生急性输血,正常发育胎儿有继而发生大脑脑皮质损害等严重脑部损害的风险。了解该病的发病机制对临床医生理解该病、探索有效治疗方案意义重大。本研究拟先探讨是否有临床高危因素可以预警sIUGR的发生(包括针对不同绒毛膜性),以期指导临床监护方案的制定实施。
胎儿生长受限的病因复杂,研究尚未定论,但主要集中在母体、胎儿及胎盘三方面。对于单绒毛膜双胎sIUGR来说,因胎儿具有相同的遗传物质及共同的宫内发育环境,发生选择性胎儿生长受限的主要原因可以理解为来自胎盘。目前sIUGR的病因学研究主要集中在单绒毛膜sIUGR的胎盘大体研究方面,例如：供应两胎儿胎盘面积比例不均衡,有研究表明胎盘份额相差越大,体重之间差异随之增加；生长受限胎儿常同时伴有脐带异常附着,导致静脉回流量减少并降低有功能的胎盘面积；不同类型胎盘血管吻合的存在等原因。尚缺乏相关的蛋白质组学研究。蛋白质组学作为近年蓬勃发展的新型分子生物学技术,因具有高通量和高灵敏度等优点,为临床疾病的诊断、治疗提供了新手段。本研究拟应用蛋白质组学技术,通过比较单绒毛膜sIUGR双胎中发育受限胎儿胎盘与发育正常胎儿胎盘间蛋白的差异表达,探讨单绒毛膜sIUGR发生的可能机制。
蛋白质组学是应用各种手段研究蛋白质组的一门新兴学科,目的是从整体的角度分析细胞内动态变化的蛋白质组成成分、表达水平与修饰状态,揭示蛋白质功能与生命活动规律,使人们可以深入研究生理状态下同一组织细胞在不同发育阶段或同一组织细胞在不同个体间或同一基因组在不同组织细胞间,及病理情况不同发展阶段下蛋白质表达模式和功能模式的变化,揭示一些重要的生命现象及重大疾病发生发展的规律。
胎盘是连接胎儿和母体的重要器官,在妊娠过程中起着交换物质、内分泌等作用以维持胎儿正常生长。妊娠相关的很多疾病都和胎盘的功能改变有关,蛋白质作为发挥基因功能的载体,研究胎盘的蛋白质组学具有十分重要的临床意义。然而人类胎盘蛋白质组学的发展尚属起步阶段,目前主要应用在早产、流产、子痫前期、辅助生殖技术等疾病对胎盘蛋白的影响研究。至今很多疾病尚未被很好的阐释,将蛋白质组学和胎盘有机结合,可以有利于更好地探讨胎盘源性妊娠相关疾病的病理生理过程,及进一步阐述疾病发生发展的分子机制。
双向荧光差异凝胶电泳技术(two-dimensional difference gel electrophoresis,2D-DIGE)是一种在2D电泳前进行荧光染料标记蛋白样品的方法,基本原理是蛋白质首先根据等电点在PH梯度胶中等电聚焦,然后按照分子量大小进行SDS-PAGE第二向电泳分离,通过对不同的蛋白样品用不同的荧光染料进行标记,在同一块双向凝胶中可同时分离多至三种不同的蛋白样品,并且每块胶上又引用了内标,从而能够对样品间蛋白质丰度差异进行精确分析。
质谱的方法是通过正确测定蛋白质分子的质量进而对蛋白质分子进行鉴定的研究手段,实现蛋白质组学鉴定的质的飞跃。目前质谱分析常用的方法是基质辅助激光解吸电离/飞行时间质谱测量法(MALDI-TOF),将多肽成分转换成离子信号,并根据质量/电荷之比(m/z)来对该多肽进行分析,判断该多肽来源与哪个蛋白。MALDI-TOF方法因其操作简便、敏感度高、可耐受少量杂质存在、易于自动化和通量化、同多种蛋白分离方法相匹配、且数据库中有充足的多肽质量/电荷比值的数据等优点成为目前蛋白质组学研究中最常用的蛋白质谱鉴定方法。
Western blot方法采用聚丙烯酰胺凝胶电泳技术检测蛋白水平的表达,既可以定性又可以半定量检测蛋白,本研究中主要用于验证2D-DIGE实验的结果。
目的：
1、回顾性分析比较双胎妊娠、特别是不同绒毛膜性质双胎的临床并发症以及可能影响不同绒毛膜性选择性胎儿生长受限发生的母体高危因素。
2、通过2D-DIGE技术研究胎盘源性疾病——单绒毛膜sIUGR中发育正常胎儿胎盘与发育受限胎儿胎盘间差异蛋白的表达。
3、对上述所得差异蛋白点进行质谱鉴定,期望找到兴趣蛋白。并对兴趣蛋白进行验证。
方法：
1、回顾性分析我院2009年3月至2012年6月间分娩的明确绒毛膜性双胎妊娠1144例,根据绒毛膜性分为单绒毛膜组和双绒毛膜组,对两组孕妇的一般资料、并发症及临床结局进行比较分析,并将孕妇年龄、孕产次、分娩孕周、受孕方式、绒毛膜性、妊娠期高血压疾病、妊娠期糖尿病、妊娠期肝内胆汁淤积征、胎儿畸形等指标带入Logistic回归分析,探索可能与sIUGR及不同绒毛膜性sIUGR发生有关的临床因素。
2、分别收集3例单绒毛膜选择性胎儿生长受限患者中生长受限胎儿及发育正常胎儿胎盘组织,提取其中的胎盘蛋白质,应用双向荧光差异凝胶电泳技术(2D-DIGE)检测在单绒毛膜sIUGR双胎中生长受限胎儿和发育正常胎儿两者之间胎盘组织差异表达的蛋白质(差异表达上调或下调1.5倍)。
3、对2D-DIGE实验中所得的差异蛋白质点采用时间飞行质谱(MALDI-TOF)鉴定,选取兴趣蛋白,应用Western blot方法对兴趣蛋白进行验证。
结果：
