胶质细胞源性神经营养因子及其酪氨酸激酶受体RET在不同胎龄先天性肛门直肠畸形胎鼠直肠末端的表达

Expressions of glial cell line -derived neurotrophic factor and its tyrosine kinase receptor RET in the terminal rectum of fetal rats with congenital anorectal malformations at different gestational ages

目的观察胶质细胞源性神经营养因子（GDNF）及其酪氨酸激酶受体RET在不同胎龄先天性肛门直肠畸形（ARM）胎鼠直肠末端的表达和分布情况，探讨其对ARM胎鼠直肠末端肠神经系统发育的影响。方法SD孕鼠35只按简单随机法分为9 g/L盐水对照组（10只）和乙烯硫脲实验组（25只），2组孕鼠在孕16 d、孕18 d、孕20 d取出胎鼠，肉眼及解剖显微镜下将胎鼠分为9 g/L盐水对照组、乙烯硫脲对照组（未产生ARM胎鼠）、乙烯硫脲畸形组（ARM胎鼠）。应用HE染色观察各组胎鼠直肠末端肠神经节细胞形态并计数。应用免疫组织化学染色、Western blot法观察3组胎鼠在孕16 d、孕18 d、孕20 d直肠末端GDNF及受体RET分布情况。实时荧光定量聚合酶链反应（qRT-PCR）检测3组胎鼠在孕16 d、孕18 d、孕20 d直肠末端GDNF mRNA的表达情况。结果HE染色：3组胎鼠在孕16 d时肛门直肠末端各层发育不清晰，肌层见少量散在神经丛，细胞核小，分布稀疏，轴突及胞质少；在孕18 d和孕20 d时9 g/L盐水对照组、乙烯硫脲对照组胎鼠直肠末端浆膜层、肌层、黏膜下层、黏膜层及其腺体逐渐清晰可见，肌间及黏膜下神经丛逐渐增多（孕18 d：11.400±3.134、11.200±3.425；孕20 d：66.100±4.954、67.600±5.481），而乙烯硫脲畸形组层次不清，神经丛少（孕18 d：7.800±1.989；孕20 d：25.200±3.048），与2个对照组比较差异均有统计学意义（F＝7.591、271.833，P均〈0.05）。免疫组织化学染色结果：孕16 d时3组胎鼠GDNF及RET在肠壁各层表达尚不清晰，仅观察到极少量弱阳性细胞；在孕18 d和孕20 d时9 g/L盐水对照组和乙烯硫脲对照组直肠末端黏膜层、黏膜下及肌间神经丛中均有表达，随胚胎不断发育，表达强度逐渐增强，与2个对照组比较，差异均有统计学意义（P均〈0.05）。qRT-PCR：在孕16 d 3组GDNF mRNA比较，差异无统计学意义（P〉0.05）；在孕18 d 9 g/L盐水对照组和乙烯硫脲对照组GDNF mRNA分别为103.624±27.533和105.184±19.634；孕20 d时9 g/L盐水对照组和乙烯硫脲对照组GDNF mRNA分别为151.496±33.622和150.738±21.423，与乙烯硫脲畸形组比较（孕18 d为79.169±11.697，孕20 d为94.873±11.309），差异均有统计学意义（P均〈0.05）。结论GDNF及其酪氨酸激酶受体RET在不同胎龄正常胎鼠与ARM胎鼠直肠末端表达均有一定的时间相关性；GDNF及其酪氨酸激酶受体RET在ARM胎鼠直肠末端表达的异常可能影响其肠神经系统发育。

ObjectiveTo explore the expressions and distributions of glial cell line-derived neurotrophic factor （GDNF） and itstyrosine kinase receptor RET in the terminal rectums of fetal rats with congenital anorectal malformations （ARM） at different gestationalage, and to explore their effects on the enteric nervous system in the terminal rectum of ARM fetal rats.MethodsThirty-five SD pregnancy rats were divided into a saline group （n=10） and an ethylenethiourea experiment group （n=25） by simple randomized study.The fetal rats were removed from the pregnant rats at the gestational 16 d, 18 d and 20 d. The fetal rats were divided into the saline control group, the ethylenethiourea control group （fetal rats without ARM） and the ethylenethiourea malformation group （ARM fetal rats） by the naked eye and dissecting microscope.HE staining was used to observe the morphology and the intestinal ganglion cells in the terminal rectum were counted.The immunohistochemical staining and Western blot methods were used to observe the distributions of GDNF and RET in the rectum at the gestational 16 d, 18 d and 20 d. The quantitative real-time polymerase chain reaction （qRT-PCR） was used to detect the expression of GDNF mRNA in the fetal rats in the terminal rectum at the gestational 16 d, 18 d and 20 d. ResultsHE staining： the development of anorectal terminal in 3 groups of fetal rats was unclear at the gestational 16 d. A small amount of scattered nerve plexuses were observed in the muscular layer.The nuclei were small and sparse.The axons and cytoplasms were less.The serosal layer, muscular layer, submucosa, mucosal layer and glands in the terminal rectum were gradually clear in the saline control group and the ethylenethiourea control group at the gestational 18 d and 20 d. The intermuscular submucosal nerve plexuses increased gra-dually （11.400±3.134 and 11.200±3.425 at the gestational 18 d; 66.100±4.954 and 67.600±5.481 at the gestational 20 d）. While, the layer was unclear in the ethylenethiourea malformation group and the nerve plexus was less （7.800±1.989 at the gestational 18 d, and 25.200±3.048 at the gestational 20 d）, and the difference was statistically significant compared with 2 control groups （F=7.591, 271.833, all P〈0.05）. Immunohistochemistry satning： the expressions of GDNF and RET in all layers of the intestinal wall in the 3 groups of fetal rats were unclear at the gestational 16 d and only a few positive cells were observed.The GDNF and RET were expressed in the mucosal layer and submucosa of the terminal rectum as well as intermuscular nerve plexus in the saline control group and the ethylene-thioured control group at the gestational 18 d and 20 d. With the continuous development of the embryo, their expression intensities were gradually increased.The expressions of GDNF and RET positive cells were decreased gradually in the ethylenethiourea malformation group.The difference was significant statistically compared with 2 control groups （all P〈0.05）. qRT-PCR： the expressions of GDNF mRNA showed no statistical difference among 3 groups at the gestational 16 d （P〉0.05）; the expressions of GDNF and RET protein were 103.624±27.533 and 105.184±19.634 at the gestational 18 d; 151.496±33.622 and 150.738±21.423 at the gestational 20 d in 2 control groups.Compared with the ethylenethiourea malformation group （79.169±11.697 at the gestational 18 d; 94.873±11.309 at the gestational 20 d）, and the difference were statistically significant （all P〈0.05）.ConclusionsThe expressions of GDNF and its tyrosine kinase receptor RET had a certain temporal correlation in the terminal rectum of normal fetal rats at different gestational ages and ARM.Moreover, the abnormal expressions of GDNF and its tyrosine kinase receptor RET in the distal rectum of ARM fetal rats can affect the development of enteric nervous system.