摘要目的 研究缺氧对肠上皮细胞丝切蛋白活化的影响及丝切蛋白活化与紧密连接蛋白带状闭合蛋白1(ZO-1)分布变化的关系. 方法 建立人肠上皮细胞株Caco-2单层细胞培养模型,取部分细胞按照随机数字表法分为对照组(不处理)、缺氧组(置于缺氧环境下)和常氧组(置于常氧环境下),取对照组细胞(样本数为9)及其他2组培养1、2、6、12、24 h的细胞(各时相点样本数均为9)采用蛋白质印迹法检测丝切蛋白及磷酸化丝切蛋白的表达.另取细胞,按照随机数字表法分为缺氧组和对照组(处理同前),取对照组细胞和缺氧组缺氧1、2、6、12、24 h的细胞采用激光扫描共聚焦显微镜观察纤维状肌动蛋白的形态与分布,荧光法检测纤维状肌动蛋白与球状肌动蛋白含量的比值,激光扫描共聚焦显微镜观察细胞形态及ZO-1的分布;该3个实验中对照组样本数依次为3、6、3,缺氧组各时相点样本数依次为3、6、3.对数据行重复测量方差分析、配对t检验、LSD-t检验. 结果 与对照组比较,常氧组细胞培养1 ～24 h丝切蛋白及磷酸化丝切蛋白表达量均无明显变化(t值分别为-0.385 ～1.701、0.040 ～1.538,P值均大于0.05).与对照组比较,缺氧组细胞培养1～24 h丝切蛋白表达量也无明显变化(t值为1.032 ～2.390,P值均大于0.05);但磷酸化丝切蛋白表达量均明显降低(t值为4.563 ～ 22.678,P值均小于0.01),其中培养24 h降至最低值.常氧组、缺氧组细胞各时相点丝切蛋白表达量相近(t值为-0.904 ～1.433,P值均大于0.05);缺氧组细胞培养1、2、6、12、24 h时磷酸化丝切蛋白表达量分别为0.87±0.08、0.78±0.05、0.89±0.07、0.68±0.07、0.57±0.06,均低于常氧组(0.90±0.07、0.97±0.06、1.00±0.06、1.00±0.05、0.99±0.05,t值为3.193 ～16.434,P值均小于0.01).对照组细胞胞质内纤维状肌动蛋白丰富,大多呈束状;缺氧组细胞从缺氧1h起纤维状肌动蛋白走向混乱、长度变短甚至消散.缺氧组细胞缺氧12、24 h时纤维状肌动蛋白与球状肌动蛋白含量比值分别为0.89±0.12、0.84±0.19,均明显低于对照组(1.00,t值分别为3.622、3.577,P值均小于0.01);其余时相点两者的比值与对照组接近(t值为0.447～ 1.526,P值均大于0.05).对照组细胞排列紧密,ZO-1沿细胞膜连续分布;缺氧组从缺氧2h起细胞形态不规则,其中缺氧24 h时ZO-1沿细胞膜分布不再连续,出现断裂. 结论 缺氧可能通过诱导肠上皮细胞丝切蛋白活化,引起纤维状肌动蛋白与球状肌动蛋白动态平衡失调,从而导致紧密连接蛋白ZO-1分布发生改变.

abstractsObjective To study the effect of hypoxia on cofilin activation in intestinal epithelial cells and its relation with distribution of tight junction protein zonula occludens 1 (ZO-1).Methods The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer cells.The monolayer-cell specimens were divided into control group (no treatment),hypoxic group (exposed to hypoxia),and normoxic group (exposed to normoxia) according to the random number table.Western blotting was used to detect the protein expressions of cofilin and phosphorylated cofilin (p-cofilin) of cells in normoxic group and hypoxic group exposed to normoxia or hypoxia for 1,2,6,12,and 24 h and control group,with 9 samples in control group and 9 samples at each time point in the other two groups.The other monolayer-cell specimens were divided into hypoxic group (exposed to hypoxia) and control group (no treatment) according to the random number table.Cells in hypoxic group exposed to hypoxia for 1,2,6,12,and 24 h and control group were obtained.Morphology and distribution of F-actin was observd with laser scanning confocal microscopy,the ratio of F-actin to G-actin was determined by fluorescence method,and distribution of ZO-1 and cellular morphology were observed with laser scanning confocal microscopy.The sample number of last 3 experiments was respectively 3,6,and 3 in both hypoxic group (at each time point) and control group.Data were processed with paired t test,analysis of variance of repeated measurement,and LSD-t test.Results The protein expressions of cofilin and p-cofilin of cells between normoxic group exposed to normoxia for 1 to 24 h and control group showed no significant changes (with t cofilin values from-0.385 to 1.701,t p-cofilin values from 0.040 to 1.538,P values above 0.05).There were no obvious differences in protein expressions of cofilin of cells between hypoxic group exposed to hypoxia for 1 to 24 h and control group (with t values from 1.032 to 2.390,P values above 0.05).Compared with that in control group,the protein expressions of p-cofilin of cells were greatly reduced in hypoxic group exposed to hypoxia for 1 to 24 h (with t values from 4.563 to 22.678,P values below 0.01),especially exposed to hypoxia for 24 h.The protein expressions of cofilin of cells between normoxic group and hypoxic group at each time point were close (with t values from -0.904 to 1.433,P values above 0.05).In hypoxic group,the protein expressions of p-cofilin of cells exposed to hypoxia for 1,2,6,12,and 24 h were 0.87 ±0.08,0.78 ±0.05,0.89 ±0.07,0.68 ±0.07,and 0.57 ± 0.06,respectively,significantly lower than those in normoxic group (0.90 ± 0.07,0.97 ± 0.06,1.00 ±0.06,1.00 ±0.05,and 0.99 ±0.05,with t values from 3.193 to 16.434,P values below 0.01).In control group,F-actin in the cytoplasm was abundant,most of it was in bunches.The trend of F-actin was disorderly in hypoxic group from being exposed to hypoxia for 1 h,shortened in length or even dissipated.The ratios of F-actin to G-actin of cells in hypoxic group exposed to hypoxia for 12 and 24 h (0.89± 0.12 and 0.84 ± 0.19) were obviously decreased as compared with that in control group (1.00,with t values respectively 3.622 and 3.577,P values below 0.01).There were no obvious differences in the ratios of F-actin to G-actin of cells between hypoxic group exposed to hypoxia for 1,2,and 6 h and control group (with t values from 0.447 to 1.526,P values above 0.05).In control group,cells were compact in arrangement,and ZO-1 was distributed continuously along the cytomembrane.From being exposed to hypoxia for 2 h,cells became irregular in shape in hypoxic group.ZO-1 was distributed in discontinuous fashion along the cytomembrane with breakage in hypoxic group exposed to hypoxia for 24 h.Conclusions Hypoxia may cause the disorder of dynamic balance between F-actin and G-actin by inducing cofilin activation,which in turn leads to the changes in distribution of tight junction protein ZO-1 in intestinal epithelial cells.