1、我院产科研究期间双胎妊娠的发生率为2.55%(1264/49563),1144例双胎产后明确其绒毛膜性,其中单绒毛膜双羊膜囊双胎239例(20.89%),双绒毛膜双羊膜囊双胎905例(79.11%)。单绒毛膜双胎组与双绒毛膜双胎组在孕妇年龄(28.40±4.27vs30.74±4.17岁,P<0.001)、分娩孕周(35.22±2.36vs35.90±1.91周,P<0.001)、胎儿生长受限(108vs297例,P=0.001)、胎儿生长不一致(59vs125例,P<0.001)、胎死宫内(34vs39例,P<0.001)、大小胎儿的体重(大胎：2328.54±479.06vs2510.07±432.90g,P<0.001；小胎：1983.46±545.72vs2218.88±445.06g,P<0.001)及新生儿窒息(大胎：5vs13例,P<0.001；小胎：11vs15例,P<0.001)间存在显著差异,而在孕产次、妊娠期高血压、妊娠期糖尿病、妊娠期胆汁淤积征、胎膜早破、前置胎盘、产前住院时间、产后出血及胎儿畸形方面无显著差异(P>0.05)。当纳入绒毛膜性时,孕次(OR=0.823,95%CI0.718-0.943,P=0.005)及受孕方式(OR=0.600,95%CI0.435-1.252,P=0.002)是发生选择性胎儿生长受限的保护因素,孕周(OR=1.155,95%CI1.066-1.252,P=0.005)及妊娠期高血压疾病(OR=1.670,95%CI1.167-2.390,P=0.005)则是危险因素。单绒毛膜双胎胎儿生长受限病因的多因素分析中仅孕次(OR=0.719,95%CI0.518-0.999,P=0.049)为保护因素。双绒毛膜双胎胎儿生长受限的多因素分析中孕周(OR=1.215,95%CI1.099-1.344,P=0.000)、妊娠期高血压疾病(OR=1.564,95%CI1.044-2.345,P=0.030)及胎儿畸形(OR:2.833,95%CI1.053-7.619,P=0.039)为危险因素。
2、经双向电泳分离,染色后图像扫描,得到胎盘组织总蛋白双向电泳胎盘图谱。使用DeCyder V6.5软件分析,经过胶内差异分析和生物学差异分析,在所有胶中选取得到分辨清楚的45个差异蛋白点。
3、对所得45个差异蛋白点进行MALDI-TOF/TOF分析,得到蛋白质点的肽质量指纹图,鉴定得到20个差异蛋白质,其中在sIUGR生长受限胎儿(小胎)胎盘中13个蛋白表达上调：钙调蛋白、热休克蛋白β-1、肌球蛋白调控轻多肽9、肌球蛋白调控轻链12b、原肌球蛋白α-3链、ATP合酶亚组d、硫氧还蛋白依赖过氧化氢还原酶/抗过氧化物蛋白、延伸因子Tu、谷氨酸(NMDA)受体亚单位ε2、Ⅰ型细胞骨架19、长上颚，肺癌和鼻上皮癌相关蛋白1、绒毛膜生长催乳激素、原肌球蛋白β链；另有7个蛋白表达下调：Ⅰ型细胞骨架10、Ⅱ型细胞骨架8、糖皮质激素诱导蛋白5、α1-抗胰蛋白酶、纤维蛋白原p链、纤维蛋白原丫链、HHIP-like蛋白1。Western blot方法验证小胎胎盘中钙调蛋白和PRDXⅢ表达上调,与2D-DIGE所得结果一致。
结论：
1、绒毛膜性对孕妇妊娠的临床过程及结局有重要影响,单绒毛膜双胎在分娩孕周、分娩年龄、胎儿出生体重等方面显著低于双绒毛膜双胎(P<0.05)。单绒毛膜双胎的胎儿生长受限、双胎生长不一致发生率、新生儿窒息明显高于双绒毛膜双胎(P<0.05)。而在妊娠期常见并发症(如妊娠期高血压疾病、妊娠期糖尿病、胎膜早破、前置胎盘、妊娠期肝内胆汁淤积征等)的发生率方面单绒毛膜双胎与双绒毛膜双胎之间无显著统计学差异(P>0.05),提示绒毛膜性质对孕妇情况影响不大。在双胎妊娠中,当孕次增加一次,发生sIUGR的风险变为原来的0.823倍,辅助生殖技术受孕双胎发生sIUGR的风险是自然受孕的0.600倍,而当孕周增加一周或并发妊娠期高血压疾病则发生sIUGR的风险分别增加1.155和1.670倍。在单绒毛膜双胎妊娠中,仅当孕次增加一次时,发生sIUGR的风险是原来的0.719倍。而在双绒毛膜双胎妊娠中,孕周增加一周或并发妊娠期高血压疾病或并发胎儿畸形则发生sIUGR的风险分别增加1.215、1.564及2.833倍。在孕早期通过超声明确双胎妊娠的绒毛膜性质对双胎妊娠孕期胎儿监护、临床咨询、干预处理等方面具有重要意义。
2、2D-DIGE可用于进行胎盘组织的蛋白质组学检测,单绒毛膜选择性胎儿生长受限患者的生长受限胎儿及发育正常胎儿所属胎盘间存在差异表达蛋白点。
3、单绒毛膜sIUGR双胎胎盘间存在差异表达蛋白,差异蛋白涉及细胞多种功能：细胞生长、增殖、凋亡、细胞骨架、氧化应激及凝血等方面。
4、Western blot实验证明2D-DIGE的结果正确可靠。
Background
The incidence of twin pregnancy continues to rise, which has been linked largely to the increased utilization of artificial reproductive technologies and, to a lesser extent, the increase in mean maternal age over this period of time. Twin pregnancies are more prone to complications than single pregnancies, with an increased rate of preterm birth, low birth weight, malformations, gestational hypertension, gestational diabetes and unique complications such as twin-twin transfusion syndrome (TTTs), intrauterine growth restriction (IUGR). In order to design potential strategies to reduce these high risks, more knowledge is needed about the natural course of twin pregnancies.
