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目的 探讨矽肺发病过程中SiO2 诱导的中国仓鼠肺成纤维细胞株 (CHL)细胞间隙连接通讯 (GJIC)功能下调及其连接蛋白 43(Cx43)定位改变与Cx43磷酸化状态的关系。方法 不同剂量的SiO2 刺激SD大鼠肺泡巨噬细胞 (PAM)的培养上清液 ,作用于CHL细胞。运用Western blot方法对Cx43的磷酸化状态进行分析。结果 Western blot结果表明 :CHL细胞的膜蛋白、总蛋白及核蛋白中 ,空白对照组及 0 μg/mlSiO2 剂量组只有非磷酸化的NP条带 ;50～ 50 0 μg/mlSiO2 剂量组的膜蛋白、总蛋白及核蛋白均显示NP、P1 、P2 、P3的磷酸化方式 ,并随剂量的增加 ,磷酸化P2 、P3的水平相对增加。加入蛋白激酶C(PKC)抑制剂后 ,CHL细胞的膜蛋白在SiO2 刺激PAM的培养上清液作用下 ,P2 、P3的水平相对减弱。结论 SiO2 抑制CHL细胞的GJIC功能、改变Cx43的定位 ,可能是通过改变Cx43的磷酸化形式实现的 ,其发生机制可能与佛波酯 (TPA)相似 ,即通过PKC激活途径
Objective To investigate the down-regulation of GJIC and the relationship between Cx43 localization and Cx43 phosphorylation in Chinese hamster lung fibroblast (CHL) induced by SiO2 during silicosis. Methods Different doses of SiO2 stimulated the culture supernatant of alveolar macrophages (PAMs) of SD rats and acted on CHL cells. The phosphorylation status of Cx43 was analyzed by Western blot. Results The results of Western blot showed that only non-phosphorylated NP bands were detected in blank control group and 0 μg / ml SiO 2 group in membrane protein, total protein and nucleoprotein of CHL cells. The membrane proteins of 50 ~ 50 μg / ml SiO 2 dose group , Total protein and nuclear protein showed NP, P1, P2, P3 phosphorylation, and with the increase of dose, phosphorylation of P2, P3 levels increased. After the addition of protein kinase C (PKC) inhibitor, the membrane proteins of CHL cells under the action of SiO2-stimulated PAM supernatant, the levels of P2 and P3 were relatively weakened. Conclusions SiO2 inhibits the GJIC function of CHL cells and changes the localization of Cx43 may be through changing the phosphorylated form of Cx43. The mechanism may be similar to that of phorbol ester (TPA), that is, through the pathway of PKC activation