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目的探索3种杯状细胞来源分子rTFF3、rMuc2和rIgGFcBp在结肠黏液层中的分布特点。方法雄性SD大鼠5只,采用Carnoy固定液固定结肠黏膜,AB-PAS染色验证不同黏液层的组织结构。通过轻吸、轻刮分别收集疏松层、致密层黏液和底层黏膜标本,加入不含DTT的2×SDS-PAGE上样缓冲液沸水浴提取全部蛋白。冷却至室温后,统一可溶性蛋白提取液体积,将不同黏液层等份样品进行还原或非还原SDS-PAGE,并对rTFF3、rMuc2和rIgGFcBp进行蛋白质印迹分析。结果两层肠道黏液均含rTFF3的250kD复合物和6kD单体,以疏松层明显;在疏松层和致密层黏液中未检测到55kD复合物。rIgGFcBp连续分布于黏液层,在非还原情况下,其分子量为214kD以上;在还原情况下,其分子量分别为258kD、214kD和140kD。rMuc2的分布模式与rIgGFcBp相似,以214kD以上的多聚体分布于黏液层,还原后转变为278kD和164kD的亚单位。结论rTFF3以单体和复合物的形式分布于疏松层和致密层黏液,其复合物可能与黏液的结构和功能相关;rIgGFcBp、rMuc2均以复合物形式分布于黏膜全层,推测两者均为黏液的结构性成分。
Objective To investigate the distribution of three goblet cell-derived molecules rTFF3, rMuc2 and rIgGFcBp in colonic mucosa. Methods Five male Sprague-Dawley rats were used to fix colon mucosa with Carnoy’s fixative solution. The tissue structure of different mucus layers was verified by AB-PAS staining. The loose layer, dense layer of mucus and the bottom mucosa were collected by light suction and light scraping, and the whole protein was extracted by adding 2 × SDS-PAGE loading buffer without DTT in a boiling water bath. After cooling to room temperature, the volume of the soluble protein extract was unified and aliquots of different mucus layers were subjected to reduced or non-reduced SDS-PAGE and Western blot analysis of rTFF3, rMuc2 and rIgGFcBp. Results The two layers of intestinal mucus contained rTFF3 250kD complex and 6kD monomer, respectively. The loose layer was obvious. No 55kD complex was detected in the loose layer and dense layer mucus. The rIgGFcBp is continuously distributed in the mucus layer, and its molecular weight is 214kD or more under the non-reducing condition. The molecular weight of rIgGFcBp is 258kD, 214kD and 140kD, respectively. The distribution pattern of rMuc2 is similar to that of rIgGFcBp, and is distributed in the mucus layer with a polymer of 214 kD or more and transforms to a subunit of 278 kD and 164 kD after reduction. Conclusion The rTFF3 is distributed in the form of monomer and complex in the loose layer and the dense layer of mucus. The complex of rTFF3 may be related to the structure and function of mucus. RIgGFcBp and rMuc2 are all distributed in the mucosal layer as complex, suggesting that both are The structural component of mucus.