先天性肛门直肠畸形胚胎发育、基因表达及远期生活质量的研究
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摘要
前言
先天性肛门直肠畸形是最常见的消化道畸形，其发生率约为1／5000～1／1500，是世界卫生组织常规监测的先天畸形之一。该畸形严重影响儿童的生活质量，给社会和家庭带来了沉重的负担。
肛门直肠畸形的病因至今仍不清楚。人们仅仅知道肛门直肠畸形的发生是胚胎期直肠发育发生障碍的结果，至今仍没有完全阐明正常肛门直肠胚胎发育的演变规律，对肛门直肠畸形在胚胎期发生的具体机制更是知之甚少。
目前认为，肛门直肠畸形的发生是遗传因素和环境因素共同作用的结果。流行病学和动物实验表明遗传因素在其发病过程中发挥重要作用。由于肛门直肠畸形发病机制和病理改变十分复杂，常合并其他畸形，且疾病谱广，表型多种多样，遗传方式不清楚，涉及的相关基因不止一个，因而人类肛门直肠畸形致病基因的研究非常复杂。人类肛门直肠畸形的致病基因至今没有得到定位，甚至没有明确的候选基因。因此从胚胎学和分子水平探索人类先天性肛门直肠畸形的发病机制和致病基因具有十分重要的意义。
本研究包括以下内容：①胚胎发育：应用乙烯硫脲致畸制作Wistar大鼠肛门直肠畸形的动物模型，连续动态观察胎龄12.5天到20天胎鼠肛门直肠在时空上的形态变化过程，探讨先天性肛门直肠畸形胚胎发生的演变规律。②基因表达：应用RT-PCR技术和基因表达谱芯片技术，检测肛门直肠畸形患儿和无畸形儿的直肠末端某些发育相关基因表达状况，探讨这些基因与人类先天性肛门直肠畸形发生的关系，为寻找人类先天性肛门直
肠畸形的致病基因提供线索。③生活质量调查：从身体J心理和社会等方
面，调查和评估肛门直肠畸形病人术后30年以上生活质量状况。
实验材料
动物：WIStar大鼠由中国医科大学第二临床学院医学实验动物中心提
供。
病例：①标本来源：19例经我科手术治疗的先天性肛门直肠畸形患儿，
男10例，女9例。年龄2天一3岁。术中无菌条件下取直肠末端后壁肠组
织，置于液氮中保存。术中收集后天性肛展、直肠外伤等5例无畸形患儿直
肠齿状线上约Icm以内水平处肠组织做对照，标本处理同前。②随访的8
例病人为30年前经我科手术治疗的肛门直肠畸形病人。
试剂：①原位末端标记细胞凋亡检测试剂盒购于武汉博士德公司。②
逆转录试剂盒购于大连宝生物工程公司。部张芯片由上海博星基因芯片
有限责任公司提供的发育相关基因表达谱芯片，型号：GCS。
方 法
①胚胎发育：用乙烯硫豚致畸孕鼠产生泄殖腔畸形和无肛畸形胎鼠。
胎龄 12．5天一20天的正常及泄殖腔发育畸形的胎鼠正中矢状面连续切
片，HE染色，连续动态对比观察泄殖腔及肛门直肠的发育过程。部分切片
TUNEL染色，观察细胞凋亡的分布。
②基因表达：应用RT—PCR方法检测HOXdl3基因在16例肛门直肠畸
形病人和5例无畸形儿直肠末端的表达水平。应用基因表达谱芯片技术对
3例先天性肛门直肠畸形患儿的直肠末端组织和无畸形儿直肠末端组织的
基因表达谱进行检测。
③生活质量调查：随访8例肛门直肠畸形病人（年龄33岁一41岁入应
用自行设计的肛门直肠畸形病人术后30年以上生活质量问卷评估该8例
病人和对照组（24例健康成人）的生活质量状况。包括排便功能、饮食、工
作、性生活、社交娱乐、心理情绪6方面10项内容。
·2·
结 果
①胚胎发育：泄殖腔发育过程中，胎龄15天前的正常鼠胚，尿直肠隔将
泄殖腔分为腹侧的尿生殖窦和背侧的直肠两部分，二者共同相通于泄殖腔
管。尿直肠隔下降与泄殖腔膜的距离逐渐减小，直至胚胎15天时与泄殖腔
膜融合，直肠与膀跳分离。16天时肛膜破裂，直肠与尾沟相通。ETU致畸
胎鼠中所有的无肛畸形均为泄殖腔畸形和直肠尿道瘦。ETU致畸的胎鼠
泄殖腔发育中有以下特点：①尿直肠隔始终未与泄殖腔膜融合；②泄殖腔构
型异常；③泄殖腔膜过短；④没有尾沟形成。正常胚胎13．5天时背侧泄殖
腔膜发生细胞凋亡。14．5天一15天直肠末端与尾沟处出现大量细胞凋亡。
ETU致畸的胎鼠13．5天时，短小的泄殖腔膜布满大量凋亡的细胞。14 5
天八5天直肠末端与相应的尾沟处未见细胞凋亡发生，直肠末端末与外界
相通。
②基因表达：RT－PCR检测发现：HOXd13基因mRNA在先天性无肛畸
形直肠末端表达N．32。0．26）水平明显低于无畸形儿直肠末端的表达
（0．73。0．10）（P＝0．001）。基因表达谱芯片技术检测发现，186条发育相
关基因中，在肛门直肠畸形患儿直肠末端和无畸形儿直肠末端表达差异的
基因有54条，表达上调的基因52条，表达下调的基因二条。
③生活质量调查：8例病人中，6例存在排便功能障碍。2例病人社会
工作方面因此受到影响，3例病人性生活受到限制，4例病人因排便功能障
碍经常忧愁和烦恼。根据肛门直肠畸形病人术后30年以上生活质量问卷，
病人组生活质量评分p9．6。4 8）显著低于正常对照组生活质量评分
