P2Y2受体在患有肛门直肠畸形的胎鼠终末直肠中的表达。

Expression of the P2Y2 receptor in the terminal rectum of fetal rats with anorectal malformation.

作者信息

Liu Yuan-Mei, Kong Meng, Jin Zhu, Gao Ming-Mei, Qu Yan, Zheng Ze-Bing

机构信息

Department of Pediatric General Thoracic Urology Surgery, The Affiliated Hospital of Zunyi Medical College Zunyi 563003, China.

出版信息

Int J Clin Exp Med. 2015 Feb 15;8(2):1669-76. eCollection 2015.

PMID:25932095

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4402742/

Abstract

OBJECTIVE

The expression and distribution of a subtype of purine receptors (P2Y2) in the terminal rectum of fetal rats with anorectal malformations (ARM) were examined to investigate their possible impact on the development of the enteric nervous system (ENS).

METHODS

Pregnant Sprague-Dawley rats were randomly divided into a control group (5 rats) and an experimental group (20 rats). The experimental group was treated with ethylene thiourea (ETU). On gestational day 20, the intrauterine fetal rats were collected from both groups of pregnant rats. Sagittal sections of the pelvic perinea were stained with HE. P2Y2 protein and mRNA expression in the terminal recta of the fetal rats in the control group, the ARM group, and the ETU-treated group that exhibited no malformations (the ETU group) were detected by immunohistochemistry, western blot, and qRT-PCR.

RESULTS

The fetal rats in the control group showed normal position of the anal opening, with no malformation. The incidence of ARM was 89.2% for the fetal rats in the experimental group. The immunohistochemistry results showed that P2Y2 was expressed in the cytoplasm of the cells in the terminal rectum submucosa and myenteric plexus of the fetus rats in the control group, the ETU group, and the ARM group. The average integrated optical density (IOD) value for the ARM group was significantly lower than the IOD value for the control and ETU groups (186.48 ± 23.03 vs. 493.18 ± 19.70; 186.48 ± 23.03 vs. 479.48 ± 41.71, P<0.01), while the IOD value for the ETU group was comparable to the control group IOD (493.18 ± 19.70 vs. 479.48 ± 41.71, P = 0.360). The western blot and qRT-PCR results showed that the P2Y2 protein and mRNA expressions were significantly lower in the terminal rectum of the fetal rats in the ARM group than in the control and ETU groups (0.28 ± 0.08 vs. 0.51 ± 0.10, 0.28 ± 0.08 vs. 0.48 ± 0.12; 48.91 ± 12.17 vs. 98.03 ± 15.68, 48.91 ± 12.17 vs. 92.53 ± 10.43; P<0.01), while the P2Y2 protein and mRNA levels in the control group were comparable to the ETU group (0.51 ± 0.10 vs. 0.48 ± 0.12, P = 0.494; 98.03 ± 15.68 vs. 92.53 ± 10.43, P = 0.058).

CONCLUSION

P2Y2 may participate in and affect the development of ENS in the terminal rectum of fetal rats with ARM.

摘要

目的

检测嘌呤受体亚型（P2Y2）在肛门直肠畸形（ARM）胎鼠终末直肠中的表达及分布，探讨其对肠神经系统（ENS）发育的可能影响。

方法

将妊娠的Sprague-Dawley大鼠随机分为对照组（5只）和实验组（20只）。实验组用乙硫脲（ETU）处理。在妊娠第20天，从两组妊娠大鼠中收集子宫内的胎鼠。盆腔会阴矢状切片行HE染色。采用免疫组织化学、蛋白质免疫印迹法和实时荧光定量PCR检测对照组、ARM组及未出现畸形的ETU处理组（ETU组）胎鼠终末直肠中P2Y2蛋白和mRNA的表达。

结果

对照组胎鼠肛门开口位置正常，无畸形。实验组胎鼠ARM发生率为89.2%。免疫组织化学结果显示，对照组、ETU组和ARM组胎鼠终末直肠黏膜下层和肌间神经丛细胞的细胞质中均有P2Y2表达。ARM组的平均积分光密度（IOD）值显著低于对照组和ETU组（186.48±23.03 vs. 493.18±19.70；186.48±23.03 vs. 479.48±41.71，P<0.01），而ETU组的IOD值与对照组相当（493.18±19.70 vs. 479.48±41.71，P = 0.360）。蛋白质免疫印迹法和实时荧光定量PCR结果显示，ARM组胎鼠终末直肠中P2Y2蛋白和mRNA表达显著低于对照组和ETU组（0.28±0.08 vs. 0.51±0.10，0.28±0.08 vs. 0.48±0.12；48.91±12.17 vs. 98.03±15.68，48.91±12.17 vs. 92.53±10.43；P<0.01），而对照组中P2Y2蛋白和mRNA水平与ETU组相当（0.51±0.10 vs. 0.48±0.12，P = 0.494；98.03±15.68 vs. 92.53±10.43，P = 0.058）。