木材烟雾凝集物刺激人气道上皮细胞发生转分化样改变  

Wood smoke condensate induced epithelial-mesenchymal transition in human airway epithelial cells

作　　者：李雯曦 邹威凤[1] 李冰[2] 冉丕鑫[1] 

机构地区：[1]呼吸疾病国家重点实验室广州医科大学广州医科大学附属第一医院,510120 [2]广州医科大学院实验医学研究中心

出　　处：《中华结核和呼吸杂志》2014年第1期24-29,共6页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：国家自然科学基金（81170043）;国家科技支撑计划呼吸系统疾病防治研究（2012BAl05BOO）;高等学校博士学科点专项科研基金（20104423110001）;广东省自然基金资助（S2011020002789）

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50mg／L的WSC和CSC刺激HBEC7d，空白对照组仅用细胞培养基培养刺激HBEC7d，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（10mg／L）刺激HBEC7d，观察细胞形态，用Westernblot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10mg／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一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10mg／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）；10mg／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。Objective To observe the detrimental effects of wood smoke condensate （WSC） exposure on human bronchial epithelial ceils （HBEC）, and to explore the expression of epithelial- mesenehymal 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 rag/L） and cigarette smoke condensate （CSC） （10 rag/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 cell- cell 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 ~ O. 04 vs O. 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 [ collagen, vimentin and MMP-9 between WSC and CSC exposure groups. Conclusion WSC exposure could induce EMT-like process in human airway epithelial cells.

关 键 词：肺疾病 慢性阻塞性 木材 烟雾 上皮细胞 

分 类 号：R363[医药卫生—病理学]