（33．IL3．1）。
Anorectal malformation (ARM) is the most common disorder affecting I/ 5000 to 1/1500 live births, one kind of malformations supervised by World Heahh Organization. ARM severely influences quality of life, and put great burdens on families and society.
The genetic, embryological, and pathogenic aspects of ARM are poorly understood and controversial. It has been only demonstrated that ARM resulted from impeded rectal development during embryonic stage. Developmental principles of normal anorectum remain poorly understood, much less the pathogenesis of ARM.
Genetic and environmental factors interact on each other to give rise to ARM. Epidemiological and animal experiments led to the conclusion that genetics played an important role in the occurrence of ARM. But the spectrum of ARM is fairly wide, pathogenesis and pathological changes are complex. ARM is also often associated with extraanal anomalies, and its phenotypes are rather variable, so it is very difficult to perform gene investigation. The pathogenic genes are still unlocated, even the candidate gene remains unknown. Therefore, it is significant to investigate pathogenesis of ARM on embryonic and molecular level.
In the current study, contents are as follows: ①Embryonic development: Rat embryos with ARM were obtained by treating with administration of ethyle-nethiourea (ETU) on pregnant rats. We explored the development of ARM by continuous and dynamic observation of anorectal morphogenesis in the involved embryos from gestational days 12. 5 to 20. ②Gene expression; RT - PCR and cDNA microarrays were used to detect developmental genes expression of ARM and normal terminal rectum, explore relativity of ARM to these genes and investigate pathogenic genes. ③ Quality of life; Eight patients after surgically corrected ARM were followed up for 33 to 41 years. The quality of life was assessed by using an interview and a modified Quality of Life Questionnaire.
Materials
Animals; Wistar rats were provided by Medical Animal Center, the Second Clinical College of China Medical University.