Twin pregnancies are classified according to either zygosity or chorionicity, and chorionicity rather than zygosity determines the outcome. During pregnancy, twins are classified as dichorionic(DC) or monochorionic(MC) based on placentation. MC twins have an increased perinatal mortality rate than DC twins, this can be explained by servering the individual placental anastomoses. To establish the chorionicity, the placenta and dividing membrane between twins were examined by the ultrasound between11-14gestational weeks. Ultrasound criteria used for the determination of chorionicity in the first and early second trimester were the number of placental sites, the lambda or "T" sign.
Selective intrauterine growth restriction (sIUGR) is a common condition associated with twin pregnancy. The term'selective intrauterine growth restriction' in twin pregnancies is applicable to cases where the estimated fetal weight (EFW) of the small fetus falls below the10th percentile. Significant fetal weight discordance is an important element of the clinical picture, which will often accompany this condition, but is not necessary for diagnosis. It is increasingly considered to be an important complication of MC twins, with potentially significant risks of intrauterine fetal demise (IUFD) or neurological adverse outcome for both twins. In recent years the pathophysiological insight of sIUGR has been substantially improved, although the ability to reliably predict the clinical outcome remains elusive-this will probably remain the case, as the clinical presentation and outcome seem to depend on a combination of multiple factors. The most feared complication of sIUGR in MC twins was the intrauterine demise of the growth-restricted twin, which carries an associated postmortem risk of acute feto-fetal transfusion from the normal to the deceased IUGR fetus. Indeed, death of one MC twin has been reported to be accompanied by concomitant death of the larger twin in25-30%of cases and neurological damage in30%of survivors. The principle cause for the development of sIUGR in MC twins is inadequate placental sharing. The relationship between unequal placental territory and birth weight discordance has recently been revealed in several studies showing that discordance increases with increased placental territory discordance. Extremely asymmetric distribution of placental territories is often associated with very eccentric or velamentous cord insertion, although it is unclear whether velamentous insertion is a mere consequence of the asymmetric displacement of the vascular equator or whether it has any implications in the pathophysiology of growth restriction. Aside from placental territory discordance, a second factor largely influencing fetal weight discordance and the natural history of sIUGR in MC twins is the presence of vascular anastomoses in the MC placenta. To manage twin growth disorders, it is important to detect the high-risk factors and prevent the neonatal complications. Thus, the aim of this study was to investigate the maternal risk factors for sIUGR firstly, and then using two dimensional difference gel electrophoresis (2D-DIGE) to identify novel proteins associated with the restricted(smaller) and the other normal(bigger) fetus's placenta in monochorionic selective intrauterine growth restriction (sIUGR) twins.
The placenta plays a vital role in the regulation of fetal growth and development during pregnancy. Roles include nutrient supply to the fetus, removal from the fetus of metabolic waste and hormone production. There have reports about abortion, pre-eclampsia, preterm, assistant reproductive technology using proteomics technology, but there has no research studied for sIUGR yet.
2D-DIGE is a method which label protein samples with different fluorescent dyes before2-D electrophoresis, and can separate up to three different protein samples at the same time in one two-dimensional gel. Since the most obvious advantage of DIGE system is integrating the advantages of both CyDye DIGE dye multiple labeling method and DeCyder difference2D analysis software. Base on the advantages of DIGE technology, great opportunities comes to us to discover the information of differential proteome contained in sample of the patients with sIUGR.
Based on difference gel electrophoresis, MALDI-TOF/TOF mass spectrometry was used to identify the differential protein spots. Western Blot was performed to validate representative findings from the2D-DIGE analysis. Objective
1. To evaluate the impact of chorionicity on the perinatal outcomes of twin pregnancies. And to investigate the high-risk factors in monochorionic sIUGR and dichorinic sIUGR.
