乙烯硫脲致畸胎鼠直肠末端Sonic hedgehog基因的表达

目的 探讨乙烯硫脲(ETU)致畸胎鼠直肠末端Sonic hedgehog(Shh)基因在胎鼠直肠肛门及其畸形发生过程中的表达和作用.方法 妊娠SD大鼠30只按受孕时间配对分为2组,实验组(n=15)在孕10 d予10 g/LETU(125 mg/kg)胃管注入,正常对照组(n=15)予等量蒸馏水.2组分别于孕13、14、15、16和17 d剖宫取胎鼠直肠末端1 cm提取RNA和组织蛋白,采用RT-PCR法和Western blot法检测Shh基因mRNA和蛋白表达情况.结果 在后肠发育的关键时期,实验组直肠末端Shh基因mRNA水平明显低于正常对照组,第14天实验组直肠末端的mRNA相对灰度值为0.29±0.22,正常对照组为2.23±0.54(t=5.72 P<0.05);第15天实验组直肠末端的mRNA相对灰度值为0.39±0.12,正常对照组为1.30±0.49(t=3.08 P<0.05).实验组Shh蛋白表达明显下降,胚胎第14天实验组直肠末端Shh基因蛋白相对灰度值为1.04±0.46,正常对照组为2.09±0.04(t=4.55 P<0.05);第15天,实验组直肠末端Shh基因蛋白相对灰度值为0.59±0.21,而正常对照组为0.95±0.16(t=26.0 P<0.05).结论 Shh的表达情况与直肠发育呈时间依赖性改变.Shh基因表达在肛门直肠及其畸形发生过程中发挥重要作用,ETU可能影响Shh信号转导系统和肛门直肠畸形发生.     

Gli2基因在胎鼠后肠发育后期的表达

目的探讨胎鼠后肠发育与sonichedgehog(Shh)基因转录因子Gli2基因表达水平的关系。方法48只妊娠Wistar大鼠随机分成2组,实验组(n=24)在孕11d按125mg/kg胃管注入1%乙烯硫脲(ethylenethiourea,ETU),对照组(n=24)给予等量蒸馏水。两组分别于孕12d、14d和16d各处死8只母鼠,每只母鼠取4只体重相似的胎鼠,取其直肠1cm提取RNA,采用半定量RT_PCR法检测Gli2基因表达。结果实验组胎鼠直肠Gli2基因表达水平明显低于对照组(P<0.05),实验组Gli2基因表达呈一低水平线,而对照组随妊娠时间呈一下降的曲线。结论Gli2基因表达水平在胎鼠后肠发育中具有重要的作用。     

肛门直肠畸形胎鼠直肠末端Gli2、BMP4基因的表达

目的探讨乙烯硫脲（ETU）致畸胎鼠直肠末端Gli2、BMP4基因在胎鼠直肠肛门及其畸形发生过程中的表达和作用。方法取妊娠SD大鼠30只,按受孕时间配对分成2组,实验组（n=15）于孕10d灌胃注入10g／LETU （125mg／kg）,对照组n=15）予等量蒸馏水。两组分别于孕13d、14d、15d、16d和17d剖宫取胎鼠直肠末端1cm提取RNA,采用RT—PCR和RealtimePCR法检测Gli2、BMP4基因在直肠肛门发育不同时期的表达情况。结果采用RT-PCR法检测Gli2、BMP4基冈在直肠肛门发育不同时期的表达情况．发现在胎鼠第13～17天,实验组直肠末端Gli2、BMP4mRNA的表达水平均明显低于正常胎鼠直肠末端（P〈0．05）。而Realtime PCR检测Gli2、BMP4mRNA表达时,发现实验组Gli2的表达在胚胎第13天、第14天、第15天分别为（0．73＋0．07、0．60＋0．09、0．81＋0．06）,勺对照组相比显著降低（P〈0．05）,而在孕第16天、第17天实验组Gli2表达比对照组虽有下降,但无统计学意义;实验组BMP4的表达在孕第3～17天均较对照组下降,差异有统计学意义。结论直肠肛门的正常发育需要Shh信号通路的正常表达,Shh信号通路中转录因子Gli2和靶基囚BMP4的异常表达在肛门直肠畸形发生过程中起重要作用,ETU可能影响Shh信号转导通路和肛门直肠畸形发生。     

先天性无肛畸形Gli2基因表达的研究

目的：探讨先天性肛门直肠畸形直肠末端SHH转录反应因子Gli2基因表达水平与畸形的关系。方法：采用RT－PCR方法检测16例不同类型先天性肛门直肠畸形直肠后壁末端及8例正常直肠后壁末端Gli2的表达水平,应用t检验比较正常组与畸形组、不同畸形类型之间Gli2表达水平的差异。结果:肛门直肠畸形直肠后壁末端Gli2的表达水平明显低于正常直肠（P＝0．01）,各不同类型畸形之间Gli2表达水平无明显差异（P＞0．05）。结论：Gli2表达水平减低与肛门直肠畸形的发生有关,还有其他机制参与该畸形的发生。     

