肿瘤坏死因子-α对大鼠肺泡上皮细胞紧密连接蛋白ZO-1、Claudin-4表达的影响

Effect of tumor necrosis factor-α on the expression of tight junction proteins ZO-1 and Claudin-4 in rat alveolar epithelial cells

目的探究肿瘤坏死因子-α(TNF-α)对大鼠肺泡Ⅱ型上皮细胞(alveolar epithelial type Ⅱ cells, AEC-Ⅱ)及紧密连接蛋白ZO-1、Claudin-4表达的影响。方法体外培养大鼠AEC-Ⅱ细胞,将细胞分为正常对照组,TNF-α24 h、48 h、72 h组。对照组用含胎牛血清的DME F12培养液培养细胞,TNF-α各组用TNF-α(10 ng/mL)分别刺激细胞24、48、72 h。共培养结束后采用透射电镜鉴定并观察细胞超微结构的变化。MTT法测定细胞增殖抑制情况。流式细胞仪测定细胞早期凋亡率。免疫荧光法观察各组细胞紧密连接蛋白ZO-1、Claudin-4表达分布,实时荧光定量PCR法测定各组细胞ZO-1、Claudin-4 mRNA水平的变化,Western blot测定紧密连接蛋白ZO-1、Claudin-4表达的变化。多组样本间均数比较采用单因素方差分析,组间两两比较采用SNK-q检验。方差不齐时则用秩转换的非参数检验。以P<0.05为差异有统计学意义。结果透射电镜下可见对照组大鼠AEC-Ⅱ细胞内含有大小不等的特征性板层小体,TNF-α各组板层小体逐渐排空,视野下可见凋亡细胞。TNF-α各组细胞的早期凋亡率及增殖抑制率均较对照组降低(均P<0.05)。共聚焦显微镜下观察到ZO-1沿细胞膜呈线状分布,Claudin-4沿细胞膜呈散点状分布,TNF-α各组ZO-1的荧光强度减弱,连续性被破坏,线性结构断裂,甚至消失。Claudin-4的荧光强度减弱,密度降低。TNF-α24 h组ZO-1的转录及表达均低于对照组,但差异无统计学意义(P>0.05)。TNF-α48 h及72 h组细胞ZO-1的mRNA(0.28±0.06;0.13±0.07)与对照组比较明显降低(P<0.01),且两组蛋白水平(0.44±0.09;0.2±0.01)均低于对照组(0.69±0.12)。与对照组比较,TNF-α各组细胞Claudin-4转录(24 h:0.16±0.03,48 h:0.04±0.01,72 h:0.01±0.00 vs 1.00±0.00)及表达水平(24 h:0.49±0.08,48 h:0.34±0.05,72 h:0.04±0.01 vs 0.96±0.13)明显降低(均P<0.05),且随着时间延长呈递减趋势。结论TNF-α通过损伤大鼠AEC-Ⅱ和下调细胞紧密连接蛋白ZO-1、Claudin-4的表达分布,损伤肺上皮屏障。

Objective To investigate the effect of TNF-α on the expressions of tight junction protein ZO-1 and Claudin-4 in rat alveolar epithelial typeⅡ cells (AEC-Ⅱ). Methods Rat AEC-Ⅱ cells were cultured in vitro and divided into the control group, and TNF-α 24 h, 48 h, 72 h groups. The control group was cultured with DME F12 medium containing fetal bovine serum, and the TNF-α groups were intervened by TNF-α with a concentration of 10 ng/mL for 24 h, 48 h, and 72 h, respectively. After co-culture, the transmission electron microscopy was used to identify and observe the ultrastructural changes of rat AEC-Ⅱ cells. The cell inhibition rate was determined by MTT assay, and the cell apoptosis rate was measured by flow cytometry. The expression and distribution of tight junction protein ZO-1 and Claudin-4 in each group were observed by immunofluorescence method. The transcriptional levels of ZO-1 and Claudin-4 were detected by real-time fluorescent quantitative PCR. The expressions of ZO-1 and Claudin-4 were detected by Western blot. One-way analysis of variance (ANOVA) was used for comparisons among groups and SNK-q test was used for pairwise comparisons between groups. The non-parametric test of rank transformation was used when homogeneity of variance were not met. The value of P<0.05 was considered significantly different. Results Transmission electron microscopy showed that the AEC-Ⅱ cells in the control group obtained characteristic lamellar bodies of different sizes. The lamellar bodies in each TNF-α group were gradually emptied and apoptotic cells were observed under the visual field. The cell inhibition rate and early apoptosis rate of each TNF-α group were significantly higher than those of the control group (all P<0.05). Confocal microscopy showed that ZO-1 was linearly distributed along the cell membrane, and Claudin-4 was scattered along the cell membrane. In the TNF-α groups, the fluorescence intensity of ZO-1 was weakened and the continuity was broken as well as the linear structure. The fluorescence intensity and density of Claudin-4 were decreased in the TNF-α groups. Besides, the transcription and expression of ZO-1 in the TNF-α 24 h group were lower than those in the control group, but there was no significant difference (P>0.05). However, the mRNA level of ZO-1 in the TNF-α 48 h group (0.28±0.06) and 72 h group (0.13±0.07) were significantly lower than those in the control group. And their protein levels of TNF-α 48 h group (0.44±0.09) and of TNF-α 72 h group (0.2±0.01) were lower than that of the control group (0.69±0.12). Compared with the control group, the transcription level (24 h: 0.16±0.03;48 h: 0.04±0.01;72 h: 0.01±0.00 vs 1.00±0.00) and expression (24 h: 0.49±0.08;48 h: 0.34±0.05;72 h: 0.04±0.01 vs 0.96±0.13) of Claudin-4 in each TNF-α group were significantly decreased, and showed a decreasing trend with time (all P<0.05). Conclusions TNF-α can damage the pulmonary epithelial barrier by damaging alveolar epithelial typeⅡ cells and down-regulating the expression and distribution of ZO-1 and Claudin-4.