缺氧肠上皮细胞自噬的变化及意义

目的：研究缺氧条件下肠上皮细胞自噬的变化。方法将培养的人肠上皮细胞株Caco-2分为常氧处理组（正常对照组）及缺氧组，缺氧组细胞分别缺氧培养0.5、1、2、6、12及24h。采用蛋白质免疫印迹法检测自噬相关（Beclin1）、微管相关蛋白1轻链3（LC3）及P62蛋白表达，透射电子显微镜镜观察细胞自噬溶酶体变化，绿色荧光蛋白（GFP）-LC3B融合蛋白示踪肠上皮细胞自噬体形成的变化。结果与正常对照相比较，肠上皮细胞Beclin1和LC3的蛋白表达水平在缺氧0.5h即开始逐渐增加，12h达高峰，24h仍显著高于正常； P62蛋白表达则逐渐降低，24h降至最低；透射电镜下观察到缺氧后肠上皮细胞自噬溶酶体显著增多； GFP-LC3B融合蛋白示踪也显示缺氧后肠上皮细胞自噬体形成非常明显。结论缺氧后肠上皮细胞自噬作用显著增强，可能参与缺氧引起肠上皮细胞损害的调控。

Changes and significance of autophagy in hypoxic intestinal epithelial cells

Objective To study the changes of autophagy in intestinal epithelial cells under hypoxic condi-tion.Methods The cultured human intestinal epithelial cell lines Caco-2 were divided into normoxia group ( nor-mal control group) and hypoxia group.In the hypoxia group,cells were exposed to hypoxia for 0.5,1,2,6, 12 and 24 hours,respectively.The protein expression of Beclin1,mircrotublue-assoliated protein 1 light chain 3( LC3) and P62 was assayed by Western blotting.The cellular autophagy lysosomals were detected by transmission electron mi-croscopy.GFP-LC3B fusion protein was employed to visualize autophagosome formation in intestinal epithelial cells.Results The protein expression of both Beclin1 and LC3 began to increase at 0.5h in hypoxic intestinal epi-thelial cells when compared with those in normal control group,peaked at 12h,and was still higher than those in nor-mal control group at 24h.The expression of P62 decreased gradually after hypoxia,with the lowest at 24h.Under transmission electron microscopy, it was observed that the number of autophagy lysosomal increased significantly in hy-poxic intestinal epithelial cells.GFP-LC3B fusion protein tracer also showed that autophagosome formation was quite evident in hypoxic intestinal epithelial cells.Conclusion The autophagy in intestinal epithelial cells is significantly enhanced by hypoxia and may contribute to the regulation of hypoxia-induced damage in intestinal epithelial cells.