Samples; Among 19 congenital. ARM infants operated in our department, 10 were males, and 9 were females., aged from 2 days to 3 years. Tissues were removed from terminal pouch of rectum and stored immediately in fluid nitrogen. In the control group, tissues were obtained at point 1 cm to dentate line in acquired anal fistula and rectal trauma infants.
Reagent; ①TUNEL kit was provided by Boster Biological Technology Co. , Ltd. Wuhan. ②Bca BEST RNA PCR kit verl. 1 ( AMV) was provided by Taka-ra Biotechnology Co. , Ltd. Dalian.③Gene chips were provided by BioStar Ge-nechip Inc. , Shanghai. Type: GCs.
Methods
Embryonic development; Cloaca malformation and ARM rats were produced by treating with administration of ETU on pregnant rats. Normal rat embryos and embryos with ARM from gestational days 12. 5 to 20 were sectioned serially and sagittally and stained with HE to observe development of cloaca and anorectum continually and contrastively. Some slices were stained with TUNEL to identify the distribution of apoptotic cells.
Gene expression; RT -PCR was used to investigate Hoxdl3 gene expression in rectal terminus of 16 infants with ARM and 5 normal infants, and cDNA microarrays to investigate mRNA in that of 3 infants with ARM and normal infants.
Quality of life; Eight patients (aged 33 to 41 years old) after surgically corrected ARM were followed up for 33 to 41, years. The quality of life was assessed by using an interview and a modified Quality of Life Questionnaire for the adult patients after surgically corrected ARM including ten items in six areas of
everyday life ( bowel function, food, occupational life, social activity, sexual life, and psychological aspects ). Twenty - four healthy people with similar age, sex and education level distributions served as controls.
Results
Embryonic development; During normal cloaca development process in rats, before gestational days 15, the urorectal septum (URS) developed and clearly demarcated th
先天性肛门直肠畸形是最常见的消化道畸形，其发生率约为1／5000～1／1500，是世界卫生组织常规监测的先天畸形之一。该畸形严重影响儿童的生活质量，给社会和家庭带来了沉重的负担。
肛门直肠畸形的病因至今仍不清楚。人们仅仅知道肛门直肠畸形的发生是胚胎期直肠发育发生障碍的结果，至今仍没有完全阐明正常肛门直肠胚胎发育的演变规律，对肛门直肠畸形在胚胎期发生的具体机制更是知之甚少。
