应用基因表达谱芯片研究MNNG诱致小鼠胚胎畸形肢体基因表达的变化
摘要
一般估计，大约3％的新生儿出生时有严重的先天畸形。我国出生监测资料显示每年有30—40万缺陷儿出生。出生缺陷发生原因包括遗传因素和环境因素,环境因素中化学物致畸日益受到人们重视。化学物致畸是可以采取措施加以控制和预防的，因此高通量地筛检致畸物，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，其核心是致畸作用分子机理的问题。胚胎发育中，发育基因选择性表达决定胚胎的生长和发育过程，同样，基因的表达谱改变将导致发育异常。所以建立理想的动物模型，从全基因组水平比较正常和异常发育的基因表达谱可望为阐明化学物所致异常发育的分子机制、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（MNNG）作为受试物，诱发小鼠肢体的异常发育表型，然后用基因表达谱芯片观察畸形肢体和相应正常器官的基因表达谱改变。目的是为筛检特异畸形的相关基因、为化学物致畸作用的分子机制提供科学依据。
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，本研究采用ICR小鼠为受试动物，通过观察不同剂量MNNG和不同給药时间胎鼠畸形率、畸形类型等指标，建立了化学物诱发的特异的肢体畸形模型。其最适诱发条件为孕期第12天一次以40mg/kg体重给予MNNG，胎鼠畸形类型以肢体畸形为主，且以短指（趾）和缺指（趾）最为常见。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。此模型的建立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、应用基因芯片研究MNNG致小鼠畸形肢体中基因表达谱改变
本研究应用含有8192个小鼠靶基因的表达谱芯片对孕期第14、16天不同发育时间的小鼠胚胎畸形肢体和相应对照组正常肢体的基因表达情况进行观察。通过组织mRNA分离纯化、表达谱探针制备、杂交、荧光扫描和结果的K-means聚类方法等统计分析。结果：1、观察到第14天中实验组与对照组相比有287条差异表达基因，第16天组织有88条差异表达基因；2、第14天和第16天差异表达基因中均为下调基因占大多数，这与肢体异常主要表现为缺、短指、趾和短肢的结果是一致的；3、通过对某些已知功能研究基因的分析，发现受影响导致基因表达改变的多数为与细胞周期、转录调控、结构蛋白相关基因，4、发现有DNA损伤修复基因和参与p53介导细胞凋亡的基因表达增高，说明MNNG诱发的肢体畸形中有DNA损伤和细胞凋亡的增加；5、发现有些与精子生成有关和睾丸中特异性表达的基因在正常和异常肢体发育中有表达，而且在肢体异常发育中有表达改变，提示这些基因的差异表达与肢体异常发育有关。另外，还筛选了有些未知功能的肢体异常发育相关的基因。本研究结果为揭示肢体异常发育的分子机制和今后进一步的研究提供了科学依据。
It is estimated that about 3% infants have birth defects.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 others are obscure and can be contributed to the interaction of genetic and environmental factors. With rapid increase in the number and category of chemicals, their potential teratogenecity can not be ignored.Understanding the molecular control of abnormal development can provid enormous groundwork for detection and extrapolation among species by identifying genes involved in the congenital malformation. 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 the cellular and molecular interaction that determine morphological pattern during embryonic development.MNNG is a known teratogen that can induce 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.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 forelimbs.
To analyse the gene expression profiles in abnormal and normal developmental limbs of mice GD14 and GD16 embryo.We employed cDNA microarry method.The PCR products of 8192 genes were spotted onto a kind of chemical-material-coated-glass slide in array.Both the mRNAs from abnormal and normal tissues were reversely transcribed to cDNAs.With the incorporation of fluorescent-labeled dUTP to prepare the hybridization probes.After hybridization, microarray were scanned for the fluorescent intensity.Teratogenicity-related gene
expression was screened through the analysis of difference in gene expression profile.Among 8192 target genes,there were 287 genes whose expression level differed between normal and abnormal tissure of GD14 embryo and 88 genes of GD16 embryo, mainly involved in cell cycle，transcription and metabolism. A few genes of spermatogenesis associated and testis-specfic were expressed in limb development and associated with limb abnormal development. Further analysis of these differentially expressed genes will be helpful for undersdanding the molecular mechanism of teratogenicity.
