N-甲基-N'-硝基-N-甲基亚硝基胍致小鼠肢体异常发育差异表达基因的筛选和鉴定
摘要
一般估计，大约3％的新生儿出生时有严重的先天畸形，另有6％—7％1岁
婴儿和12％-14％学龄儿童表现有不同程度的出生缺陷。我国目前共有各种畸形
者3000多万人，每年大约30—40万缺陷婴儿出生，并且有半数左右的缺陷婴
儿存活，参加人类繁衍。事实上出生缺陷的发生率比肿瘤高8倍，比心血管病
高5倍，已经是新生儿围生期死亡的主要原因，并逐渐成为人类的主要死因之
一。出生缺陷发生原因主要包括环境因素和遗传因素。随着化学物合成速度的
加快，动物致畸物的种类和数量也在逐年增加，其对人体的潜在致畸性不容忽
视。由于化学致畸因素是可以采取措施加以预防的，因此高通量的筛检致畸物、
阐明化学致畸作用的分子机制，建立快速准确鉴定人类致畸物的方法和种间外
推方法，是发育毒理学面临的迫切任务。目前对化学物致畸分子机制的研究大
多数局限在个别已知基因，所采用的模型主要是无效突变体动物模型。由于畸
形的发生往往是多个基因共同作用的结果，单基因研究不能全面诠释其发生的
分子机制。而无效突变体由于基因功能的丰余性和动物不能发育到晚期即死亡，
使研究者不能观察到预期的表型，并且该模型不能对畸形发生的非突变性基因
改变（调控异常）进行研究。因此建立理想的动物模型，从全基因组水平了解化学
物所致异常发育的分子机制，有望为早期检测、种间外推和防治找到突破口。
本研究采用可导致肢体畸形的已知致畸物N-甲基-N′-硝基-N-甲基亚硝基胍
（N-methyl-N-nitro-N-nitrosoguanidine MNNG）作为受试物，诱发小鼠肢体的异
常发育表型，然后用抑制消减杂交技术（SSH）筛选该模型畸形肢体和相应正常器
官的差异表达基因。目的是为化学物致畸分子机制的研究提供科学依据，并为
异常发育相关基因的筛选研究提供新的思路。
一．MNNG致小鼠肢体异常发育模型的建立
脊椎动物肢体由于其特殊的形态结构，被认为是研究胚胎发育过程中形态
模式形成的细胞和分子机制的最有效模型之一。MNNG为一已知的致突变性致
畸物，可诱发小鼠的多脏器畸形包括肢体畸形，但不同时间和剂量暴露所诱发
的主要畸形类型和发生率不同，为了寻求其导致肢体畸形的最佳条件，本研究
较系统地观察了不同剂量和不同给药时间，母鼠流产率、胎鼠畸形率、畸形类
型等指标。结果发现，母鼠流产率和胎鼠畸形发生率均随着剂量的增加而增高，
并有剂量一反应趋势。胎鼠的主要畸形类型包括胯裂、肢体畸形和卷尾等。在
孕期第 12天一次给与 MN’NG 40mg／kg体重时，母鼠流产率较低，与对照组比较
无显著性差别O＜0刀5卜胎鼠畸形类型主要以肢体畸形为主，少数并发卷尾，肢
体畸形似活胎计）的发生率为33．7％，以窝计为33．7％，肢体畸形占畸胎鼠的构
成比为 100％，且以短指（趾）和缺指（趾）最为常见，其次还有多指（趾）和短肢等。
畸形发生的部位存在左右及前后不对称性，四肢畸形的发生率和畸形严重程度
依次为左后＞左前＞右后＞右前。对肢体畸形胚胎骨骼染色之后发现，掌路骨缺失
和骨化不全发生率较高，此外还有径胖骨的缺失和骨化不全，四肢发生畸形的
严重程度与大体形态观察结果相同。特别是发现并证实大体形态所见短肢是径
胖骨缺失所致；肋骨的异常发育（缺肋、短肋、肋骨分叉等）同肢体相似，也存在
明显的左右不对称性，这些发现尚未见国内外有报道。综上所述，通过本研究，
成功地建立了化学物诱发的特异的肢体畸形模型，其最适诱发条件是小鼠妊娠
第 12天一次腹腔注射给予 40wig MNNG。进一步揭示了 MNNG对小鼠的致
畸作用特点，并发现MNNG所致后肢短肢是径胖骨缺失所致，肋骨异常也存在
左右不对称性。该研究不仅为研究肢体异常发育而且也为胚胎异常发育过程中
形态学模式形成的分子机制研究提供了良好的模型。
二、MNNG致小鼠肢体异常发育差异表达基因的筛选和鉴定
本研究应用SSH技术对孕期第18天的小鼠胚胎畸形肢体和相应对照组正常
肢体的差异表达基因进行筛选。然后通过克隆、兰白斑筛选、点杂交等技术对
筛选的差异表达片断进行验证，对杂交有差异的克隆进一步测序和数据库查询，
确定肢体畸形相关基因。兰白斑筛选之后，共挑选白色菌落275个，菌液PCR
扩增之后，得到有插人片断的菌落235个，转化效率为85．4％。将插入片断大于
100hp的 172个克隆的 PCR产物点膜，与实验组和对照组的原始 CDNA库杂交，
放射自显影之后得到 25个差异片断，对其中的 12个进行测序。1个克隆的插入
片断只有 52hp，未进行比对，其余 11条序列先后进行 GeneBank blastn／nr和
blasin／dbEST查询之后，发现有2个核昔酸序列完全相同，其余均为不同序列，
4
说明克隆重复挑选的概率不大。对剩余10条比对分析，结果发现，l条为已知
基因，与 Mus musculus pmxisomal biogmesis facor 3 （Pex3，rnRNA的同源性较
高【分值为 369 bits（186hp），e－100，Id
Congenital malformation is one of the major causes in infant death. Our
knowledge about the cause and mechanisms through which these defects are
manifested is limited. Current estimated about 20% of all birth defects are due to
genetic factors and 10% are due to environmental factors. The advent of
combinatorial chemistry and computer-aid drug design has led to a recent upsurge in
