缺氧诱导因子1α调控claudin-1蛋白表达对肠道粘膜屏障功能的影响

徐卫华，高胜东

孝昌县第一人民医院消化内科(湖北 孝昌 432900)



缺氧诱导因子1α调控claudin-1蛋白表达对肠道粘膜屏障功能的影响

徐卫华，高胜东

孝昌县第一人民医院消化内科(湖北 孝昌 432900)

目的观察缺氧诱导因子1α(HIF1α)对紧密连接蛋白claudin-1及肠道屏障功能的影响。方法在常氧和缺氧条件下培养大鼠小肠上皮细胞系IEC-6细胞，Elisa法检测HIF1α的分泌，RT-PCR及免疫印迹法观察claudin mRNA和蛋白表达，肠上皮细胞跨上皮电阻检测通透性。结果与常氧组比较，缺氧可明显增加HIF1α分泌，减少claudin-1蛋白表达，减小跨上皮电阻(P<0.01；P<0.001)。以重组HIF1α处理常氧状态下IEC-6细胞24～72 h，可明显抑制claudin-1的mRNA表达(P<0.01；P<0.001)。结论缺氧状态下IEC-6细胞分泌HIF1α增多，抑制claudin-1表达，进而增强肠上皮细胞通透性，导致屏障功能受损。

缺氧诱导因子1α；Claudin-1；缺氧；肠道屏障功能

【Abstract】Objective To observe the effects of Human hypoxia-inducible factor 1α (HIF1α) on claudin-1 expression and intestinal epithelial barrier function.Methods Intestinal epithelial cell line IEC-6 was cultured under the condition of normal oxygen and hypoxia. HIF1α secretion from IEC-6 was measured by Elisa and the expression of claudin-1 mRNA and protein was detected by reverse transcription-polymerase chain reaction and western blot,respectively.Transepithelial electrical resistance was measured by resistance test instrument.Results Compared with the normoxia group,HIF1α secretion significantly increased in the hypoxia group and suppressed claudin-1 protein expression.Moreover,transepithelial electrical resistance was also reduced under hypoxia.IEC-6 cells under normoxia condition with recombinant HIF1α suppressed claudin-1 mRNA expression.Conclusions Under the hypoxia condition, HIF1α was increasingly secreted from IEC-6 cells and it suppressed claudin-1 expression,which in turn enhanced intestinal permeability and resulted in intestinal epithelial barrier dysfunction.

【Key words】Human hypoxia-inducible factor 1α;Claudin-1;hypoxia；intestinal epithelial barrier function

肠黏膜机械屏障主要由紧密连接(tight junction，TJ)这一结构组成，为选择性屏障，可阻止大多数亲水性溶质透过[1]。TJ主要由相关蛋白复合物组成，成员包括claudin、 occludin家族、ZO(zonula occludens)家族、连接黏附分子等，其中最主要的结构蛋白为claudin-1[2]。大量研究表明，肠黏膜屏障功能调控与局部缺氧关系密切。缺氧可导致缺氧诱导因子1α(HIF1α)稳定表达[3]。然而HIF1α是否参与调控claudin-1表达，进而影响肠黏膜屏障功能尚不清楚。本研究通过体外细胞实验观察HIF1α在缺氧条件下对claudin-1表达以及肠上皮屏障功能的调控作用。

1　材料与方法

1.1主要试剂及仪器大鼠小肠上皮细胞系 IEC-6细胞购自中国科学院细胞库；HIF1α购自艾美捷科技有限公司；HIF1α ELISA试剂盒购自R&D Systems中国分公司；胎牛血清、胰蛋白酶购自美国Sigma 公司；抗大鼠闭合蛋白(claudin-1)购自美国Abcam公司；Transwell小室、Millicell ERS-2电阻测定仪、化学发光检测试剂盒、PVDF膜均购自美国Millipore公司；电泳仪、垂直电泳仪购自美国 BioRad公司；Trizol，RT-PCR引物及RT-PCR相关试剂盒购自上海捷瑞生物工程有限公司。……