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，本研究采用ICR小鼠为受试动物，通过观察不同剂量MNNG和不同給药时间胎鼠畸形率、畸形类型等指标，建立了化学物诱发的特异的肢体畸形模型。其最适诱发条件为孕期第12天一次以40mg/kg体重给予MNNG，胎鼠畸形类型以肢体畸形为主，且以短指（趾）和缺指（趾）最为常见。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。此模型的建立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、应用基因芯片研究MNNG致小鼠畸形肢体中基因表达谱改变
本研究应用含有8192个小鼠靶基因的表达谱芯片对孕期第14、16天不同发育时间的小鼠胚胎畸形肢体和相应对照组正常肢体的基因表达情况进行观察。通过组织mRNA分离纯化、表达谱探针制备、杂交、荧光扫描和结果的K-means聚类方法等统计分析。结果：1、观察到第14天中实验组与对照组相比有287条差异表达基因，第16天组织有88条差异表达基因；2、第14天和第16天差异表达基因中均为下调基因占大多数，这与肢体异常主要表现为缺、短指、趾和短肢的结果是一致的；3、通过对某些已知功能研究基因的分析，发现受影响导致基因表达改变的多数为与细胞周期、转录调控、结构蛋白相关基因，4、发现有DNA损伤修复基因和参与p53介导细胞凋亡的基因表达增高，说明MNNG诱发的肢体畸形中有DNA损伤和细胞凋亡的增加；5、发现有些与精子生成有关和睾丸中特异性表达的基因在正常和异常肢体发育中有表达，而且在肢体异常发育中有表达改变，提示这些基因的差异表达与肢体异常发育有关。另外，还筛选了有些未知功能的肢体异常发育相关的基因。本研究结果为揭示肢体异常发育的分子机制和今后进一步的研究提供了科学依据。
It is estimated that about 3% infants have birth defects.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 others are obscure and can be contributed to the interaction of genetic and environmental factors. With rapid increase in the number and category of chemicals, their potential teratogenecity can not be ignored.Understanding the molecular control of abnormal development can provid enormous groundwork for detection and extrapolation among species by identifying genes involved in the congenital malformation. 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 the cellular and molecular interaction that determine morphological pattern during embryonic development.MNNG is a known teratogen that can induce 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.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 forelimbs.
To analyse the gene expression profiles in abnormal and normal developmental limbs of mice GD14 and GD16 embryo.We employed cDNA microarry method.The PCR products of 8192 genes were spotted onto a kind of chemical-material-coated-glass slide in array.Both the mRNAs from abnormal and normal tissues were reversely transcribed to cDNAs.With the incorporation of fluorescent-labeled dUTP to prepare the hybridization probes.After hybridization, microarray were scanned for the fluorescent intensity.Teratogenicity-related gene
expression was screened through the analysis of difference in gene expression profile.Among 8192 target genes,there were 287 genes whose expression level differed between normal and abnormal tissure of GD14 embryo and 88 genes of GD16 embryo, mainly involved in cell cycle，transcription and metabolism. A few genes of spermatogenesis associated and testis-specfic were expressed in limb development and associated with limb abnormal development. Further analysis of these differentially expressed genes will be helpful for undersdanding the molecular mechanism of teratogenicity.
引文
1 李勇、 张天宝.发育毒理学研究方法和实验技术，北京：北京医科大学出版社，2000.4
2 Beckman D.A. & Brent R.L. Mechanisms of teratogenesis. Ann. Rev. Pharmacol. Toxicol .1984, 24: 483-580
3 Cohn MJ. & Bright PE. Molecular control of vertebrate limb development evolution and comgenital malformation.Cell & Tissue Research. 1999,296(1):3
4 杨胜利.生物芯片未来发展趋势即及对策.第二军医大学学报.2000,21(9):801.
5 International Agency for Research on Cancer:IARC monpgraphs on the Evaluation of Carcinogenic Risks to Humans Suppl 1987,7:248.
6 Manson JM ,.Miller ML.Contribution of mesenchymal cell death and mitotic altertion to asymmetric limb malformation induced by MNNG. Tertogen.Carciogen.mutagen.1983:335-353.
7 International Agency for Research on Cancer:IARC monpgraphs on the Evaluation of Carcinogenic Risks of chemicals to humans 1978,17:227-230.
8 Ng JK, Tamura K,Buscher D,et al. Molecular and cellular basis of pattern
formation during vertebrate limb development.Curr DevBiol.1999,41:37-66.
9 Hinchliffe JR,Johnson DR.The development of the vertebrate limb,dev Biol,180:566-57
10 Gibson-Brown JJ. et al.Involvement of T-box genes Tbx-2、Tbx-5 in vertebrate limb specification and development. Development .1998; 125:2499-2509
11 Khan J,Simon R,Bittner ML,et al.Gene expression profiling of alveolar rhabdomyosarcoma with cDNA icroarrys.Canceres,1998,58(22):5009-5013.
12 赵国屏 等.生物信息学,北京:科学出版社, 2002.4。
13 Hu B,Burkhart R,Schulte D,et,al.The PI family:a new class of nuclear mammalian proteins related to the yeast Mcm replication proteins.Nucleic Acids Res 1993,25;21(23):5289-93.
14 Hu B,Burkhart R,Schulte D,et,al.The PI family:a new class of nuclear mammalian proteins related to the yeast Mcm replication proteins.Nucleic Acids Res 1993,25;21(23):5289-93.
15 Susin SA, Lorenzo HK, Zamzami N,et al. Molecular characterization of mitochondrial apoptosis-inducing factor.Nature,1999 ,Feb 4,397(6718)441-6.
