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2018, 35(9):795-800.doi:10.13213/j.cnki.jeom.2018.18134

Image analysis on skeletal malformation in SD rat embyros induced by retinoic acid

1. Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China2. Shanghai Sippr-BK Laboratory Animal Co. Ltd., Shanghai 201203, China3. Shanghai Lab. Animal Research Center, Shanghai 201203, China

Accepted: 2018-01-10;  Published: 2018-11-05

Corresponding Author: WANG Xiao-dong, Email: dongxw113@163.com   HU Jing-ying, Email: hujingying@aliyun.com  

[Objective] To supplement the DevTox database with various embryonic skeletal malformation images of SD rats by constructing a rat model using retinoic acid (RA) and assessing the dose-response relationship between RA and malformation

[Methods] Pregnant SD rats were administered via gavage on gestational day (GD) 10 with RA at doses of 50, 100, and 150 mg/kg or with corn oil. After the animals were executed on GD 20, the details of embryo implantation and development in each group were observed and recorded. After embryo bones were stained by alizarin red for identifying skeletal malformation in each body part, the incidence of bone deformity was calculated and the identified malformation site images were taken under anatomical microscope.

[Results] Multiple abnormalities in embryo bones were clearly observed in all three RA-treated groups with teratogenetic rates up to 100%. The main abnormalities included dagnathia, fusion of maxilla and zygomatic arch, sternothyma, deficiency or fusion of thoracic vertebra section 10-13, deficiency or fusion of lumbar vertebra, deficiency of pelvic girdle and caudal vertebra. The phenotype and occurrence rate of abnormalities in the three RA-treated groups were highly consistent and repeatable, the rates of embryo abnormalities among the three dose groups were all 100%, but the degrees of abnormalities increased with higher doses. The collected images of normal and abnormal bones were clear and free from bubbles and reflections with high resolution.

[Conclusion] Severe skeletal malformations are identified in SD rat embryos after maternal exposure to RA on GD10 at 50 mg/kg and above, showing a high malformation rate and various abnormality types. The images of skeletal malformation could supplement the DevTox database.

Key Words: DevTox database;  retinoic acid;  SD rat;  skeletal malformation;  embryo 

表 1


Table 1 Embryo implantation and development of SD rats treated with RA

表 2


Table 2 Fetal skeletal malformation of SD rats treated with RA

图 1


Figure 1 Fetal skull bone malformations of SD rats treated with RA

[注]A:对照组;B:维甲酸50 mg/kg组;C:维甲酸100 mg/kg组;D:维甲酸150 mg/kg组。1:额骨;2:顶骨;3:顶间骨;4:上枕骨;5:外枕骨;6:鼻骨;7:前颌骨;8:上颌骨;9:颧弓;10:下颌骨;11:鼓环;12:鳞状骨。白色箭头:不完全骨化;黑色箭头:骨骼融合;黑色方框:下颌骨缺失;白色方框:鼓环缺失;蓝色方框:鳞状骨缺失。 [Note]A: Control; B: RA 50mg/kg; C: RA 100mg/kg; D: RA 150mg/kg.1: Frontal; 2: Parietal; 3: Interparietal; 4: Supraoccipital; 5: Exoccipital; 6: Nasal; 7: Premaxilla; 8: Maxilla; 9: Zygomatic arch; 10: Mandible; 11: Tympanic annulus; 12: Squamosal.White arrow: incomplete ossification; Black arrow: fused; Black box: mandible absent; White box: tympanic annulus absent; Blue box: squamosal absent.
图 2


Figure 2 Forlimb, hindlimb, and sternebra malformations of SD rats treated with RA

