NeurexinⅡ基因在不同胎龄大鼠胚胎肠神经系统的表达及意义

Expression and significance of Neurexin Ⅱ in enteric nervous system of rats at different embryonic days

目的观察不同胎龄大鼠胚胎肠神经系统（enteric nervous system，ENS）中NeurexinⅡ基因的表达，探讨NeurexinⅡ基因与ENS发育的时间相关性及与肠神经元发育异常性疾病的可能关系。方法清洁级健康Wistar大鼠用随机数字表法随机分组后，单独交配受孕。麻醉后处死孕龄16d、18d和20d孕鼠及出生第1天乳鼠，手术取胚胎第16天（E16组）、18天（E18组）、20天（E20组）和出生第1天（Ep0组）大鼠近回盲部约1．0cm结肠样本，E16组、E18组、E20组、Ep0组样本数量分别为78、82、69和60个。运用免疫组织化学染色、Western-blotting及qRT-PCR实验方法检测各标本NeurexinⅡ基因在蛋白和mRNA水平的表达。结果免疫组织化学染色结果显示NeurexinⅡα蛋白主要表达于结肠肌间神经丛，在黏膜下神经丛亦有少量表达，其表达量自胚胎第16天至出生第1天，随胚胎逐渐发育表达逐渐增加。Western-blotting结果显示NeurexinⅡα蛋白相对含量在E16、E18、E20和Ep0组分别为0．054±0．009、0．106±0．020、0．215±0．013和0．280±0．030，相对表达量随胚胎发育逐渐增加，组间差异比较与两两比较，差异均有统计学意义（P〈0．05），与免疫组化结果趋势一致。qRT-PCR结果显示NeurexinⅡ基因在mRNA水平E16、E18、E20和Ep0组中相对表达分别为0．390±0．029、0．552±0．054、0．748±0．065和1．000±0．000，与蛋白水平表达趋势一致，组间差异比较与两两比较，差异均有统计学意义（P〈0．05）。结论NeurexinⅡ基因与ENS发育具有时间相关性；若其在ENS发育过程中的某一时间点出现异常，可能导致肠神经元发育异常性疾病的发生。

Objective To elucidate the expressions of Neurexin Ⅱ in enteric nervous system （ENS） in rats at different embryonic days and explore the correlation of expressions with the development of ENS and neuronal intestinal malformations. Methods The 16, 18, 20-day pregnant Wistar rats and the newborns within 24 h were sacrificed after anesthesia. And 78, 82, 69 and 60 specimens at 1.0 cm near the appendix were harvested at E16, El8, E20, Ep0 respectively. Then the expressions of NeurexinⅡα and mRNA were verified by ilmnunohistochemical staining, Western blot and quantitative reverse transcription-polymerase chain reaction （ qRT-PCR ）. Results Immunohistochemical staining revealed that Neurexin Ⅱα protein positive cells were observed in myenteric nerve plexus of colon specimens and its expression level increased gradually from embryonic day 16 to newborns within 24 h. Western blot also revealed that the Neurexin Ⅱ a protein expression levels of El6, El8, E20 and Ep0 （0. 054 ± 0. 009, 0. 106 ± 0. 020, 0. 215 ± 0. 013,0. 280 ± 0. 030） increased gradually with embryonic growth （P〈0. 05）. And qRT-PCR revealed that the tendency of Neurexinll rnRNA expression level at E16, E18, E20 and Ep0 （0. 390 ± 0. 029, 0. 552 ± 0. 054, 0. 748 ± 0. 065, 1. 000 ± 0. 000, respectively） was ansistent with that of protein（P〈0. 05）. Conclusions Neurexin Ⅱ may have temporal correlation with the development of ENS during whichneuronal intestinal malformations occur due to dysplasia.