目前认为，肛门直肠畸形的发生是遗传因素和环境因素共同作用的结果。流行病学和动物实验表明遗传因素在其发病过程中发挥重要作用。由于肛门直肠畸形发病机制和病理改变十分复杂，常合并其他畸形，且疾病谱广，表型多种多样，遗传方式不清楚，涉及的相关基因不止一个，因而人类肛门直肠畸形致病基因的研究非常复杂。人类肛门直肠畸形的致病基因至今没有得到定位，甚至没有明确的候选基因。因此从胚胎学和分子水平探索人类先天性肛门直肠畸形的发病机制和致病基因具有十分重要的意义。
本研究包括以下内容：①胚胎发育：应用乙烯硫脲致畸制作Wistar大鼠肛门直肠畸形的动物模型，连续动态观察胎龄12.5天到20天胎鼠肛门直肠在时空上的形态变化过程，探讨先天性肛门直肠畸形胚胎发生的演变规律。②基因表达：应用RT-PCR技术和基因表达谱芯片技术，检测肛门直肠畸形患儿和无畸形儿的直肠末端某些发育相关基因表达状况，探讨这些基因与人类先天性肛门直肠畸形发生的关系，为寻找人类先天性肛门直
肠畸形的致病基因提供线索。③生活质量调查：从身体J心理和社会等方
面，调查和评估肛门直肠畸形病人术后30年以上生活质量状况。
实验材料
动物：WIStar大鼠由中国医科大学第二临床学院医学实验动物中心提
供。
病例：①标本来源：19例经我科手术治疗的先天性肛门直肠畸形患儿，
男10例，女9例。年龄2天一3岁。术中无菌条件下取直肠末端后壁肠组
织，置于液氮中保存。术中收集后天性肛展、直肠外伤等5例无畸形患儿直
肠齿状线上约Icm以内水平处肠组织做对照，标本处理同前。②随访的8
例病人为30年前经我科手术治疗的肛门直肠畸形病人。
试剂：①原位末端标记细胞凋亡检测试剂盒购于武汉博士德公司。②
逆转录试剂盒购于大连宝生物工程公司。部张芯片由上海博星基因芯片
有限责任公司提供的发育相关基因表达谱芯片，型号：GCS。
方 法
①胚胎发育：用乙烯硫豚致畸孕鼠产生泄殖腔畸形和无肛畸形胎鼠。
胎龄 12．5天一20天的正常及泄殖腔发育畸形的胎鼠正中矢状面连续切
片，HE染色，连续动态对比观察泄殖腔及肛门直肠的发育过程。部分切片
TUNEL染色，观察细胞凋亡的分布。
②基因表达：应用RT—PCR方法检测HOXdl3基因在16例肛门直肠畸
形病人和5例无畸形儿直肠末端的表达水平。应用基因表达谱芯片技术对
3例先天性肛门直肠畸形患儿的直肠末端组织和无畸形儿直肠末端组织的
基因表达谱进行检测。
③生活质量调查：随访8例肛门直肠畸形病人（年龄33岁一41岁入应
用自行设计的肛门直肠畸形病人术后30年以上生活质量问卷评估该8例
病人和对照组（24例健康成人）的生活质量状况。包括排便功能、饮食、工
作、性生活、社交娱乐、心理情绪6方面10项内容。
·2·
结 果
①胚胎发育：泄殖腔发育过程中，胎龄15天前的正常鼠胚，尿直肠隔将
泄殖腔分为腹侧的尿生殖窦和背侧的直肠两部分，二者共同相通于泄殖腔
管。尿直肠隔下降与泄殖腔膜的距离逐渐减小，直至胚胎15天时与泄殖腔
膜融合，直肠与膀跳分离。16天时肛膜破裂，直肠与尾沟相通。ETU致畸
胎鼠中所有的无肛畸形均为泄殖腔畸形和直肠尿道瘦。ETU致畸的胎鼠
泄殖腔发育中有以下特点：①尿直肠隔始终未与泄殖腔膜融合；②泄殖腔构
型异常；③泄殖腔膜过短；④没有尾沟形成。正常胚胎13．5天时背侧泄殖
腔膜发生细胞凋亡。14．5天一15天直肠末端与尾沟处出现大量细胞凋亡。
ETU致畸的胎鼠13．5天时，短小的泄殖腔膜布满大量凋亡的细胞。14 5
天八5天直肠末端与相应的尾沟处未见细胞凋亡发生，直肠末端末与外界
相通。
②基因表达：RT－PCR检测发现：HOXd13基因mRNA在先天性无肛畸
形直肠末端表达N．32。0．26）水平明显低于无畸形儿直肠末端的表达
（0．73。0．10）（P＝0．001）。基因表达谱芯片技术检测发现，186条发育相
关基因中，在肛门直肠畸形患儿直肠末端和无畸形儿直肠末端表达差异的
基因有54条，表达上调的基因52条，表达下调的基因二条。
③生活质量调查：8例病人中，6例存在排便功能障碍。2例病人社会
工作方面因此受到影响，3例病人性生活受到限制，4例病人因排便功能障
碍经常忧愁和烦恼。根据肛门直肠畸形病人术后30年以上生活质量问卷，
病人组生活质量评分p9．6。4 8）显著低于正常对照组生活质量评分
（33．IL3．1）。
Anorectal malformation (ARM) is the most common disorder affecting I/ 5000 to 1/1500 live births, one kind of malformations supervised by World Heahh Organization. ARM severely influences quality of life, and put great burdens on families and society.
