乙烯硫脲致畸胎鼠直肠末端Sonic hedgehog基因的表达

Expression of Sonic Hedgehog Gene in Distal Rectum of Rat Fetus with Malformation Caused by Ethylenethiourea

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。

Objective To investigate the expression and effective of Sonic hedgehog（Shh） gene during anorectum development in ethyle- nethiourea （ETU） -exposed embryos with anorectal malformations. Methods Thirty time - mated pregnant Sprague - Dawley （SD） rats were divided into control group and experimental group. The experimental rats（ n = 15 ） were received 10 g/L ETU （125 mg/kg） by garage 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 day13 - 17. Reverse transeriptase - polymerase chain reaction（ RT - PCR） and Western blot were done to determine the expression of Shh in each sample. Results In the critical period of hind gut development, mRNA of Shh expression levels of ETU exposed rat embryos were significantly decreased on day 14 and 15 in the developing hindguts as compared with normal control group（0.29 ±0.22 vs 2.23 ±0.54 andO. 39±0. 12vs1.30±0. 49,respectively）（t=5.72,3.08 Pa〈 0.05 ）. Protein of Shh expression levels of ETU exposed rat embryos were also significantly decreased on day 14 and 15 in the developing hindguts as compared with normal control group（ 1.04 ± 0.46 vs 2.09±0.04 and0.59±0.21 vs0.95 ±0. 16,respectively）（t=4.55,26.0 Pa 〈0.05）. Conclusions Shh expression shows time -dependent changes in the developing normally separates into the rectum and urethra occurs in the rat fetus. The misregulated expression of Shh in the distal rectum of ETU exposed rat embryos suggests that Shh plays a crucial role in the development of anorectum and ETU may interfere with Shh signalling and the development of congenital anorectal malformations.