研究肠道糖基化修饰的荧光凝集素免疫组化方法建立

糖基化修饰是生命体中最重要的一种蛋白质翻译后修饰之一,对生命体起着非常重要的作用。糖蛋白上的糖基可以作为凝集素的结合位点,因此凝集素可以用来鉴别和分析糖链结构,作为糖基化分析方法如质谱法的重要补充。本试验旨在建立和优化研究猪肠道糖基化修饰的荧光凝集素免疫组化方法,并应用该方法研究猪不同肠段糖基化修饰的差异以及断奶对仔猪肠道糖基化修饰的影响。结果表明：①荧光凝集素免疫组化方法最适条件为：切片脱蜡后,封闭切片30 min,采用5 μg/mL FITC标记植物凝集素（FITC-Lectin）室温孵育1 h,以含有DAPI的封片剂进行封片。该方法具有灵敏度高、特异性好以及定量更为多元化的特点。②生长猪回肠和结肠中糖基化修饰模式不同,回肠主要以岩藻糖、N-乙酰葡萄糖胺为主,而结肠中主要以N-乙酰葡萄糖胺、岩藻糖和甘露糖为主。除甘露糖分布在非杯状细胞的肠上皮细胞和固有层外,岩藻糖和N-乙酰葡萄糖胺主要分布在肠绒毛表面和杯状细胞中。③22~28日龄仔猪回肠中N-乙酰葡萄糖胺的含量随着仔猪日龄的增加而增加（P<0.05）,断奶显著降低了28日龄仔猪回肠中N-乙酰葡萄糖胺的含量（P<0.05）。综上所述,本研究成功建立猪肠道糖基化修饰的研究方法,并通过此方法发现仔猪回肠中N-乙酰葡萄糖胺可作为肠道屏障功能完善的标志之一。

Establishment of Fluorescent Lectin-based Immunohistochemical Method to Investigate Glycosylation in the Intestine

Glycosylation is one of the most important post-translational modifications of proteins in life and plays a very important role in life.Oligosaccharides on glycoproteins can serve as binding sites for lectins,so lectins can be used to identify and analyze sugar chain structures,as an important complement to glycosylation analysis methods such as mass spectrometry.The purpose of this study was to establish and optimize the fluorescence lectin-based immunohistochemical method to study the glycosylation modification in the pig intestines,and to apply this method to study the differences in glycosylation modification in different intestinal segments and the effect of weaning on intestinal glycosylation modification in piglets.The results showed that:①The optimal conditions for fluorescence lectin-based immunohistochemical method were:deparaffinized sections were blocked with carbohydrate blocking solution for 30 min,sections were then incubated with 5 μg/mL FITC-lectin for 1 h at room temperature,and mounted with DAPI mounting medium.This method was characterized with high sensitivity,good specificity and more diversification regarding quantification.②Different glycosylation modification patterns were observed in the ileum and colon of growing pigs.The ileum was enriched with fucose and N-acetylglucosamine,while the colon was enriched with N-acetylglucosamine,fucose and mannose.Except that mannose was distributed in the non-goblet cells in the intestinal epithelium and lamina propria,fucose and N-acetylglucosamine were mainly distributed in the surface of intestinal villi and goblet cells.③The content of N-acetylglucosamine in the ileum increased along with the age of 22-28 days old piglets (P<0.05),and the content of N-acetylglucosamine in the ileum of piglets was significantly reduced by weaning at day 28 (P<0.05).In conclusion,a method to investigate glycosylation modification in pig intestine was successfully established in this study,and it was found that N-acetylglucosamine in the ileum could be used as a biomarker to indict a health intestinal barrier function in piglets by this method.