The genetic, embryological, and pathogenic aspects of ARM are poorly understood and controversial. It has been only demonstrated that ARM resulted from impeded rectal development during embryonic stage. Developmental principles of normal anorectum remain poorly understood, much less the pathogenesis of ARM.
Genetic and environmental factors interact on each other to give rise to ARM. Epidemiological and animal experiments led to the conclusion that genetics played an important role in the occurrence of ARM. But the spectrum of ARM is fairly wide, pathogenesis and pathological changes are complex. ARM is also often associated with extraanal anomalies, and its phenotypes are rather variable, so it is very difficult to perform gene investigation. The pathogenic genes are still unlocated, even the candidate gene remains unknown. Therefore, it is significant to investigate pathogenesis of ARM on embryonic and molecular level.
In the current study, contents are as follows: ①Embryonic development: Rat embryos with ARM were obtained by treating with administration of ethyle-nethiourea (ETU) on pregnant rats. We explored the development of ARM by continuous and dynamic observation of anorectal morphogenesis in the involved embryos from gestational days 12. 5 to 20. ②Gene expression; RT - PCR and cDNA microarrays were used to detect developmental genes expression of ARM and normal terminal rectum, explore relativity of ARM to these genes and investigate pathogenic genes. ③ Quality of life; Eight patients after surgically corrected ARM were followed up for 33 to 41 years. The quality of life was assessed by using an interview and a modified Quality of Life Questionnaire.
Materials
Animals; Wistar rats were provided by Medical Animal Center, the Second Clinical College of China Medical University.
Samples; Among 19 congenital. ARM infants operated in our department, 10 were males, and 9 were females., aged from 2 days to 3 years. Tissues were removed from terminal pouch of rectum and stored immediately in fluid nitrogen. In the control group, tissues were obtained at point 1 cm to dentate line in acquired anal fistula and rectal trauma infants.
Reagent; ①TUNEL kit was provided by Boster Biological Technology Co. , Ltd. Wuhan. ②Bca BEST RNA PCR kit verl. 1 ( AMV) was provided by Taka-ra Biotechnology Co. , Ltd. Dalian.③Gene chips were provided by BioStar Ge-nechip Inc. , Shanghai. Type: GCs.
Methods
Embryonic development; Cloaca malformation and ARM rats were produced by treating with administration of ETU on pregnant rats. Normal rat embryos and embryos with ARM from gestational days 12. 5 to 20 were sectioned serially and sagittally and stained with HE to observe development of cloaca and anorectum continually and contrastively. Some slices were stained with TUNEL to identify the distribution of apoptotic cells.
Gene expression; RT -PCR was used to investigate Hoxdl3 gene expression in rectal terminus of 16 infants with ARM and 5 normal infants, and cDNA microarrays to investigate mRNA in that of 3 infants with ARM and normal infants.
Quality of life; Eight patients (aged 33 to 41 years old) after surgically corrected ARM were followed up for 33 to 41, years. The quality of life was assessed by using an interview and a modified Quality of Life Questionnaire for the adult patients after surgically corrected ARM including ten items in six areas of
everyday life ( bowel function, food, occupational life, social activity, sexual life, and psychological aspects ). Twenty - four healthy people with similar age, sex and education level distributions served as controls.
Results
Embryonic development; During normal cloaca development process in rats, before gestational days 15, the urorectal septum (URS) developed and clearly demarcated th
引文
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2. Johnson RJ, Palken M, Derrick W, et al. The embryology of high anorectal and associated genitourinary anomalies in the female. Surg Gynecol Obstet, 1972, 135(5): 759-762.
3. de Vries PA, Friedland GW. The staged sequential development of the anus and rectum in human embryos and fetuses. J Pediatr Surg, 1974,9(5):755-69.
4. van der Putte SC. Normal and abnormal development of the anorectum. J Pediatr Surg, 1986, 21(5): 434-40.