the number of chemicals. Their potential teratogenecity cannot be ignored.
Understanding the molecular control of abnormal development can provide enormous
groundwork for detection and extrapolation among species by identifying candidate
genes and some known genes involved in the congenital malformation bad been
identified. With the establishment of a series of molecular strategy, a detailed
understanding of the genome-wide gene expression of abnormal development is
becoming a reality.
The vertebrate limb is a powerful model system for studying the cellular and
molecular interaction that determine morphological pattern during embryonic
development. MNNG is a known teratogen that can induced limb malformation. In
this study, we used MNNG as test material to induce ICR mice embryo limb
malformation. The result of experiment show that a spectrum of malformation
involving the palate, ribs and limbs is produced. Limb defects are prominent with
exposure on day 12, When the type of limb malformation are examined, an unusual
pattern was observed. Limbs on the left side were more frequently malformed than
limb on the right side and bindlimbs were more frequently malformed than forelimbs.
The phenotypes of affected limb include oligodactyly, polydactyly and truncated limb
mainly. Skeletal preparation show defect of metacarpals. The loss of tibia and fibia is
account for the truncated limbs. Left-right asymmetry of ribs defect has been
observed especially. To identify altered gene expression in abnormal and normal
developmental limbs of mice GD 18 embryo, we employed a suppression subtractive
hybridization(SSH) technique to generate two subtracted cDNA libraries. A set of
eDNA clones include 172 SSH inserts amplified by PCR was screened, and 25 clones
were shown by dotblotting to be differentially expressed in the malformed limb. 12 of
25 clones were set to be sequenced analysis and 10 significant clones were aligned in
GeneBank. One clone with increased expression were identical to mus musculus
peroxisomal biogenesis factor 3 (Pex3) mRNA and the others contain limited
sequence homology to the previously reported mus musculus or 8-12 days embryo
mus musculus expressed sequence tag. This work thus provide a starting point for
understanding a molecular profile of abnormal, limbs development and chemical
teratogenic mechanism that could ultimately provide an essential aid to developing
strategies or the prevention, early detection of birth defect.