2. To compare the differences of protein expression of the restricted(smaller) and normal(bigger) fetus's placenta in monochorionic sIUGR in hopes of finding associatied proteins of sIUGR by using2D-DIGE, so as to elucidate the pathogenic mechanism of sIUGR.
3. Based on difference gel electrophoresis, MALDI-TOF/TOF mass spectrometry was used to identify the differential protein spots. Western Blot analysis was performed to validate representative findings from the2D-DIGE analysis. Methods
1. In total,1264pairs of twins were enrolled from March2009to June2012.According to the chorionicity,1144pairs of twins were divided into monochorionic twins and dichorionic twins. Maternal characteristics, complications, and perinatal outcomes were analyzed among the two groups. Logistic regression was performed to identify the interrelationships between independent maternal risk factors for sIUGR in a multivariable model.
2. The total proteins extracted from placenta tissues of the smaller and bigger fetuses were separated by means of2D-DIGE. The first dimension was carried by IPG PH gradient isoelectric focusing system, the second dimension was separated by vertical SDS-PAGE.
3. Differentially expressed proteins spots in2D-DIGE maps were chosed and incised after in-gel by trypsin, and the candidate proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF). The peptide mass spectrometry maps and acid sequences were searched from SEQUEST databases. And representative findings from the2D-DIGE analysis were confirmed by western blot analysis. Results
1. The overall twin pregnancy incidence was2.55%. Monochorionic twin pregnancies was more earlier in maternal age and delivery age than dichorionic twin pregnancies (P<0.05). And monochorionic twin pregnancies had more sIUGR, discordance growth, intrauterine death and neonatal asphyxia than dichorionic twin pregnancies (P<0.05). The co-twins'birth weights of monochorionic twin pregnancies were lower than that of dichorionic twin pregnancies (P<0.05). Gravidity and mode of conception were protective factors, hypertensive disorders of pregnancy and delivery age were high risk factors for all sIUGR. Gravidity was the protective factors for the MC sIUGR. Whereas hypertensive disorders of pregnancy, delivery age and fetal malformation were high risk factors for the DC sIUGR.
2. Compared with the bigger fetus' placenta,45protein spots with1.5folds of difference statistically in smaller fetus'placenta were found.
3.20proteins were identified by MALDI-TOF/TOF, including13up-regulated and7down-regulated proteins spots. Among them, Fibrinogen beta chain, Fibrinogen gamma chain, Keratin(type I cytoskeletal10), Keratin(type II cytoskeletal8), Glucocorticoid-Inducible protein5, Alpha-1-antitrypsin, HHIP-like protein1were down regulated in restricted fetuses' placentas compared with normal fetuses' placentas, and Elongation factor Tu, Keratin(type I cytoskeletal19), Tropomyosin beta chain, Tropomyosin alpha-3Chain, Glutamate receptor Subunit epsilon-2, Heat shock protein beta-1, Thioredoxin-dependent peroxide reductase, Chorionic somatomammotropin hormone, ATP synthase subunit d, Myosin regulatory light polypeptide9, Myosin regulatory light chain12B, Calmodulin, Long palate,lung and nasal epithelium carcinoma-associated were up regulated in restricted fetuses' placentas. Western blot analysis of calmodulin and PRDXIII confirmed that these two proteins were significantly up-regulated in smaller fetuses's placentas. Conclusion
1. There was a trend towards worse outcomes in MC pregnancies compared with DC pregnancies. The gravity, mode of conception, delivery age, hypertensive disorders of pregnancy and fetal malformation were related with the sIUGR. It is recommended that the chorionicity should be examined by the ultrasound between11-14gestational weeks every twin pregnancy.
2. Proteomic analysis of placenta reveals differential expression of several proteins between fetuses of monochorionic sIUGR.
3. These proteins are implicated in a variety of cellular functions such as cell survival and proliferation, cell apoptosis regulation, cytoskeletal structure, oxidative stress and coagulation.
