CAJ | 학술논문

目的观察不同胎龄大鼠胚胎肠神经系统(enteric nervous system,ENS)中Neurexin Ⅱ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16 d、18 d和20 d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1.0 cm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Neurexin Ⅱα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.
목적 관찰불동태령대서배태장신경계통(enteric nervous system,ENS)중Neurexin Ⅱ기인적표체,탐토NeurexinⅡ기인여ENS발육적시간상관성급여장신경원발육이상성질병적가능관계.방법 청길급건강Wistar대서용수궤수자표법수궤분조후,단독교배수잉.마취후처사잉령16 d、18 d화20 d잉서급출생제1천유서,수술취배태제16천(E16조)、18천(E18조)、20천(E20조)화출생제1천(Ep0조)대서근회맹부약1.0 cm결장양본,E16조、E18조、E20조、Ep0조양본수량분별위78、82、69화60개.운용면역조직화학염색、Western-blotting급qRT-PCR실험방법검측각표본NeurexinⅡ기인재단백화mRNA수평적표체.결과 면역조직화학염색결과현시Neurexin Ⅱα단백주요표체우결장기간신경총,재점막하신경총역유소량표체,기표체량자배태제16천지출생제1천,수배태축점발육표체축점증가.Western-blotting결과현시NeurexinⅡα단백상대함량재E16、E18、E20화Ep0조분별위0.054±0.009、0.106±0.020、0.215±0.013화0.280±0.030,상대표체량수배태발육축점증가,조간차이비교여량량비교,차이균유통계학의의(P＜0.05),여면역조화결과추세일치.qRT-PCR결과현시NeurexinⅡ기인재mRNA수평E16、E18、E20화Ep0조중상대표체분별위0.390±0.029、0.552±0.054、0.748±0.065화1.000±0.000,여단백수평표체추세일치,조간차이비교여량량비교,차이균유통계학의의(P＜0.05).결론 NeurexinⅡ기인여ENS발육구유시간상관성;약기재ENS발육과정중적모일시간점출현이상,가능도치장신경원발육이상성질병적발생.
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, E18, E20, Ep0 respectively.Then the expressions of Neurexin Ⅱ α and mRNA were verified by immunohistochemical staining, Western blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR).Results Immunohistochemical staining revealed that Neurexin Ⅱ α 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 Ⅱα protein expression levels of E16, E18, 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 NeurexinⅡ mRNA 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 which neuronal intestinal malformations occur due to dysplasia.