紧密连接蛋白Occludin在鼻息肉中的表达及调节

Expression and regulation of tight junction protein Occludin in nasal polyps

目的 探讨紧密连接蛋白Occludin在鼻息肉发病中的作用.方法 采用免疫组化和实时荧光定量聚合酶链反应检测鼻息肉组织（n=20）和健康对照组钩突组织（n=15）中紧密连接蛋白Claudin-1、Occludin及ZO-1蛋白及其mRNA的表达.并通过离体细胞培养模型检测白细胞介素（interleukin,IL）6、IL-13、IL-17、γ干扰素（interferon-y,IFN-γ）、转化生长因子β（transforming growth factor-β、TGF-β）、肿瘤坏死因子α（tumor necrosis factor-α,TNF-α）刺激对鼻黏膜上皮细胞表达Occludin mRNA和蛋白的调节作用.以SPSS 16.0软件进行统计学分析.结果 紧密连接蛋白Claudin-1、Occludin和ZO-1在鼻息肉与对照组钩突组织中均有表达,主要表达部位在鼻黏膜上皮细胞的细胞膜与细胞质.鼻息肉组Claudin-1、Occludin及ZO-1的平均吸光度值分别为0.187±0.076、0.172 ±0.109、0.098±0.035,均较对照组的0.312 ±0.101、0.220±0.069、0.233±0.093明显减少,差异有统计学意义（t值分别为9.345、3.301、13.323,P值均＜0.01）.鼻息肉组织中Occludin mRNA相对表达量为0.000 117 ±0.000 035,低于对照组的0.000464±0.000 134,差异具有统计学意义（Z=-5.0,P＜0.05）,而Claudin-1和ZO-1 mRNA在两组之间表达差异无统计学意义（P值均＞0.05）.IL-13、IL-17和IFN-γ等促炎因子刺激鼻黏膜上皮细胞后,Occludin mRNA相对表达量分别为0.631±0.039、0.581±0.029、0.648±0.040,与未刺激对照组相比,差异有统计学意义（f值分别为16.299、24.669、14.995,P值均＜0.05）.蛋白免疫印迹结果显示上述因子刺激组与未刺激对照组相比Occludin蛋白表达升高,差异有统计学意义（t值分别为9.880、8.442、7.310,P值均＜0.05）.结论 紧密连接蛋白Occludin表达降低可能是鼻息肉发生的原因之一.

Objective To evaluate the possible role of tight junction protein Occludin in nasal polyps. Methods The expression of Claudin-1, Oeeludin and ZO-1 in nasal polyps （ n = 20） and healthy uncinate mucosa （n = 15 ） were examined using immunohistochemieal staining, real-time quantitative polymerase chain reaction （PCR） and Western blot analysis. The regulatory effects of proinflammatory cytokines （IFN-γ, IL-13, IL-17, TGF-β, TGF-α） on the expression of Occludin in cultured human nasal epithelial cells were investigated. Results The immunohistochemieal results showed that Claudin-1, Oecludin and ZO-1 were detected both in the nasal polyp group and the control group. The expression sites were the cell membrane and cytoplasm of nasal mueosa epithelial cells. The mean optical density of Claudin- 1, Occludin and ZO-1 were 0. 187 ±0. 076,0. 172 ±0. 109 and 0. 098 ± 0. 035 respectively in the nasal polyp group and were significantly lower than those in the control group （0. 312 ±0. 101,0. 220 ±0. 069 and 0. 233 ± 0. 093 respectively） , the differences were significant （ t = 9. 345, t = 3. 301, t = 13. 323, all P 〈 0. 01 ）. RT-PCR results showed that the relative expression of Occludin mRNA was 0. 000 117±0. 000 035 in the nasal polyp group and was significantly lower than that in the control group（0. 000 464 ±0. 000 134）, and the difference was significant（ Z = -5. 0, P 〈 0. 01 ）. There was no statistically significant difference inthe relative expression of Claudin-1 and ZO-1 mRNA between the nasal polyp group and the control group（P 〉 0. 05）. After the cultured human nasal epithelial cells were stimulated by IL-β, IL-17, IFN-γand other proinflammatory cytokines,the relative expression of Occludin mRNA was 0. 631 ±0. 039, 0. 581 ±0. 029 and 0. 648 ± 0. 040, respectively. Compared with the unstimulated control group, the differences were statistically significant （t = 16. 299, 24. 669 and 14. 995 respectively, all P 〈 0. 05 ）. Western blot analyse showed that the relative grayscale in the above proinflammatory cytokines stimulation groups was 0. 650 ± 0. 061,0. 482 ± 0. 106 and 0. 536 ± 0. 109, respectively. Compared with the unstimulated control group, the differences were statistically significant （t = 9.880, 8.442 and 7.310 respectively, all P〈0.05）. Coneluslons The reduced expression of Occludin might be involved in the pathogenesis of nasal polyps. [ Key words ] Nasal polyps; Connexins ; Occudin ; Cytokines ; Real-time polymerase chain reaction