4. Western blot analysis of calmodulin and PRDXIII confirmed the findings from the2D-DIGE.
随着辅助生殖技术的广泛开展及女性生育年龄的延后,近年多胎妊娠发生率正逐年上升。我国的多胎妊娠率虽缺少文献报道,但其发生呈上升趋势是无需置疑的事实。多胎妊娠尤其是最常见的双胎妊娠属于高危妊娠,可并发多种妊娠常见并发症,以及一些双胎特有的复杂并发症,例如：双胎输血综合征(twin-to-twin transfusion syndrome, TTTs)、选择性胎儿生长受限(selective intrauterine growth restriction, sIUGR)、双胎贫血-红细胞增多序列征(twin anemia-polycythemia sequence, TAPS)、单羊膜囊双胎、不一致生长双胎等复杂性双胎。目前对复杂性双胎的发病机制、理想的治疗方案、规范化监护、科学的咨询等多方面仍存在很多研究空间,引发世界范围内临床专业医生的关注。
双胎妊娠可分别依据受精卵性质或绒毛膜性进行分类,其中绒毛膜性较受精卵性质更能影响临床结局。绒毛膜性质的判断在双胎妊娠的产前监护中意义重大,对于产前咨询及临床干预有重要的指导意义。2011年加拿大妇产科医师协会(Society of Obstetricians and Gynaecologists of Canada, SOGC)指南指出,应对所有怀疑双胎妊娠孕妇在孕早期进行超声检查,重点确定孕周、胎儿是否成活、是否合并先天畸形及双胎绒毛膜性及羊膜性。推荐所有双胎在孕10-14周期间进行超声检查判定其绒毛膜性质,主要依靠胎儿胎盘数及胎盘λ或T征来明确绒毛膜性质。
选择性胎儿生长受限(sIUGR)是双胎妊娠的一个较为常见的并发症,不仅仅影响胎儿围生期结局,还对新生儿乃至成人后的心血管系统、内分泌代谢系统的功能有长远的影响,但其病因及科学规范的治疗原则尚未明确,是目前复杂性双胎亟待解决的热点问题。目前选择性胎儿生长受限概念应用比较广泛的是：双胎之一胎儿的超声检查估计体重(estimated fetal weight, EFW)小于相应孕周第十百分位。sIUGR临床预后不良,特别是其中单绒毛膜双胎因共用一个胎盘,99%拥有吻合血管支,生长受限胎儿发生宫内死亡之后,发育正常的胎儿有发生严重神经系统后遗症、甚至继发胎死宫内的风险；即便双胎均存活,仍有发生生长受限胎儿在出现短暂心动过缓或血压过低时,双胎间通过动脉-动脉(A-A)吻合支发生急性输血,正常发育胎儿有继而发生大脑脑皮质损害等严重脑部损害的风险。了解该病的发病机制对临床医生理解该病、探索有效治疗方案意义重大。本研究拟先探讨是否有临床高危因素可以预警sIUGR的发生(包括针对不同绒毛膜性),以期指导临床监护方案的制定实施。
胎儿生长受限的病因复杂,研究尚未定论,但主要集中在母体、胎儿及胎盘三方面。对于单绒毛膜双胎sIUGR来说,因胎儿具有相同的遗传物质及共同的宫内发育环境,发生选择性胎儿生长受限的主要原因可以理解为来自胎盘。目前sIUGR的病因学研究主要集中在单绒毛膜sIUGR的胎盘大体研究方面,例如：供应两胎儿胎盘面积比例不均衡,有研究表明胎盘份额相差越大,体重之间差异随之增加；生长受限胎儿常同时伴有脐带异常附着,导致静脉回流量减少并降低有功能的胎盘面积；不同类型胎盘血管吻合的存在等原因。尚缺乏相关的蛋白质组学研究。蛋白质组学作为近年蓬勃发展的新型分子生物学技术,因具有高通量和高灵敏度等优点,为临床疾病的诊断、治疗提供了新手段。本研究拟应用蛋白质组学技术,通过比较单绒毛膜sIUGR双胎中发育受限胎儿胎盘与发育正常胎儿胎盘间蛋白的差异表达,探讨单绒毛膜sIUGR发生的可能机制。
蛋白质组学是应用各种手段研究蛋白质组的一门新兴学科,目的是从整体的角度分析细胞内动态变化的蛋白质组成成分、表达水平与修饰状态,揭示蛋白质功能与生命活动规律,使人们可以深入研究生理状态下同一组织细胞在不同发育阶段或同一组织细胞在不同个体间或同一基因组在不同组织细胞间,及病理情况不同发展阶段下蛋白质表达模式和功能模式的变化,揭示一些重要的生命现象及重大疾病发生发展的规律。
胎盘是连接胎儿和母体的重要器官,在妊娠过程中起着交换物质、内分泌等作用以维持胎儿正常生长。妊娠相关的很多疾病都和胎盘的功能改变有关,蛋白质作为发挥基因功能的载体,研究胎盘的蛋白质组学具有十分重要的临床意义。然而人类胎盘蛋白质组学的发展尚属起步阶段,目前主要应用在早产、流产、子痫前期、辅助生殖技术等疾病对胎盘蛋白的影响研究。至今很多疾病尚未被很好的阐释,将蛋白质组学和胎盘有机结合,可以有利于更好地探讨胎盘源性妊娠相关疾病的病理生理过程,及进一步阐述疾病发生发展的分子机制。
双向荧光差异凝胶电泳技术(two-dimensional difference gel electrophoresis,2D-DIGE)是一种在2D电泳前进行荧光染料标记蛋白样品的方法,基本原理是蛋白质首先根据等电点在PH梯度胶中等电聚焦,然后按照分子量大小进行SDS-PAGE第二向电泳分离,通过对不同的蛋白样品用不同的荧光染料进行标记,在同一块双向凝胶中可同时分离多至三种不同的蛋白样品,并且每块胶上又引用了内标,从而能够对样品间蛋白质丰度差异进行精确分析。
质谱的方法是通过正确测定蛋白质分子的质量进而对蛋白质分子进行鉴定的研究手段,实现蛋白质组学鉴定的质的飞跃。目前质谱分析常用的方法是基质辅助激光解吸电离/飞行时间质谱测量法(MALDI-TOF),将多肽成分转换成离子信号,并根据质量/电荷之比(m/z)来对该多肽进行分析,判断该多肽来源与哪个蛋白。MALDI-TOF方法因其操作简便、敏感度高、可耐受少量杂质存在、易于自动化和通量化、同多种蛋白分离方法相匹配、且数据库中有充足的多肽质量/电荷比值的数据等优点成为目前蛋白质组学研究中最常用的蛋白质谱鉴定方法。
Western blot方法采用聚丙烯酰胺凝胶电泳技术检测蛋白水平的表达,既可以定性又可以半定量检测蛋白,本研究中主要用于验证2D-DIGE实验的结果。
目的：
1、回顾性分析比较双胎妊娠、特别是不同绒毛膜性质双胎的临床并发症以及可能影响不同绒毛膜性选择性胎儿生长受限发生的母体高危因素。
2、通过2D-DIGE技术研究胎盘源性疾病——单绒毛膜sIUGR中发育正常胎儿胎盘与发育受限胎儿胎盘间差异蛋白的表达。
3、对上述所得差异蛋白点进行质谱鉴定,期望找到兴趣蛋白。并对兴趣蛋白进行验证。
方法：
1、回顾性分析我院2009年3月至2012年6月间分娩的明确绒毛膜性双胎妊娠1144例,根据绒毛膜性分为单绒毛膜组和双绒毛膜组,对两组孕妇的一般资料、并发症及临床结局进行比较分析,并将孕妇年龄、孕产次、分娩孕周、受孕方式、绒毛膜性、妊娠期高血压疾病、妊娠期糖尿病、妊娠期肝内胆汁淤积征、胎儿畸形等指标带入Logistic回归分析,探索可能与sIUGR及不同绒毛膜性sIUGR发生有关的临床因素。
2、分别收集3例单绒毛膜选择性胎儿生长受限患者中生长受限胎儿及发育正常胎儿胎盘组织,提取其中的胎盘蛋白质,应用双向荧光差异凝胶电泳技术(2D-DIGE)检测在单绒毛膜sIUGR双胎中生长受限胎儿和发育正常胎儿两者之间胎盘组织差异表达的蛋白质(差异表达上调或下调1.5倍)。
