PM2.5对正常人鼻黏膜上皮细胞防御功能的影响

PM2.5 undermines the barrier function of cultured normal human nasal epithelium

目的通过体外实验评估PM2.5刺激对正常人鼻黏膜紧密连接功能的影响。方法选取因非炎性疾病(如鼻中隔偏曲、泡状中鼻甲、阻塞性睡眠呼吸暂停低通气综合征、脑脊液鼻漏等)而拟行相关手术的成年患者。鼻黏膜标本来自全麻鼻内镜手术中所得到的中下鼻甲、钩突或筛窦黏膜。通过鼻黏膜上皮细胞气液交界培养,在培养基中分别加入不同浓度的PM2.5,测量比较培养细胞跨上皮电阻(trans-epithelium electrical resistant,TER)和异硫氰酸荧光素(fluorescein isothiocyanate,FITC)渗透性与对照组的差异,以反映细胞间连接紧密程度的改变。通过荧光定量PCR,观察PM2.5对培养细胞紧密连接分子mRNA表达量的影响;通过免疫荧光标记,在共聚焦激光显微镜下观察PM2.5对培养细胞紧密连接蛋白表达情况的影响。结果加入PM2.5后,随着刺激时间延长,反映培养鼻黏膜上皮细胞之间紧密连接完整性的TER与对照组相比明显降低,并且PM2.5刺激的浓度越高,TER下降的越明显。而反映上皮渗透性的FITC通过率与PM2.5刺激的浓度呈正相关,PM2.5浓度越高,通过培养上皮的FITC越多。与对照组相比,100 μg/ml PM2.5间断刺激3天后,培养鼻黏膜上皮细胞的claudin-1 mRNA表达量显著下降;培养鼻黏膜上皮细胞的紧密连接蛋白claudin-1、occludin和ZO-1的分布连续性和荧光强度均显著减低。结论PM2.5刺激可通过破坏鼻黏膜上皮细胞膜上紧密连接分子的表达,进而影响正常人鼻黏膜的屏障功能。

OBJECTIVE To investigate the impact of PM2.5 exposure on the barrier function of normal human nasal epithelium and to explore the underlying mechanism. METHODS Mucosa of middle turbinate,inferior turbinate, uncinate process, or ethmoid sinus was obtained from adult patients undergoing endoscopic surgery for non-inflammatory diseases, such as septal deviation,obstructive sleep apnea syndrome, bullous middle turbinate,and cerebrospinal fluid leak etc. Air-liquid interface(ALI)cultures were established and exposed to different doses of PM2.5 collected from downtown Beijing. Transepithelium resistance(TER) and paracellular permeability of fluorescein isothiocyanate-dextran(FITC) were measured to reflect the barrier function of cultured epithelium. The expression of tight junction(TJ) molecules was examined by means of real-time quantitative PCR and immunofluorescence staining. RESULTS Compared to control group, TER decreased in response to PM2.5 exposure in a concentrationand time-dependent manner, while FITC permeability increased. Incubation with 100 μg/ml PM2.5 significantly decreased the expression of claudin-1 mRNA in ALI cultures. In PM2.5-treated cultures, confocal images of claudin-1, occludin, and ZO-1 demonstrated significant loss of fluorescent intensity and continuity comparing to the control cultures. CONCLUSION PM2.5 undermines the barrier function of normal human nasal mucosa, by means of breaking down the expression of tight junction molecules in nasal epithelium.