维生素D调节坏死性小肠结肠炎新生大鼠肠道上皮occludin蛋白表达的研究

Regulatory effect of vitamin D on the expression of occludin in the intestinal epithelial cells of neonatal rats with necrotizing enterocolitis

目的检测坏死性小肠结肠炎（necrotizing enterocolitis，NEC）新生大鼠肠道上皮occludin蛋白的定位及表达情况，探索维生素D处理对NEC新生大鼠的保护作用及对occludin蛋白表达的影响。方法SPF级不同窝别新生48h的Wistar大鼠60只，采用随机数字表法随机分为4组：母乳喂养＋对照组10只，母乳喂养＋维生素D组10只，NEC十对照组20只，NEC＋维生素D组20只。NEC模型：将生后48h的仔鼠与母鼠隔离，采用鼠乳代乳品人工喂养＋缺氧＋寒冷刺激处理；维生素D处理：分别于NEC造模前30min，造模后1d、2d腹腔注射活性维生素D（帕立骨化醇）。各组大鼠分别在实验72h后处死取材，选取回肠组织采用HE染色观察肠腔结构改变并做Nadler病理评分，采用免疫荧光染色观察occludin蛋白在肠上皮的定位和表达情况，采用Westernblot方法检测肠道黏膜组织occludin蛋白的表达量并做统计学分析。结果HE染色可见NEC大鼠的肠腔组织结构破坏，黏膜下或肌层分离，部分绒毛脱落，甚至肠绒毛消失，肠上皮脱落伴肠坏死，Nadler病理评分为（2．90±0．23）分；维生素D处理的NEC大鼠上述病理表现较NEC大鼠减轻，Nadler病理评分为（1．70±0．21）分，两者比较差异有统计学意义（P〈0．01）。免疫荧光显示NEC大鼠肠道上皮细胞间occludin表达明显减少、稀疏，呈现不连续或点状表达，维生素D处理的NEC大鼠肠道上皮细胞间occludin表达较NEC对照组大鼠明显增多，基本呈均匀、连续的表达。Westernblot显示NEC大鼠肠道黏膜组织的occludin表达明显减少，维生素D处理可以明显增加occludin的表达（P〈0．01）。结论NEC模型大鼠肠道上皮紧密连接相关蛋白occludin表达明显降低，维生素D处理可以通过上调肠道上皮occludin的表达来抑制NEC的发生发展。

Objective To investigate the expression of occludin in the intestinal epithelial cells of neonatal rats with necrotizing enterocolitis （NEC）. The influence of vitamin D on NEC and the expression of occhidin was also explored. Methods Sixty neonatal Wistar rats from different litters were divided into four groups 48 hours after birth：10 in the breast-fed ＋ control group, 10 in the breast-fed ＋ vitamin D group,20 in the NEC ＋ control group and 20 in the NEC ＋ vitamin D group. In groups with NEC, neonatal rats were separated from their mothers and treated with rat milk substitute ＋ hypoxia ＋ hypothermia. In groups with vitamin D treatment, paricalcitol was given through intraperitoneal injection 30 minutes before, one day and two days after the induction of NEC. The tissue was harvested from the ileum of the rats 72 hours after induction of NEC. Microscopically,the tissue was assessed according to the Nadler scoring system. The expression and location of occludin was observed after immunofluorescence staining. Western blotting was performed and quantified to compare the expression of occludin among groups. Results In NEC rats,the intestinal structure was destructed. Separation of the submucosal or muscular layer, discharge or disappearance of the villa was accompanied by necrosis of the intestinal cells. Vitamin D treatment could significantly alleviate those changes ,with the Nadler score 1.70 ± 0. 21 compared to 2. 90 ±0. 23 without vitamin D treatment（ P 〈 0. 01 ）. The expression of occludin was decreased in NEC rats and vitamin D could partly rescue this decrease （P 〈0. 01）. The distribution of occhidin was sparse and discontinuous in NEC rats while after vitamin D treatment, the distribution was maintained even and continuous. Conclusion The expression of occludin in intestinal epithelial cells is significantly reduced in NEC rat. Vitamin D treatment may inhibit the development of NEC by maintaining the expression of occludin.