[注]A、E、I:对照组;B、F、J:维甲酸50 mg/kg组;C、G、K:维甲酸100 mg/kg组;D、H、L:维甲酸150 mg/kg组。1:肩胛骨;2:肱骨;3:三角肌粗隆;4:桡骨;5:尺骨;6:掌骨;7:股骨;8:腓骨;9:胫骨;10:跖骨;11:锁骨;12:胸骨节。黑色方框:上肢或下肢短小;黑色箭头:缺趾;白色箭头:桡骨弯曲;黑色双箭头:锁骨偏小;白色方框:胸骨节缺失;蓝色箭头:胸骨节裂开、哑铃状骨化或非对称性骨化。 [Note]A, E, I: Control; B, F, J: RA 50mg/kg; C, G, K: RA 100mg/kg; D, H, L: RA 150 mg/kg. 1: Scapula; 2: Humerus; 3: Deltoid tuberosity; 4: Radius; 5: Ulna; 6: Metacarpal; 7: Femur; 8: Fibula; 9: Tibia; 10: Metatarsal; 11: Clavicle; 12: Sternebra. Black box: forelimb or hindlimb small; Black arrow: phalanx or hind paw phalanx absent; White arrow: radius bent; Black double arrow: clavicle small; White box: sternebra absent; Blue arrow: sternebra split, dumbbell ossification, or asymmetric ossification.
图 3


Figure 3 Axial skeleton and pelvic girdle malformations of SD rats treated with RA

[注]A、B:对照组;C、D:维甲酸50mg/kg组;E、F:维甲酸100mg/kg组;G、H:维甲酸150 mg/kg组。1:上枕骨;2:外枕骨;3:颈椎;4:胸椎;5:肋骨;6:腰椎;7:髂骨;8:坐骨;9:骶椎;10:股骨;11:耻骨。黑色方框:第10~13节胸椎与肋骨畸形;白色方框:腰椎、骶椎和尾椎畸形;蓝色方框:骨盆带畸形。 [Note]A, B: Control; C, D: RA 50 mg/kg; E, F: RA 100 mg/kg; G, H:RA 150 mg/kg.1: Supraoccipital; 2: Exoccipital; 3: Cervical vertebra; 4:Thoracic vertebra; 5: Rib; 6: Lumbar vertebra; 7: Ilium; 8:Ischium; 9: Sacral vertebra; 10: Femur; 11: Pubis.Black box: section 10-13 of thoracic vertebra and ribs abnormalities; White box: Lumbar vertebra, sacral vertebra, and caudal vertebra abnormalities; Blue box: pelvic girdle abnormalities.



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实验动物胚胎发育异常图像数据库(DevTox数据库,www.devtox.org/index_en.php)是研究胚胎发育形态异常图像的数据库,它呈现了实验动物胚胎(新生幼仔)的畸形和变异的图片、说明、分类和规范术语[1-2]。该数据库由国际畸形学学会联合会(International Federation of Teratology Societies,IFTS)组织,汇集了欧洲、北美和日本的相关科研机构共同参与汇编[1-2]。DevTox数据库旨在为发育毒理学领域的科研人员提供基础而宝贵的学术资源,是面向全球的、非营利性的、开放共享的发育毒理学资源库。DevTox数据库目前有2 500多幅详细的图片,并配有描述、术语和统一分类,是迄今毒理学领域最大的图像资源库,它极大地促进了发育毒理学研究中形态异常描述术语的统一和规范化[3]


维甲酸(retinoic acid,RA)是维生素A的一种活性代谢产物,在脊椎动物胚胎发育中起着重要的信号作用。它调节着许多由蛋白质生长因子控制的发育过程,包括神经发生、心脏生成、身体轴延伸、前肢芽、前肠和眼的发育等,维甲酸的过度暴露将引起胚胎发育多器官畸形[6-8]。因此,在引进DevTox数据库的同时,采用维甲酸构建多种胚胎畸形动物模型(包括大鼠、小鼠、家兔、斑马鱼及小型猪等),详尽收集整理畸形图片,并以数据库统一的术语来描述胚胎骨骼畸形,上传至数据库,对该数据库进行完善和补充。本研究报告了大鼠试验结果,相关图片可为发育/生殖毒理研究提供参考。

1   材料与方法

1.1   实验动物

SPF级SD大鼠,购自上海西普尔-必凯实验动物有限公司,实验动物合格证编号2008001668188,许可证号:SCXK(沪)2013-0016。动物房饲养条件:温度为20~25℃,通风,相对湿度为40%~70%,每日光照12 h,自由摄食饮水。