3、对2D-DIGE实验中所得的差异蛋白质点采用时间飞行质谱(MALDI-TOF)鉴定,选取兴趣蛋白,应用Western blot方法对兴趣蛋白进行验证。
结果：
1、我院产科研究期间双胎妊娠的发生率为2.55%(1264/49563),1144例双胎产后明确其绒毛膜性,其中单绒毛膜双羊膜囊双胎239例(20.89%),双绒毛膜双羊膜囊双胎905例(79.11%)。单绒毛膜双胎组与双绒毛膜双胎组在孕妇年龄(28.40±4.27vs30.74±4.17岁,P<0.001)、分娩孕周(35.22±2.36vs35.90±1.91周,P<0.001)、胎儿生长受限(108vs297例,P=0.001)、胎儿生长不一致(59vs125例,P<0.001)、胎死宫内(34vs39例,P<0.001)、大小胎儿的体重(大胎：2328.54±479.06vs2510.07±432.90g,P<0.001；小胎：1983.46±545.72vs2218.88±445.06g,P<0.001)及新生儿窒息(大胎：5vs13例,P<0.001；小胎：11vs15例,P<0.001)间存在显著差异,而在孕产次、妊娠期高血压、妊娠期糖尿病、妊娠期胆汁淤积征、胎膜早破、前置胎盘、产前住院时间、产后出血及胎儿畸形方面无显著差异(P>0.05)。当纳入绒毛膜性时,孕次(OR=0.823,95%CI0.718-0.943,P=0.005)及受孕方式(OR=0.600,95%CI0.435-1.252,P=0.002)是发生选择性胎儿生长受限的保护因素,孕周(OR=1.155,95%CI1.066-1.252,P=0.005)及妊娠期高血压疾病(OR=1.670,95%CI1.167-2.390,P=0.005)则是危险因素。单绒毛膜双胎胎儿生长受限病因的多因素分析中仅孕次(OR=0.719,95%CI0.518-0.999,P=0.049)为保护因素。双绒毛膜双胎胎儿生长受限的多因素分析中孕周(OR=1.215,95%CI1.099-1.344,P=0.000)、妊娠期高血压疾病(OR=1.564,95%CI1.044-2.345,P=0.030)及胎儿畸形(OR:2.833,95%CI1.053-7.619,P=0.039)为危险因素。
2、经双向电泳分离,染色后图像扫描,得到胎盘组织总蛋白双向电泳胎盘图谱。使用DeCyder V6.5软件分析,经过胶内差异分析和生物学差异分析,在所有胶中选取得到分辨清楚的45个差异蛋白点。
3、对所得45个差异蛋白点进行MALDI-TOF/TOF分析,得到蛋白质点的肽质量指纹图,鉴定得到20个差异蛋白质,其中在sIUGR生长受限胎儿(小胎)胎盘中13个蛋白表达上调：钙调蛋白、热休克蛋白β-1、肌球蛋白调控轻多肽9、肌球蛋白调控轻链12b、原肌球蛋白α-3链、ATP合酶亚组d、硫氧还蛋白依赖过氧化氢还原酶/抗过氧化物蛋白、延伸因子Tu、谷氨酸(NMDA)受体亚单位ε2、Ⅰ型细胞骨架19、长上颚，肺癌和鼻上皮癌相关蛋白1、绒毛膜生长催乳激素、原肌球蛋白β链；另有7个蛋白表达下调：Ⅰ型细胞骨架10、Ⅱ型细胞骨架8、糖皮质激素诱导蛋白5、α1-抗胰蛋白酶、纤维蛋白原p链、纤维蛋白原丫链、HHIP-like蛋白1。Western blot方法验证小胎胎盘中钙调蛋白和PRDXⅢ表达上调,与2D-DIGE所得结果一致。
结论：
1、绒毛膜性对孕妇妊娠的临床过程及结局有重要影响,单绒毛膜双胎在分娩孕周、分娩年龄、胎儿出生体重等方面显著低于双绒毛膜双胎(P<0.05)。单绒毛膜双胎的胎儿生长受限、双胎生长不一致发生率、新生儿窒息明显高于双绒毛膜双胎(P<0.05)。而在妊娠期常见并发症(如妊娠期高血压疾病、妊娠期糖尿病、胎膜早破、前置胎盘、妊娠期肝内胆汁淤积征等)的发生率方面单绒毛膜双胎与双绒毛膜双胎之间无显著统计学差异(P>0.05),提示绒毛膜性质对孕妇情况影响不大。在双胎妊娠中,当孕次增加一次,发生sIUGR的风险变为原来的0.823倍,辅助生殖技术受孕双胎发生sIUGR的风险是自然受孕的0.600倍,而当孕周增加一周或并发妊娠期高血压疾病则发生sIUGR的风险分别增加1.155和1.670倍。在单绒毛膜双胎妊娠中,仅当孕次增加一次时,发生sIUGR的风险是原来的0.719倍。而在双绒毛膜双胎妊娠中,孕周增加一周或并发妊娠期高血压疾病或并发胎儿畸形则发生sIUGR的风险分别增加1.215、1.564及2.833倍。在孕早期通过超声明确双胎妊娠的绒毛膜性质对双胎妊娠孕期胎儿监护、临床咨询、干预处理等方面具有重要意义。
2、2D-DIGE可用于进行胎盘组织的蛋白质组学检测,单绒毛膜选择性胎儿生长受限患者的生长受限胎儿及发育正常胎儿所属胎盘间存在差异表达蛋白点。
3、单绒毛膜sIUGR双胎胎盘间存在差异表达蛋白,差异蛋白涉及细胞多种功能：细胞生长、增殖、凋亡、细胞骨架、氧化应激及凝血等方面。
4、Western blot实验证明2D-DIGE的结果正确可靠。
Background
The incidence of twin pregnancy continues to rise, which has been linked largely to the increased utilization of artificial reproductive technologies and, to a lesser extent, the increase in mean maternal age over this period of time. Twin pregnancies are more prone to complications than single pregnancies, with an increased rate of preterm birth, low birth weight, malformations, gestational hypertension, gestational diabetes and unique complications such as twin-twin transfusion syndrome (TTTs), intrauterine growth restriction (IUGR). In order to design potential strategies to reduce these high risks, more knowledge is needed about the natural course of twin pregnancies.
