目的 探讨结核性胸腔积液中白介素(IL)-22和干扰素(IFN)-γ对胸膜间皮细胞上皮-间质转化过程的影响及其机制.方法 2013年7月至2014年6月在佛山市第一人民医院呼吸内科住院并且经胸腔镜取胸膜活检由病理学确诊的结核性胸膜炎患者22例,其中男12例,女10例,年龄22 ~64岁,平均38.4岁.从患者胸腔积液中的分离纯化出原代胸膜间皮细胞,以单纯培养基培养条件为对照组,实验组则加入重组人IL-22、IFN-γ或两者组合进行培养,在此基础上的部分实验中加入细胞内信号转导和转录激活因子(STAT)1或STAT3的抑制剂以阻断相应信号转导过程.使用流式细胞仪检测胸膜间皮细胞在经过IL-22和IFN-γ刺激后STAT的通路活化情况;显微镜下观察胸膜间皮细胞的形态学变化,用流式细胞仪检测细胞角蛋白8、E-钙黏蛋白及间质细胞标志物波形蛋白、α-平滑肌肌动蛋白(α-SMA)的表达.结果 与对照组相比,IFN-γ能够激活胸膜间皮细胞的STAT1通路,促使该细胞形态由上皮向间质转化,活化STAT3通路的IL-22未引起这种上皮-间质转化,但IL-22可逆转由IFN-γ诱导的间质化形态学变化;对照组的细胞角蛋白8表达水平为(43.8±2.8)%,E-钙黏蛋白为(43.8±1.9)%,波形蛋白为(41.2±2.4)%,α-平滑肌肌动蛋白为(55.8±2.0)%.IFN-γ组的细胞角蛋白8为(14.3±1.5)%,E-钙黏蛋白为(13.4±1.2)%,两者表达显著下调(t值为8.1和9.7,均P<0.05),而波形蛋白为(70.6±3.6)%,α-平滑肌肌动蛋白为(80.6±2.9)%,两者表达显著上调(t值为8.5和7.2,均P<0.05);与IFN-γ组相比,IFN-γ+IL-22组的细胞角蛋白8为(62.4±3.1)%,E-钙黏蛋白为(46.5±3.6)%,显著上调(t值为13.2和10.5,均P<0.05),而波形蛋白[(36.7±2.8)%]和α-平滑肌肌动蛋白[(35.2±2.5)%]显著下调(t值为9.8和13.2,均P<0.05);与IFN-γ+IL-22组相比,IFN-γ+IL-22+STAT3组的细胞角蛋白8[(16.7±0.7)%]和E-钙黏蛋白[(14.4±0.9)%]显著下调(t值为12.5和10.2,均P<0.05),而波形蛋白[(68.0±2.5)%]和α-平滑肌肌动蛋白[(79.2±5.7)%]显著上调(t值为9.0和12.8,均P<0.05).结论 IL-22可通过激活STAT3通路,逆转由IFN-γ诱导的上皮-间质转化过程,可能在结核性胸膜炎的抗胸膜纤维化过程起到保护作用.
Objective To investigate the effects of interleukin (IL)-22 and interferon IFN)-γ on epithelial-mesenchymal transition (EMT) of pleural mesothelial cells,and to explore the relevant signal transduction pathways,in tuberculous pleural effusion.Methods Twenty-two patients (12 males and 10 females,age range 22-64 years,mean 38.4 years) with tuberculous pleurisy hospitalized in department of respiratory medicine of the First People' s Hospital of Foshan were recruited from July 2013 to June 2014.Diagnosis was confirmed by pathology.Freshly isolated pleural mesothelial cells (PMCs) from tuberculous pleural effusion were cultured either in medium alone as control,or stimulated with IL-22 and/or IFN-γ,and the phosphorylated signal transducers and activators of transcription (STAT) signalings in PMCs were determined by flowcytometry.In some experiments,STAT inhibitors were added into coculture with IL-22 and/or IFN-γ,and then morphological changes of PMCs were observed,and the expressions of epithelial markers such as cytokeratin-8 and E-cadherin as well as mesenchymal markers such as vimentin and αr-Smooth muscle actin (SMA) were also determined by flow cytometry.Results As compared with the control group,IFN-γinduced epithelial-mesenchymal transition of PMCs via a STAT1 pathway as evidenced by down-regulation of cytokeratin-8 [(43.8 ±2.8)% vs (14.3 ± 1.5)%,t =8.1,P <0.05] and E-cadherin [(43.8 ± 1.9) % vs (13.4 ± 1.2) %,t =9.7,P < 0.05],and by up-regulation of vimentin [(41.2±2.4)% vs (70.6 ±3.6)%,t =8.5,P<0.05] and α-SMA [(55.8 ±2.0)% vs (80.6± 2.9)%,t =7.2,P <0.05].IL-22 not only maintained the epithelial property of PMCs,but also reverted IFN-γ-induced EMT [cytokeratin-8 (62.4 ± 3.1) %,E-cadherin (46.5 ± 3.6) %,vimentin (36.7 ± 2.8) %,and α-SMA (35.2 ± 2.5) % in ‘ IFN-γ + IL-22’ group,all P < 0.05 as compared with those of IFN-γ group].Whereas addition of STAT3 inhibitor significantly abrogated such anti-EMT effect of IL-22 on PMCs [cytokeratin-8 (16.7 ± 0.7) %,E-cadherin (14.4 ± 0.9) %,vimentin (67.9 ± 2.5) %,and α-SMA (79.2 ± 5.7) % in ‘ IFN-γ + IL-22 + STAT3 inhibitor’ group,all P < 0.05 as compared with those of ‘IFN-γ + IL-22’ group].Conclusion The IL-22-STAT3 signal pathway could revert IFN-γ-induced EMT of PMCs,and might play a protective role in anti-pleural fibrosis in tuberculous pleurisy.