1.2   药品与试剂


1.3   动物分组和给药

选择体重200~250 g的SD大鼠,按雌雄比例1:1合笼,次日早晨查见阴栓,定为孕第0天(GD0)。取孕鼠40只,随机分成4组,即对照组和维甲酸低、中、高3个剂量组。实验组在大鼠孕第10天(GD10),依据大鼠体重,分别用50、100、150mg/kg维甲酸灌胃1次,对照组在大鼠孕第10天用0.3 mL/只玉米油灌胃1次。

1.4   胎鼠骨骼标本制备


1.5   统计学分析

所有数据均输入SPSS 18.0软件进行统计处理。计量资料以x±s表示,各组均数比较采用方差分析;计数资料采用行列表资料的卡方检验。检验水准α= 0.05。

2   结果

2.1   维甲酸对SD大鼠胚胎植入与发育情况的影响

用玉米油和50、100、150 mg/kg维甲酸灌胃孕鼠,各组孕鼠未见明显的外观异常,被毛有光泽,活动正常,摄食与排泄无异常。处死解剖后,各组孕鼠的活胎数与活胎发育情况见表 1。观察胚胎体重、顶臀径和尾长,并与DevTox数据库对比鉴定其外观形态后发现:对照组胚胎体型完好,而维甲酸50、100、150 mg/kg组胚胎体型明显小于对照组(P<0.05),均存在外观畸形。与对照组相比,50 mg/kg组胚胎总数与活胎数均无差异,未出现吸收胎和死胎情况;维甲酸100 mg/kg组活胎数明显减少;维甲酸150 mg/kg组胚胎总数明显减少,活胎数则更少。经茜素红染色后,对照组胚胎骨骼形态完整,无可见异常(少量胸骨节缺失,考虑为正常的偶发现象),而维甲酸50、100、150 mg/kg组均发现明显的胚胎骨骼畸形,各组内胚胎骨骼畸形种类高度一致,畸形率均为100%,见表 2



Table1.Embryo implantation and development of SD rats treated with RA



Table2.Fetal skeletal malformation of SD rats treated with RA

2.2   维甲酸对胚胎颅骨形态的影响

对照组胚胎颅骨中,各个骨骼形态完整,界限清晰,与之相比,各维甲酸用药组胚胎颅骨普遍偏小(图 1)。在维甲酸用药组中,额骨、顶骨、顶间骨、前颌骨均出现程度不一的不完全骨化(图 1中白色箭头);上颌骨、颧弓界限不明,相互融合(图 1中黑色箭头),下颌骨大部分缺失,且程度随维甲酸剂量升高而加剧(图 1中黑色方框);鼓环均缺失(图 1中白色方框);维甲酸50 mg/kg组鳞状骨畸形,而100、150 mg/kg组的鳞状骨则缺失(图 1中蓝色方框)。

图 1


2.3   维甲酸对胚胎四肢和胸骨骨骼的影响

对照组胚胎肩胛骨、上肢(包括肱骨、桡骨、尺骨和掌骨)与下肢(包括股骨、腓骨、胫骨和跖骨)各部位骨骼形态完整,无明显异常(图 2AEI)。各维甲酸用药组上肢与下肢长度比对照组短小(图 2BCDFGH中黑色方框)。对照组掌骨与跖骨染色后显示为4个,而维甲酸用药组显示为3个(图 2BCDFGH中黑色箭头);维甲酸150 mg/kg组胚胎中,部分出现桡骨弯曲(图 2D中白色箭头),而另外两个维甲酸用药组中未出现此情况。对照组锁骨形态正常,胸骨节形态与数目无明显异常,而各维甲酸组锁骨偏小(图 2JKL中黑色双箭头),胸骨节都存在程度不一的缺失(图 2JKL中白色方框)、裂开(图 2JKL中蓝色箭头)及哑铃状骨化(图 2JL中蓝色箭头),部分胚胎也存在胸骨节非对称性骨化(图 2K中蓝色箭头)。