婴儿和12％-14％学龄儿童表现有不同程度的出生缺陷。我国目前共有各种畸形
者3000多万人，每年大约30—40万缺陷婴儿出生，并且有半数左右的缺陷婴
儿存活，参加人类繁衍。事实上出生缺陷的发生率比肿瘤高8倍，比心血管病
高5倍，已经是新生儿围生期死亡的主要原因，并逐渐成为人类的主要死因之
一。出生缺陷发生原因主要包括环境因素和遗传因素。随着化学物合成速度的
加快，动物致畸物的种类和数量也在逐年增加，其对人体的潜在致畸性不容忽
视。由于化学致畸因素是可以采取措施加以预防的，因此高通量的筛检致畸物、
阐明化学致畸作用的分子机制，建立快速准确鉴定人类致畸物的方法和种间外
推方法，是发育毒理学面临的迫切任务。目前对化学物致畸分子机制的研究大
多数局限在个别已知基因，所采用的模型主要是无效突变体动物模型。由于畸
形的发生往往是多个基因共同作用的结果，单基因研究不能全面诠释其发生的
分子机制。而无效突变体由于基因功能的丰余性和动物不能发育到晚期即死亡，
使研究者不能观察到预期的表型，并且该模型不能对畸形发生的非突变性基因
改变（调控异常）进行研究。因此建立理想的动物模型，从全基因组水平了解化学
物所致异常发育的分子机制，有望为早期检测、种间外推和防治找到突破口。
本研究采用可导致肢体畸形的已知致畸物N-甲基-N′-硝基-N-甲基亚硝基胍
（N-methyl-N-nitro-N-nitrosoguanidine MNNG）作为受试物，诱发小鼠肢体的异
常发育表型，然后用抑制消减杂交技术（SSH）筛选该模型畸形肢体和相应正常器
官的差异表达基因。目的是为化学物致畸分子机制的研究提供科学依据，并为
异常发育相关基因的筛选研究提供新的思路。
一．MNNG致小鼠肢体异常发育模型的建立
脊椎动物肢体由于其特殊的形态结构，被认为是研究胚胎发育过程中形态
模式形成的细胞和分子机制的最有效模型之一。MNNG为一已知的致突变性致
畸物，可诱发小鼠的多脏器畸形包括肢体畸形，但不同时间和剂量暴露所诱发
的主要畸形类型和发生率不同，为了寻求其导致肢体畸形的最佳条件，本研究
较系统地观察了不同剂量和不同给药时间，母鼠流产率、胎鼠畸形率、畸形类
型等指标。结果发现，母鼠流产率和胎鼠畸形发生率均随着剂量的增加而增高，
并有剂量一反应趋势。胎鼠的主要畸形类型包括胯裂、肢体畸形和卷尾等。在
孕期第 12天一次给与 MN’NG 40mg／kg体重时，母鼠流产率较低，与对照组比较
无显著性差别O＜0刀5卜胎鼠畸形类型主要以肢体畸形为主，少数并发卷尾，肢
体畸形似活胎计）的发生率为33．7％，以窝计为33．7％，肢体畸形占畸胎鼠的构
成比为 100％，且以短指（趾）和缺指（趾）最为常见，其次还有多指（趾）和短肢等。
畸形发生的部位存在左右及前后不对称性，四肢畸形的发生率和畸形严重程度
依次为左后＞左前＞右后＞右前。对肢体畸形胚胎骨骼染色之后发现，掌路骨缺失
和骨化不全发生率较高，此外还有径胖骨的缺失和骨化不全，四肢发生畸形的
严重程度与大体形态观察结果相同。特别是发现并证实大体形态所见短肢是径
胖骨缺失所致；肋骨的异常发育（缺肋、短肋、肋骨分叉等）同肢体相似，也存在
明显的左右不对称性，这些发现尚未见国内外有报道。综上所述，通过本研究，
成功地建立了化学物诱发的特异的肢体畸形模型，其最适诱发条件是小鼠妊娠
第 12天一次腹腔注射给予 40wig MNNG。进一步揭示了 MNNG对小鼠的致
畸作用特点，并发现MNNG所致后肢短肢是径胖骨缺失所致，肋骨异常也存在
左右不对称性。该研究不仅为研究肢体异常发育而且也为胚胎异常发育过程中
形态学模式形成的分子机制研究提供了良好的模型。
二、MNNG致小鼠肢体异常发育差异表达基因的筛选和鉴定
本研究应用SSH技术对孕期第18天的小鼠胚胎畸形肢体和相应对照组正常
肢体的差异表达基因进行筛选。然后通过克隆、兰白斑筛选、点杂交等技术对
筛选的差异表达片断进行验证，对杂交有差异的克隆进一步测序和数据库查询，
确定肢体畸形相关基因。兰白斑筛选之后，共挑选白色菌落275个，菌液PCR
扩增之后，得到有插人片断的菌落235个，转化效率为85．4％。将插入片断大于
100hp的 172个克隆的 PCR产物点膜，与实验组和对照组的原始 CDNA库杂交，
放射自显影之后得到 25个差异片断，对其中的 12个进行测序。1个克隆的插入
片断只有 52hp，未进行比对，其余 11条序列先后进行 GeneBank blastn／nr和
blasin／dbEST查询之后，发现有2个核昔酸序列完全相同，其余均为不同序列，
4
说明克隆重复挑选的概率不大。对剩余10条比对分析，结果发现，l条为已知
基因，与 Mus musculus pmxisomal biogmesis facor 3 （Pex3，rnRNA的同源性较
高【分值为 369 bits（186hp），e－100，Id
Congenital malformation is one of the major causes in infant death. Our
knowledge about the cause and mechanisms through which these defects are
manifested is limited. Current estimated about 20% of all birth defects are due to
genetic factors and 10% are due to environmental factors. The advent of
combinatorial chemistry and computer-aid drug design has led to a recent upsurge in
the number of chemicals. Their potential teratogenecity cannot be ignored.
