肛门直肠畸形胎鼠直肠末端Gli2、BMP4基因的表达

Expression of Gli2,BMP4 gene in distal rectum in rat fetus with anorectal malformations.

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。

Objective To investigate the pattern of expression of Gli2,BMP4 gene during anorectum development in ethylenethiourea （ETU3-exposed embryos with anorectal malformations. Methods Thirty time-mated pregnant Sprague-Dawley （SD） rats were divided into control and experimental groups. The experimental rats （n = 15） were received 10 g/L ETU （125 rag/kg） by gavage on gestational day 10, and control rats （ n = 15） were received an equal dose of distilled water. Their embryos were harvested by caesarean section on gestational day 13 - 17. RT-PCR were done to determine the expression of Gli2 and BMP4 in each sample. Results In the critical period of hind gut development, mRNA of Gli2 and BMP4 expression levels of ETU exposed rat embryos were significantly decreased on day 13 to 17 in the developing hindguts as compared to controls （P〈0.05）. Relative quantitation PCR demonstrated the expression of Gli2 levels of ETU exposed rat embryos were significantly decreased on day 13,14 and 15 （0.73＋0.07,0.60＋0.09 and 0.81＋0.06, respectively） in the developing hindguts as compared to controls （P〈0.05）. On day 16 and 17, mRNA expression of Gli2 levels of ETU exposed rat embryos were diminished but without reaching significant levels compared with controls; The expression of BMP4 levels of ETU exposed rat embryos were all significantly decreased on day 13 to 17 in the developing hindguts as compared to controls （P〈0.05）. Conclusions Shh signaling pathway is essential for the development of the distal hindgnl. The misregnlated expression of transcription factor Gli2,target gene BMP4 plays a crucial role in the development of anorectum and malformation, Ethylenethiourea may interfere with expression of Shh signaling pathway and form all kinds ofanorectal malformations.