EphB2在先天性肛门直肠畸形直肠末端表达的研究

目的 研究酪氨酸蛋白激酶受体--EphB2在先天性肛门直肠畸形直肠末端组织中的mRNA表达和蛋白表达的水平,探讨EphB2与先天性肛门直肠畸形发生的关系.方法 RT-PCR方法和Western蛋白印迹方法,检测31例不同类型先天性肛门直肠畸形直肠后壁末端、5例后天瘘及8例正常直肠后壁末端EphB2在蛋白水平和mRNA水平的表达情况,应用单因素方差分析比较正常组和畸形组、不同类型的畸形组之间EphB2表达水平的差异.结果 ①先天性肛门直肠畸形末端肠壁EphB2基因mRNA相对表达量明显低于正常对照组(0.99±0.11,P=0.00).不同类型的畸形组之间,高位组(0.43±0.07)和中位组(0.47±0.10)之间差异无统计学意义(P=0.33),但二者明显低于低位组(0.64±0.06,P=0.00).后天瘘组(0.97±0.09)与正常对照组比较差异没有统计学意义(P=0.63);②Western蛋白印迹结果显示,先天性肛门直肠畸形高位组(0.21±0.05)、中位组(0.24±0.04)和低位组蛋白表达水平均明显低于后天瘘组(0.51±0.05)和正常对照组(0.52±0.03,P=0.00).不同类型的畸形之间,高位组和中位组之间无明显差异(P=0.11),但二者明显低于低位组(P＜0.01).后天瘘组与正常对照组比较差异没有统计学意义(P=0.67).结论 EphB2在先天性肛门直肠畸形直肠末端低表达.EphB2的表达和肛门直肠畸形的发生有关,EphB2在先天性肛门直肠畸形的发生中可能具有重要作用.     

乙烯硫脲对胎鼠的致畸作用：肛门闭锁动物模型建立

应用致畸性药物乙烯硫脲诱导妊娠中的大白鼠，使胎鼠产生肛门直肠畸形。结果表明：肛门直肠畸形出现率为４０．６％，雄性发病率高，高位肛门闭锁占绝大多数，肛提肌均未见到明显的发育障碍。认为畸形发生与用药剂量有关，畸形种类与用药时间有关。     

先天性肛门直肠畸形胎鼠直肠末端NGF及受体TrkA与p75的表达

目的 观察神经生长因子(NGF)及其受体TrkA、p75在先天性肛门直肠畸形(ARM)动物模型胎鼠直肠末端的表达及分布.方法 选用成年SD孕鼠16只,实验组10只,对照组6只,实验组在孕11d及13d时分别以1％乙烯硫脲(125mg/kg)灌胃制作ARM动物模型,对照组灌入等量生理盐水,两组孕鼠在孕20 d手术取出宫内胎鼠,进行形态学观察及畸形率统计,并取胎鼠盆腔、会阴部矢状面大体标本HE染色及直肠末端石蜡包埋切片,免疫组化检测NGF及其受体TrkA、p75在正常胎鼠和ARM胎鼠直肠末端的分布情况并半定量分析.结果 正常组:胎鼠存活率100％,肛门开口正常,实验组:存活率80.7％,实验组ARM发生率98.7％、短尾畸形5.1％、无尾畸形94.8％、双足内翻6.4％、脊柱裂6.4％,盆腔会阴部矢状切面HE染色显示:ARM胎鼠肛门为一闭合盲端,无正常肛门结构,直肠盲端为增生鳞状上皮组织及增厚的肌层,未见肠腺分布,直肠末端黏膜下及肌间神经丛数量:ARM组为46.40±16.08,对照组为96.33±18.57,两组比较,差异有统计学意义(P＜0.01);免疫组化结果显示:NGF、TrkA、p75主要分布在胎鼠直肠末端黏膜下、肌间神经丛,其中NGF主要分布在细胞核,TrkA分布在细胞质,p75在细胞核、细胞质均有分布,其积分光密度(IOD值):对照组分别为731.75±232.18、262.82±101.72、134.67±67.13,ARM组分别为17.97±74.95、33.15±11.13、113.01±36.47,两组比较,差异有统计学意义(P＜0.01).结论 NGF及其受体TrkA、p75可能共同参与ARM的发生、发展过程.     

