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目的:检测γ-氨基丁酸(gamma-aminobutyricacid,GABA)和谷氨酸脱羧酶(glutamicaciddecarboxylase,GAD)在大鼠降结肠上皮的表达及分布特征,并探讨GABA与上皮细胞分化增殖的关系.方法:用免疫荧光及激光共聚焦显微扫描技术,检测GABA、GAD65及GAD67在大鼠降结肠上皮中的表达,并以麦芽凝聚素组织化学染色与免疫荧光结合的双重染色显示GABA和GAD65表达细胞的分布特征.同时,用RT-PCR和原位分子杂交方法检测GADmRNA的表达.此外,用3H-胸腺嘧啶放射自显影法显示降结肠上皮的增殖带.结果:RT-PCR显示降结肠黏膜中GAD65及GAD67mRNA均阳性,原位杂交显示阳性杂交信号主要分布在上皮细胞的隐窝和腔面,且GAD65信号较GAD67强.GABA及GAD65免疫反应阳性细胞主要分布在降结肠的腔面和隐窝的上1/3上皮细胞的胞质,而GAD67阳性细胞仅分布腔面,此外,GABA及GAD65阳性染色也见于黏膜固有层.双重染色显示杯状细胞中GABA及GAD65均阴性.3H-胸腺嘧啶标记阳性细胞主要在隐窝的中下段.结论:GABA及GAD65分布在大鼠降结肠上皮的成熟带及功能带,GABA系统可能参与上皮细胞的分化与增殖的调节.
OBJECTIVE: To detect the expression and distribution of gamma-aminobutyric acid (GABA) and glutamic acid decarboxylase (GAD) in the descending colon epithelium of rats and to explore the relationship between GABA and epithelial cell differentiation and proliferation.Methods : The expression of GABA, GAD65 and GAD67 in the rat descending colon epithelium was detected by immunofluorescence and confocal laser scanning microscopy. The expression of GABA and GAD65 in the ascending colon of the rat was detected by double staining with the combination of immunofluorescence and hemagglutinin chemiluminescence The expression of GAD mRNA was detected by RT-PCR and in situ hybridization.Additionally, 3H-thymidine autoradiography was used to detect the proliferation zone of descending colon epithelium.Results: RT-PCR showed that the expression of GAD mRNA in the descending colon mucosa GAD65 and GAD67mRNA were all positive.The in situ hybridization showed that the positive hybridization signal mainly distributed in the crypt and cavity surface of epithelial cells and the GAD65 signal was stronger than GAD67.The GABA and GAD65 immunoreactive positive cells were mainly distributed in the descending colon cavity and crypt Of the upper third of the epithelial cell cytoplasm, and GAD67 positive cells only distributed cavity surface, in addition, GABA and GAD65 positive staining is also found in lamina propria. Double staining showed goblet cells GABA and GAD65 were negative.3H-thymidine positive cells mainly in the middle and lower crypt.Conclusion: GABA and GAD65 are distributed in the mature zone and function zone of the descending colon epithelium in rats, GABA system may be involved in epithelial cell differentiation and proliferation Adjustment.