图 2


2.4   维甲酸对胚胎中轴骨骨骼的影响

图 3显示:对照组上枕骨与外枕骨形态完整,无明显异常。维甲酸用药组上枕骨存在典型的双位点骨化,而外枕骨未见明显异常。对照组颈椎形态完整,各维甲酸用药组也未见明显异常。对照组胸椎数目为13,胸弓与胸椎体及肋骨形态正常,排列整齐,维甲酸用药组普遍出现第10~13节胸椎与肋骨异常(图 3CDEFGH黑色方框),包括胸弓融合、畸形、缺失,胸椎体非对称性骨化、分裂、半椎体、哑铃状骨化、融合、畸形、缺失,肋骨融合、缺失、畸形、弯曲、结节状等。对照组腰椎6节、骶椎4节、尾椎形态正常,排列整齐,维甲酸用药组腰椎弓融合、缺失、畸形,腰椎体缺失、非对称性骨化、异位,骶椎与尾椎则完全缺失(图 3DFH白色方框)。对照组骨盆带各部分形态完整,包括髂骨、坐骨与耻骨,维甲酸用药组髂骨出现畸形、短小,坐骨短小、缺失,耻骨则完全缺失(图 3DFH蓝色方框)。以上异常均随着维甲酸剂量升高而加剧。

图 3


3   讨论

在胚胎中,细胞内的维甲酸是由维生素A代谢而来,必须经过两个氧化步骤:第一步由乙醇/视黄醇脱氢酶催化,第二步是由视黄醛脱氢酶催化。维甲酸的降解则由细胞色素P450 CYP26酶催化[9-10]。在靶细胞中,维甲酸充当核维甲酸受体的配体,它与维甲酸X受体形成异质二聚体。该复合物与调节DNA片段、维甲酸的应答元件结合,以控制维甲酸目标基因的转录[11-12]。维甲酸是脊椎动物器官形成过程中重要的细胞信号组成部分。它能与多个发育相关基因交互作用,在脊椎动物早期胚胎发育和器官形成中扮演重要角色,胚胎中过高或过低水平的维甲酸均可导致多种器官(前脑、后脑、四肢以及神经管等)的先天畸形[7, 13-14],而各种胚胎畸形最终可在骨骼发育上有所表现。因此为了保证获得多种胚胎骨骼畸形图片,在诱导大鼠胚胎发育畸形时,设置了较高的维甲酸用药剂量。低维甲酸用药组(50 mg/kg)已出现100%致畸率,因此不能完全捕捉从正常骨骼到畸形骨骼之间的全部状态,这也较为遗憾。在3个维甲酸用药组中,虽然每组畸形率均为100%,但从形态学可见在畸形程度上随维甲酸剂量升高而加剧。随着维甲酸剂量增高,大鼠胚胎各发育参数逐渐降低。胚胎骨骼染色后,分类统计骨骼畸形时发现,维甲酸致胚胎骨骼畸形主要集中于颅骨与中轴骨,具体表现为下颌骨缺失、上颌骨与颧弓融合;胸骨节缺失;第10~13节胸椎融合、缺失;腰椎融合、缺失;骨盆带骨骼与尾椎缺失等。随着维甲酸剂量的增高,骨骼由轻微的单类型畸形向多样化畸形发展,由部分缺失向完全缺失发展,由单一骨骼缺失向多部位缺失发展。也正因此,本研究获得了许多不同畸形类型、不同畸形程度的胚胎骨骼畸形图片。

DevTox数据库对图片质量的要求很高,为了拍摄足够清晰精致的图片,本研究在解剖镜下拍摄骨骼图片的时候,将标本浸没于70%甘油中,能有效去除灯光引起的反光;此外,拍摄之前超声波震荡,能有效去除吸附在标本上的微小气泡,排除气泡对某些细微表型的干扰。采用上述改进方法后,拍摄的骨骼畸形图片无气泡和反光干扰,清晰度高,与相应对照图片比较后,畸形表型清楚、明确。目前,本研究已收集了2 000多幅正常及畸形胚胎图片,筛选整理后,将这些图片上传至DevTox数据库。在图片上传后,数据库工作人员按照第二版术语集对图片进行解剖学定位命名和畸形命名,再交由三名国际畸形学会专家审阅,一致同意后,在术语集汇总表中增加图片内容,增加内容再经过两名专家审阅通过后,可由数据库正式收录[1]。目前,本研究项目组提交的图片中已有40多幅图片被数据库正式收录。



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