5. Stephens FD. Embryology of the cloaca and embryogenesis of anorectal malformations. Birth Defects Original articles series, 1988, 24: 197-209.
6. Kluth D, Hillen M, Lambrecht W. The principles of normal and abnormal hindgut development. J Pediatr Surg, 1995, 30(8): 1143-1147.
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9. Paidas CN, Morreale RF, Holoski KM, et al. Septation and differentiation of the embryonic human cloaca. J Pediatr Surg, 1999, 34(5): 877-884.
10. Qi BQ, Williams A, Beasley S, et al. Clarification of the process of separation of the cloaca into rectum and urogenital sinus in the rat embryo. J Pediatr Surg, 2000, 35(12): 1810-1816.
11. Qi BQ, Beasley SW, Williams AK, et al. Does the urorectal septum fuse with the cloacal membrane? J Urol, 2000, 164(6): 2070-2072.
12. Hirai Y, Kuwabara N. Transplacentally induced anorectal malformations in rats. J Pediatr Surg, 1990, 25(7): 812-816.
13. Hung GF. Experimentally induced axial dysraphism and anorectal malformation in male rat fetuses by intragastric administration of ethylenethiourea. J Formosan Med Assoc, 1992, 91: 1166-1169.
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24. Qi BQ, Beasley SW, Williams AK, et al. Apoptosis during regression of the tailgut and septation of the cloaca. J Pediatr Surg, 2000, 35(11): 1556-1561.
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11. Qi BQ, Beasley SW, Williams AK, et al. Does the urorectal septum fuse with the cloacal membrane? J Urol, 2000, 164(6): 2070-2072.
12. Hirai Y, Kuwabara N. Transplacentally induced anorectal malformations in rats. J Pediatr Surg, 1990, 25(7): 812-816.
13. Hung GF. Experimentally induced axial dysraphism and anorectal malformation in male rat fetuses by intragastric administration of ethylenethiourea. J Formosan Med Assoc, 1992, 91: 1166-1169.
14. Hall PA, Coates PJ, Ansari B, et al. Regulation of cell number in the mammalian gastrointestinal tract: the importance of apoptosis. J Cell Sci, 1994, 107(Pt 12): 3569-3577.
15. Endo M, Hayashi A, Ishihara M, et al. Analysis of 1,992 patients with anorectal malformations over the past two decades in Japan. Steering Committee of Japanese Study Group of Anorectal Anomalies. J Pediatr Surg, 1999, 34(3): 435-441.
16. Cuschieri A. Descriptive epidemiology of isolated anal anomalies: a survey of 4.6 million births in Europe. Am J Med Genet, 2001, 103(3):207-215.
17. Kimmel SG, Mo R, Hui CC, et al. New mouse models of congenital anorectal malformations. J Pediatr Surg, 2000, 35(2): 227-230.
18. Wheeler PG, Weaver DD. Partial urorectal septum malformation sequence: a report of 25 cases. Am J Med Genet, 2001, 103(2): 99-105.
19. Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol, 1980, 68: 251-306.
20. Zhou B, Hutson JM, Farmer PJ, et al. Apoptosis in tracheoesophageal embryogenesis in rat embryos with or without adriamycin treatment. J Pediatr Surg, 1999, 34(5): 872-875.
21. Orford J, Manglick P, Cass DT, et al. Mechanisms for the development of esophageal atresia. J Pediatr Surg, 2001, 36(7): 985-994.
22. Miller SA, Briglin A. Apoptosis removes chick tail gut and remnant of the primitive streak. Development Dynamics, 1996, 206: 212-218
23. Kubota Y, Shimotake T, Yanagihara J, et al. Development of anorectal malformations using etretinate. J Pediatr Surg, 1998, 33(1): 127-129.
24. Qi BQ, Beasley SW, Williams AK, et al. Apoptosis during regression of the tailgut and septation of the cloaca. J Pediatr Surg, 2000, 35(11): 1556-1561.
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