邻苯二甲酸二丁酯致雄性仔鼠肛门直肠畸形与相关基因表达的研究

目的 邻苯二甲酸二丁酯(di-n-butyl phthalate,DBP)孕晚期染毒诱导子代雄性大鼠发生肛门直肠畸形(anorectal malformations,ARMs),探讨形态学变化及相关基因的表达.方法 孕鼠20只,随机分为两组,在怀孕天数第12～18天,每日分别给予DBP及大豆油850mg/kg灌胃.出生后(PND)1d统计雄性仔鼠肛门闭锁发生率,测量雄性仔鼠体重和肛门生殖器距离(AGD),观察肛门闭锁雄性仔鼠末端直肠及肛周病理学改变.Real-time PCR法检测雄性仔鼠末端直肠组织中Shh、Gli2、Gli3、Bmp4、Wnt5a、Hoxa13、Hoxd13、Fgf10、Fgfr2基因mRNA的表达水平.结果 与对照组相比,DBP染毒组母鼠孕期体重增量减少,孕期延长,仔鼠产量降低(P＜0.05),PND 1d肛门闭锁雄性仔鼠体重及AGD较对照组明显降低(P＜0.05).染毒组雄性仔鼠肛门闭锁发生率大约为39.5％.肛门闭锁雄性仔鼠PND1末端直肠及肛周组织病理显示典型的ARMs.与对照组相比,肛门闭锁雄性仔鼠PND1末端直肠中Shh、Gli2、Gli3、Bmp4、Wnt5a、Hoxa13、Hoxd13、Fgf10、Fgfr2的mRNA表达水平较对照组也明显降低(P＜0.05).结论 DBP孕晚期染毒能明显诱导雄性仔鼠ARMs发生,并对其生长发育具有不良影响,其末端直肠组织中与肛门直肠分化发育相关的基因Shh、Gli2、Gli3、Bmp4、Wnt5a、Hoxa13、Hoxd13、Fgf10、Fgfr2的mRNA的表达水平降低可能促进ARMs的发生.     

TrkA、p75NTR mRNA在肛门直肠畸形胎鼠直肠末端的表达

目的通过检测肛门直肠畸形(ARM)胎鼠直肠末端神经生长因子受体TrkA、p75NTR mRNA的表达水平,探讨ARM直肠末端肠神经系统发育情况。方法成年SD孕鼠16只。其中实验组10只,对照组6只。实验组孕鼠在孕11 d、13 d 2次给予10 g.L-1乙烯硫脲(125 mg.kg-1)灌胃,制作先天性ARM动物模型;对照组灌胃注入等量9 g.L-1盐水。2组孕鼠在孕20 d时行剖宫手术取出胎鼠,取其直肠末端并保存于-80℃冰箱;采用反转录/实时定量PCR(RT-PCR)方法检测正常胎鼠和ARM胎鼠直肠末端TrkA、p75NTR mRNA表达情况。结果 TrkA、p75NTR mRNA在正常胎鼠直肠末端的相对表达量分别是162.221±104.675,129.778±51.976,在ARM胎鼠直肠末端中表达分别是78.937±33.425,87.145±25.812,两组比较差异均有统计学意义(Pa<0.05);琼脂糖凝胶电泳显示TrkA、p75NTR基因扩增后产物cDNA片段与引物设计的扩增片段完全吻合,TrkA、p75NTR基因条带亮度对照组均高于实验组。结论 TrkA、p75NTR mRNA在ARM胎鼠直肠末端的表达异常,提示神经生长因子及受体TrkA、p75NTR可能参与ARM肠神经系统的发育。     

先天性肛门直肠畸形基础及临床研究进展

先天性肛门直肠畸形基础及临床研究进展李正近年来，有关先天性肛门直肠畸形的病因研究有很大进展。有人发现肛门直肠畸形出现在正常家鼠的ＳＤ基因突变型鼠，称此鼠为ＳＤ鼠。ＳＤ基因以半显性方式遗传，影响直肠、泌尿生殖系统和中轴骨骼系统的发育。用杂合子ＳＤ鼠可繁...     

Diaphragmatic Hernia Induced in Rat Fetuses by Administration of Bisdiamine

Administration of N, N'-bis (dichloroacetyl)-l,8-octamethylenediamine, bisdiamine, to pregnant Donryu rats on a single day of gestation induced unilateral and bilateral diaphragmatic hernias in fetuses with high incidence. The protruded liver was not covered with a serous membrane or a muscular layer. Incidence of unilateral diaphragmatic hernia on the left side was high when bisdiamine was administered on day 9 or 13 of gestation, and that on the right side was high when administered on day 12 of gestation. Incidence of bilateral diaphragmatic hernia was high when bisdiamine was administered on day 12 of gestation. Differences in sensitivity to hernia formation according to day of bisdiamine administration between right and left sides may reflect differences in developmental chronology between the two sides. Two distinct times for induction of left diaphragmatic hernia might be attributed to at least two different mechanisms. The present model is expected to help analyzing not only anatomical characteristics of congenital diaphragmatic hernia but also possible mechanisms responsible for their development.     