16 Maisonpietin PC,Suri C,Jones PF,et al.Angiopietin-2 ,a natural antagonist for Tie2 that disrupts in vivo angiogenesis.Science,1997,Jul 4;277(5322)：55－60.
17 Paria BC,Das SK,Andrews Gk, et al. Expression of the epidermal growth fator receptor gene is regulated in mouse blastocysts during delayed implantation.Proc Natl Acad Sci USA.1993,90(1):55-9.
18 Kobayashi A,Yamagiwa H,Hoshino H,et,al.Acombinatorial code for gene expression generated by transcription factor Bach2 and MAZR(MAZ-related factor) through the BTB/POZ domain.Mol Cell Biol 2000 20(50)1733－46.
19 Liu Y,Black J,Kisiel N,et,al. SPAF,a new AAA-proyein specific to early spermatogenesis and malignant conveersion.Oncogene 2000 ,19(12):1579-88.
20 Iguchi N,Tanaka H,Fujii T,et al. Molecular cloning of haploid germ cell-specific tektin cDNA and analysis of the protein in mouse testis.FEBS Lett 1999,456(2):315-21.
21 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
22 梁国栋 主编，最新分子生物学实验技术.北京：科学出版社，2001.2385-389.
23 张天宝. 基因芯片及其在一些研究中的应用.第二军医大学学报，2000（21）：91－94.
2 Beckman D.A. & Brent R.L. Mechanisms of teratogenesis. Ann. Rev. Pharmacol. Toxicol .1984, 24: 483-580
3 Cohn MJ. & Bright PE. Molecular control of vertebrate limb development evolution and comgenital malformation.Cell & Tissue Research. 1999,296(1):3
4 杨胜利.生物芯片未来发展趋势即及对策.第二军医大学学报.2000,21(9):801.
5 International Agency for Research on Cancer:IARC monpgraphs on the Evaluation of Carcinogenic Risks to Humans Suppl 1987,7:248.
6 Manson JM ,.Miller ML.Contribution of mesenchymal cell death and mitotic altertion to asymmetric limb malformation induced by MNNG. Tertogen.Carciogen.mutagen.1983:335-353.
7 International Agency for Research on Cancer:IARC monpgraphs on the Evaluation of Carcinogenic Risks of chemicals to humans 1978,17:227-230.
8 Ng JK, Tamura K,Buscher D,et al. Molecular and cellular basis of pattern
formation during vertebrate limb development.Curr DevBiol.1999,41:37-66.
9 Hinchliffe JR,Johnson DR.The development of the vertebrate limb,dev Biol,180:566-57
10 Gibson-Brown JJ. et al.Involvement of T-box genes Tbx-2、Tbx-5 in vertebrate limb specification and development. Development .1998; 125:2499-2509
11 Khan J,Simon R,Bittner ML,et al.Gene expression profiling of alveolar rhabdomyosarcoma with cDNA icroarrys.Canceres,1998,58(22):5009-5013.
12 赵国屏 等.生物信息学,北京:科学出版社, 2002.4。
13 Hu B,Burkhart R,Schulte D,et,al.The PI family:a new class of nuclear mammalian proteins related to the yeast Mcm replication proteins.Nucleic Acids Res 1993,25;21(23):5289-93.
14 Hu B,Burkhart R,Schulte D,et,al.The PI family:a new class of nuclear mammalian proteins related to the yeast Mcm replication proteins.Nucleic Acids Res 1993,25;21(23):5289-93.
15 Susin SA, Lorenzo HK, Zamzami N,et al. Molecular characterization of mitochondrial apoptosis-inducing factor.Nature,1999 ,Feb 4,397(6718)441-6.
16 Maisonpietin PC,Suri C,Jones PF,et al.Angiopietin-2 ,a natural antagonist for Tie2 that disrupts in vivo angiogenesis.Science,1997,Jul 4;277(5322)：55－60.
17 Paria BC,Das SK,Andrews Gk, et al. Expression of the epidermal growth fator receptor gene is regulated in mouse blastocysts during delayed implantation.Proc Natl Acad Sci USA.1993,90(1):55-9.
18 Kobayashi A,Yamagiwa H,Hoshino H,et,al.Acombinatorial code for gene expression generated by transcription factor Bach2 and MAZR(MAZ-related factor) through the BTB/POZ domain.Mol Cell Biol 2000 20(50)1733－46.
19 Liu Y,Black J,Kisiel N,et,al. SPAF,a new AAA-proyein specific to early spermatogenesis and malignant conveersion.Oncogene 2000 ,19(12):1579-88.
20 Iguchi N,Tanaka H,Fujii T,et al. Molecular cloning of haploid germ cell-specific tektin cDNA and analysis of the protein in mouse testis.FEBS Lett 1999,456(2):315-21.
21 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
22 梁国栋 主编，最新分子生物学实验技术.北京：科学出版社，2001.2385-389.
23 张天宝. 基因芯片及其在一些研究中的应用.第二军医大学学报，2000（21）：91－94.