Understanding the molecular control of abnormal development can provide enormous
groundwork for detection and extrapolation among species by identifying candidate
genes and some known genes involved in the congenital malformation bad been
identified. With the establishment of a series of molecular strategy, a detailed
understanding of the genome-wide gene expression of abnormal development is
becoming a reality.
The vertebrate limb is a powerful model system for studying the cellular and
molecular interaction that determine morphological pattern during embryonic
development. MNNG is a known teratogen that can induced limb malformation. In
this study, we used MNNG as test material to induce ICR mice embryo limb
malformation. The result of experiment show that a spectrum of malformation
involving the palate, ribs and limbs is produced. Limb defects are prominent with
exposure on day 12, When the type of limb malformation are examined, an unusual
pattern was observed. Limbs on the left side were more frequently malformed than
limb on the right side and bindlimbs were more frequently malformed than forelimbs.
The phenotypes of affected limb include oligodactyly, polydactyly and truncated limb
mainly. Skeletal preparation show defect of metacarpals. The loss of tibia and fibia is
account for the truncated limbs. Left-right asymmetry of ribs defect has been
observed especially. To identify altered gene expression in abnormal and normal
developmental limbs of mice GD 18 embryo, we employed a suppression subtractive
hybridization(SSH) technique to generate two subtracted cDNA libraries. A set of
eDNA clones include 172 SSH inserts amplified by PCR was screened, and 25 clones
were shown by dotblotting to be differentially expressed in the malformed limb. 12 of
25 clones were set to be sequenced analysis and 10 significant clones were aligned in
GeneBank. One clone with increased expression were identical to mus musculus
peroxisomal biogenesis factor 3 (Pex3) mRNA and the others contain limited
sequence homology to the previously reported mus musculus or 8-12 days embryo
mus musculus expressed sequence tag. This work thus provide a starting point for
understanding a molecular profile of abnormal, limbs development and chemical
teratogenic mechanism that could ultimately provide an essential aid to developing
strategies or the prevention, early detection of birth defect.
引文
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2 中国出生缺陷监测协作组(肖坤泽主编)。中国出生缺陷地图集。成都地图出版社, 1992;1-8
3 Beckman DA. & Brent RL. Mechanisms of teratogenesis. Ann. Rev. Pharmacol. Toxicol .1984; 24:483-580
4 John CR. et al. Use of suppresion-PCR subtraction hybridication to identify genes that demonstrate altered expression in male rat and guinea pig livers following exposure to wy-14643, a peroxisomeproliferator and no-genotoxic hepatacarcino-gen. Toxicology. 2000; 144: 13-29
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24 Xu XL et al. Fribroblast growth factor receptors(FGFRs)and their roles in limb development. Cell Tissue Res 1999; 296: 33-43
25 Cohn MJ.et al. Fibroblast growth factors induce additionallimb development from the foank of chick embryos. Cell;1995:80:739-746
26 Ohuchi H. et al Anadditional limb can be induced from the flank of the chick embryo by FGF-4． Biochem Biophys Res Commun 1995;209:809-816
27 Dietrich S. et al. Specification of the hypaxial musculature. Development. 1998; 125:2235-2249
28 Brand-Saberi B. & Christ B. Genetic and epigenetic control of muscle
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29 Nisander L.Limb mutants: what can they tell us about normal limb development? Curr Opin Genet Dev 1997; 7:530-536
30 Muragaki Y.et al. Altered growth and branching patterns in the synpolydactyly caused by mutations in HOXD13． Sciense 1996; 272: 548-551
31 Mortlock DP & Innis JW. Mutation of HOXal3 in hand-foot-genital syndrome. Nat Genet. 1997; 156:179-180
32 Kang S. et al. GL13 frame-shift mutations cause autosomal dominant Pallister-Hall syndrome. Nat Genet 1997; 15:266-268
33 Wild A et al. Pount mutations in human GLI3 cause Greig syndrome,Hum Mol Genet 1997;6:1079-1984
34 Basson CT. et al. Mutation in human TBX3 alter limb, apocrine and genital development in ulnar-mammary syndrome. Nature Genet. 1997; 16:311-315
2 中国出生缺陷监测协作组(肖坤泽主编)。中国出生缺陷地图集。成都地图出版社, 1992;1-8
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