低位无肛术术中完整保留瘘口组织的必要性和可行性

目的探讨低位肛门直肠畸形术术中保留瘘口组织的必要性和可行性。方法对67例无肛前庭及会阴瘘患儿的临床资料进行回顾性分析。其中女童无肛前庭瘘59例,男童无肛会阴瘘8例。年龄3个月~16岁,平均10个月。患儿多以排便困难或肛门位置异常就诊。4例无肛前庭瘘患儿曾在婴儿期行瘘口后切术。患儿均采用完整保留瘘口的前矢状入路肛门成形术治疗。结果患儿术后3及6个月常规来院复诊,最长随访8年。65例(97.0%)患儿会阴部矢状切口一期愈合,会阴及肛门外观良好;另2例(男、女各1例)术中直肠破损患儿,自修补处穿孔导致会阴部矢状切口感染(占直肠破损修补术的40%),最终形成直肠会阴瘘,但肛门外观良好。67例均采用肛门功能临床评分标准评估患儿排便功能,64例(95.5%)患儿排便功能良好,总评分为5~6分;另3例(男1例,女2例)顽固便秘,需开塞露协助排便。结论低位无肛术中完整保留瘘口组织非常必要。     

Atrioventricular Septal Defect in Rat Fetuses Induced by Administration of Bisdiamine

ABSTRACT A high incidence of atrioventricular septal defect was induced in rat fetuses by administration of N, N -his (dichloroacetyl)-1, 8-octamethylenediamine, bisdiamine, to pregnant Donryu rats. Induction of atrioventricular septal defect was highly dependent on day of bisdiamine administration. When bisdiamine was administered singly on day 9.4 of gestation or consecutively on days 9 and 10 of gestation, the incidence of atrioventricular septal defect in fetuses increased. Survival rate decreased with increase in the amount of bisdiamine administered. Normalized incidence ⁵ for atrioventricular septal defect, which was calculated as a product of incidence and survival rate, was as high as 54.5% when 175 mg bisdiamine was administered on day 9.4 of gestation. Atrioventricular septal defect thus formed was of the complete type associated with either persistent truncus arteriosus or tetralogy of Fallot, indicating that the conotruncal portion was unexceptionally malformed. The present model is useful for analysis of development and pathogenesis of atrioventricular septal defect and also contributes to clarifying the normal development of atrioventricular valves and septa.     

先天性肛门直肠畸形Hoxd 13基因表达的研究(英文)

目的：检测Hoxd 13基因在肛门直肠畸形病人和正常人的直肠末端表达状况 ,探讨Hoxd 13基因与人类先天性肛门直肠畸形发生的关系。方法：应用RT -PCR方法检测Hoxd 13基因在 16例肛门直肠畸形患儿和 5例正常儿童直肠末端的表达水平。结果：Hoxd 13基因mRNA在先天性无肛畸形直肠末端表达水平明显低于正常直肠末端的表达 (0 .32± 0 .36vs 0 .73± 0 .10 )。 (P <0 .0 1)。结论：Hoxd 13基因表达可能与人类先天性肛门直肠畸形的发生有关     

Congenital Hydrocephalus Induced in Rats by Prenatal Administration of Ethylenethiourea

Abstract: Pregnant Wistar rats were given a single i.p. injection of 30 mg/kg ethylenethiourea (ETU) on one of gestational days 8.5 to 20.5. After rearing to postnatal day 20, the offspring were sacrificed and their brains were excised. Numerous brains removed from the pups prenatally treated with ETU on any day from gestational days 12.5 to 20.5 showed dilatation of lateral ventricle in various degrees. Histological examination revealed forking and stenosis of third ventricle in these brains. Since the degree of the third-ventricular stenosis roughly corresponded to the degree of dilatation of lateral ventricle, this anomaly is considered to be the main cause of congenital hydrocephalus induced in rats by prenatal ETU-treatment. The embryological study showed that the ependymal lining of the third ventricle was partially denuded in the fetuses 24–48 hours after ETU-treatment, and also that the denuded ependymal lining was never repaired during the following gestational days; instead, the ventricular walls fused laterally in the area where the ependymal lining had been denuded.     

Syndromic microphthalmia‐3 caused by a mutation on gene SOX2 in a Colombian male patient

Syndromic microphthalmia‐3 is a rare congenital syndrome associated with brain anomalies, esophageal atresia and genital anomalies. This is the case of a 4‐year‐old male with bilateral microphthalmia, short stature, neurodevelopmental delay, genital anomalies, and maternal exposition to glyphosate during pregnancy. Genetic testing detected a previously reported pathogenic heterozygous mutation in the SOX2 gene, confirming a diagnosis of syndromic microphthalmia‐3. Whenever a patient presents bilateral microphthalmia, it is necessary to determine whether it is isolated or syndromic; afterwards, genetic testing should be performed in order to offer an effective genetic counseling.