Twin pregnancies are classified according to either zygosity or chorionicity, and chorionicity rather than zygosity determines the outcome. During pregnancy, twins are classified as dichorionic(DC) or monochorionic(MC) based on placentation. MC twins have an increased perinatal mortality rate than DC twins, this can be explained by servering the individual placental anastomoses. To establish the chorionicity, the placenta and dividing membrane between twins were examined by the ultrasound between11-14gestational weeks. Ultrasound criteria used for the determination of chorionicity in the first and early second trimester were the number of placental sites, the lambda or "T" sign.
Selective intrauterine growth restriction (sIUGR) is a common condition associated with twin pregnancy. The term'selective intrauterine growth restriction' in twin pregnancies is applicable to cases where the estimated fetal weight (EFW) of the small fetus falls below the10th percentile. Significant fetal weight discordance is an important element of the clinical picture, which will often accompany this condition, but is not necessary for diagnosis. It is increasingly considered to be an important complication of MC twins, with potentially significant risks of intrauterine fetal demise (IUFD) or neurological adverse outcome for both twins. In recent years the pathophysiological insight of sIUGR has been substantially improved, although the ability to reliably predict the clinical outcome remains elusive-this will probably remain the case, as the clinical presentation and outcome seem to depend on a combination of multiple factors. The most feared complication of sIUGR in MC twins was the intrauterine demise of the growth-restricted twin, which carries an associated postmortem risk of acute feto-fetal transfusion from the normal to the deceased IUGR fetus. Indeed, death of one MC twin has been reported to be accompanied by concomitant death of the larger twin in25-30%of cases and neurological damage in30%of survivors. The principle cause for the development of sIUGR in MC twins is inadequate placental sharing. The relationship between unequal placental territory and birth weight discordance has recently been revealed in several studies showing that discordance increases with increased placental territory discordance. Extremely asymmetric distribution of placental territories is often associated with very eccentric or velamentous cord insertion, although it is unclear whether velamentous insertion is a mere consequence of the asymmetric displacement of the vascular equator or whether it has any implications in the pathophysiology of growth restriction. Aside from placental territory discordance, a second factor largely influencing fetal weight discordance and the natural history of sIUGR in MC twins is the presence of vascular anastomoses in the MC placenta. To manage twin growth disorders, it is important to detect the high-risk factors and prevent the neonatal complications. Thus, the aim of this study was to investigate the maternal risk factors for sIUGR firstly, and then using two dimensional difference gel electrophoresis (2D-DIGE) to identify novel proteins associated with the restricted(smaller) and the other normal(bigger) fetus's placenta in monochorionic selective intrauterine growth restriction (sIUGR) twins.
