创伤外科杂志
創傷外科雜誌
창상외과잡지
JOURNAL OF AUMATIC SURGERY
2015年
3期
252-255
,共4页
创伤%细胞%缺氧%自噬
創傷%細胞%缺氧%自噬
창상%세포%결양%자서
trauma%cells%hypoxia%autophagy
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。
目的：研究缺氧條件下腸上皮細胞自噬的變化。方法將培養的人腸上皮細胞株Caco-2分為常氧處理組（正常對照組）及缺氧組，缺氧組細胞分彆缺氧培養0.5、1、2、6、12及24h。採用蛋白質免疫印跡法檢測自噬相關（Beclin1）、微管相關蛋白1輕鏈3（LC3）及P62蛋白錶達，透射電子顯微鏡鏡觀察細胞自噬溶酶體變化，綠色熒光蛋白（GFP）-LC3B融閤蛋白示蹤腸上皮細胞自噬體形成的變化。結果與正常對照相比較，腸上皮細胞Beclin1和LC3的蛋白錶達水平在缺氧0.5h即開始逐漸增加，12h達高峰，24h仍顯著高于正常； P62蛋白錶達則逐漸降低，24h降至最低；透射電鏡下觀察到缺氧後腸上皮細胞自噬溶酶體顯著增多； GFP-LC3B融閤蛋白示蹤也顯示缺氧後腸上皮細胞自噬體形成非常明顯。結論缺氧後腸上皮細胞自噬作用顯著增彊，可能參與缺氧引起腸上皮細胞損害的調控。
목적：연구결양조건하장상피세포자서적변화。방법장배양적인장상피세포주Caco-2분위상양처리조（정상대조조）급결양조，결양조세포분별결양배양0.5、1、2、6、12급24h。채용단백질면역인적법검측자서상관（Beclin1）、미관상관단백1경련3（LC3）급P62단백표체，투사전자현미경경관찰세포자서용매체변화，록색형광단백（GFP）-LC3B융합단백시종장상피세포자서체형성적변화。결과여정상대조상비교，장상피세포Beclin1화LC3적단백표체수평재결양0.5h즉개시축점증가，12h체고봉，24h잉현저고우정상； P62단백표체칙축점강저，24h강지최저；투사전경하관찰도결양후장상피세포자서용매체현저증다； GFP-LC3B융합단백시종야현시결양후장상피세포자서체형성비상명현。결론결양후장상피세포자서작용현저증강，가능삼여결양인기장상피세포손해적조공。
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.
