摘要目的 观察木材烟雾凝集物(WSC)对人支气管上皮细胞(HBEC)间充质转分化(EMT)样改变的影响.方法 WSC组和烟草烟雾凝集物(CSC)组分别用5、10、20、40和50 mg/L的WSC和CSC刺激HBEC 7 d,空白对照组仅用细胞培养基培养刺激HBEC 7 d,每组细胞数为1.2×106/ml.采用细胞计数试剂盒-8检测细胞总体活性.用WSC最大影响活性剂量(10 mg/L)刺激HBEC 7 d,观察细胞形态,用Western blot法和细胞免疫荧光技术检测EMT标记物E-钙黏蛋白、Ⅰ型胶原、波形蛋白和基质金属蛋白酶-9(MMP-9)的表达水平.多组间比较采用单因素方差分析,两组间比较采用t检验.结果 10 mg/L的WSC吸光度值最高(2.36 ±0.07),部分细胞形态由鹅卵石状向长梭形、多角形或星形改变,E-钙黏蛋白表达吸光度值(0.30 ±0.05)显著低于空白对照组(0.59±0.08),差异有统计学意义(F=22.07,P＜0.05),但与10 mg/L的CSC组吸光度值(0.30±0.05)比较,差异无统计学意义(F=22.07,P＞0.05)；10 mg/L的WSC组Ⅰ型胶原相对含量吸光度值(0.58±0.04)显著高于空白对照组(0.26±0.02),差异有统计学意义(F=119.72,P＜0.05),与CSC组(0.60±0.02)比较,差异无统计学意义(F=119.72,P＞0.05)；WSC组和CSC组MMP-9表达吸光度值(0.56±0.08和0.54±0.10)均显著高于空白对照组(0.19±0.03),差异均有统计学意义(F=21.79,均P＜0.05).结论 WSC能够刺激HBEC出现转分化样改变.

abstractsObjective To observe the detrimental effects of wood smoke condensate (WSC) exposure on human bronchial epithelial cells (HBEC),and to explore the expression of epithelialmesenchymal transition (EMT) markers in HBEC exposed to WSC.Methods HBEC were exposed respectively to 5,10,20,40 and 50 mg/L of WSC/CSC for 7 days,with control groups only in cell culture medium at the same time,then the total cytoactivity was detected by cell counting kit-8.After observing the cellular morphology of WSC-stimulated HBEC.Western blot and immunofluorescence method were used to evaluate the expression levels of type Ⅰ collagen,vimentin,E-cad and MMP-9 in HBEC exposed to WSC (10 mg/L) and cigarette smoke condensate (CSC) (10 mg/L) for 7 days.Statistical evaluation of the continuous data was performed by ANOVA.Independent-Samples t-test for between-group comparisons.Results After 7 days of exposure to WSC,HBEC manifested a morphological characteristic of loss of cellcell contact and elongated shape.The level of E-cad was decreased in WSC exposure groups (Western blot:0.30 ± 0.05,F =22.07,P ＜ 0.05) compared with the groups without WSC exposure (Western blot:0.59 ± 0.08,F =22.07,P ＜ 0.05).In contrast,an upregulation in expression of type Ⅰ collagen (Western blot:0.58 ± 0.04 vs 0.26 ± 0.02,F =119.72,P ＜ 0.05) and MMP-9 (0.56 ± 0.08 vs 0.19 ± 0.03,F =21.79,P ＜ 0.05) was observed in the presence of WSC,compared with the control groups.Immunofluorescence analysis showed that after a 7-day exposure to WSC in these cells,the E-cad protein was lost whereas type Ⅰ collagen,vimentin and MMP-9 were acquired.Both Western blot and immunofluorescence analysis showed no difference in expression levels of E-cad,type Ⅰ collagen,vimentin and MMP-9 between WSC and CSC exposure groups.Conclusion WSC exposure could induce EMT-like process in human airway epithelial cells.