The placenta plays a vital role in the regulation of fetal growth and development during pregnancy. Roles include nutrient supply to the fetus, removal from the fetus of metabolic waste and hormone production. There have reports about abortion, pre-eclampsia, preterm, assistant reproductive technology using proteomics technology, but there has no research studied for sIUGR yet.
2D-DIGE is a method which label protein samples with different fluorescent dyes before2-D electrophoresis, and can separate up to three different protein samples at the same time in one two-dimensional gel. Since the most obvious advantage of DIGE system is integrating the advantages of both CyDye DIGE dye multiple labeling method and DeCyder difference2D analysis software. Base on the advantages of DIGE technology, great opportunities comes to us to discover the information of differential proteome contained in sample of the patients with sIUGR.
Based on difference gel electrophoresis, MALDI-TOF/TOF mass spectrometry was used to identify the differential protein spots. Western Blot was performed to validate representative findings from the2D-DIGE analysis. Objective
1. To evaluate the impact of chorionicity on the perinatal outcomes of twin pregnancies. And to investigate the high-risk factors in monochorionic sIUGR and dichorinic sIUGR.
2. To compare the differences of protein expression of the restricted(smaller) and normal(bigger) fetus's placenta in monochorionic sIUGR in hopes of finding associatied proteins of sIUGR by using2D-DIGE, so as to elucidate the pathogenic mechanism of sIUGR.
3. Based on difference gel electrophoresis, MALDI-TOF/TOF mass spectrometry was used to identify the differential protein spots. Western Blot analysis was performed to validate representative findings from the2D-DIGE analysis. Methods
1. In total,1264pairs of twins were enrolled from March2009to June2012.According to the chorionicity,1144pairs of twins were divided into monochorionic twins and dichorionic twins. Maternal characteristics, complications, and perinatal outcomes were analyzed among the two groups. Logistic regression was performed to identify the interrelationships between independent maternal risk factors for sIUGR in a multivariable model.
2. The total proteins extracted from placenta tissues of the smaller and bigger fetuses were separated by means of2D-DIGE. The first dimension was carried by IPG PH gradient isoelectric focusing system, the second dimension was separated by vertical SDS-PAGE.
3. Differentially expressed proteins spots in2D-DIGE maps were chosed and incised after in-gel by trypsin, and the candidate proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF). The peptide mass spectrometry maps and acid sequences were searched from SEQUEST databases. And representative findings from the2D-DIGE analysis were confirmed by western blot analysis. Results
1. The overall twin pregnancy incidence was2.55%. Monochorionic twin pregnancies was more earlier in maternal age and delivery age than dichorionic twin pregnancies (P<0.05). And monochorionic twin pregnancies had more sIUGR, discordance growth, intrauterine death and neonatal asphyxia than dichorionic twin pregnancies (P<0.05). The co-twins'birth weights of monochorionic twin pregnancies were lower than that of dichorionic twin pregnancies (P<0.05). Gravidity and mode of conception were protective factors, hypertensive disorders of pregnancy and delivery age were high risk factors for all sIUGR. Gravidity was the protective factors for the MC sIUGR. Whereas hypertensive disorders of pregnancy, delivery age and fetal malformation were high risk factors for the DC sIUGR.
2. Compared with the bigger fetus' placenta,45protein spots with1.5folds of difference statistically in smaller fetus'placenta were found.
3.20proteins were identified by MALDI-TOF/TOF, including13up-regulated and7down-regulated proteins spots. Among them, Fibrinogen beta chain, Fibrinogen gamma chain, Keratin(type I cytoskeletal10), Keratin(type II cytoskeletal8), Glucocorticoid-Inducible protein5, Alpha-1-antitrypsin, HHIP-like protein1were down regulated in restricted fetuses' placentas compared with normal fetuses' placentas, and Elongation factor Tu, Keratin(type I cytoskeletal19), Tropomyosin beta chain, Tropomyosin alpha-3Chain, Glutamate receptor Subunit epsilon-2, Heat shock protein beta-1, Thioredoxin-dependent peroxide reductase, Chorionic somatomammotropin hormone, ATP synthase subunit d, Myosin regulatory light polypeptide9, Myosin regulatory light chain12B, Calmodulin, Long palate,lung and nasal epithelium carcinoma-associated were up regulated in restricted fetuses' placentas. Western blot analysis of calmodulin and PRDXIII confirmed that these two proteins were significantly up-regulated in smaller fetuses's placentas. Conclusion
1. There was a trend towards worse outcomes in MC pregnancies compared with DC pregnancies. The gravity, mode of conception, delivery age, hypertensive disorders of pregnancy and fetal malformation were related with the sIUGR. It is recommended that the chorionicity should be examined by the ultrasound between11-14gestational weeks every twin pregnancy.
2. Proteomic analysis of placenta reveals differential expression of several proteins between fetuses of monochorionic sIUGR.
3. These proteins are implicated in a variety of cellular functions such as cell survival and proliferation, cell apoptosis regulation, cytoskeletal structure, oxidative stress and coagulation.
4. Western blot analysis of calmodulin and PRDXIII confirmed the findings from the2D-DIGE.
引文
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