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目的研究高渗条件对正常人气道上皮细胞(HBE)黏蛋白(MUC)5AC分泌的影响,以及蛋白激酶C(PKC)-热休克蛋白(HSP)70信号途径在其中的可能作用。方法 采用高渗盐水诱导培养HBE16细胞的方法复制黏液高分泌体外模型,分别用PKCμ抑制剂G 6976、PKCα抑制剂Safingol、PKCβ抑制剂LY333531和PKCδ抑制剂Rot-tlerin干预HBE16细胞。Western blot检测HSP70-2的蛋白含量;RT-PCR检测人HSP70-2转录水平;ELISA检测培养上清MUC5AC蛋白含量c各高渗组的培养上清MUC5AC蛋白含量、HSP70-2蛋白和转录水平较对照组显著升高,并随着培养时间的延长而逐渐增加(P<0.05)。G 6976处理组的上述指标显著降低(P<0.01),但仍高于对照组(P<0.05);而Safingol、LY333531和Rottlerin处理组均无改变。结论 高渗盐水可诱导人气道上皮细胞MUC5AC的高分泌,HSP70-2系通过PKC中的μ亚型在该过程中起重要作用的。
Objective To investigate the effect of hypertonic conditions on the secretion of 5AC of normal human airway epithelial cells (HBE) mucin (MUC) and the possible role of protein kinase C (PKC) - heat shock protein (HSP) 70 signal pathway. Methods Hypertonic saline-induced HBE16 cells were cultured in vitro. Mucous exudates were induced in vitro by PKCμ inhibitor G 6976, PKCα inhibitor Safingol, PKCβ inhibitor LY333531 and PKCδ inhibitor Rot-tlerin. The protein level of HSP70-2 was detected by Western blot. The transcription level of HSP70-2 was detected by RT-PCR. The content of MUC5AC in culture supernatant was determined by ELISA. The control group was significantly increased, and gradually increased with the extension of incubation time (P <0.05). The above parameters of G 6976 treatment group were significantly lower (P <0.01), but still higher than those of control group (P <0.05), while Safingol, LY333531 and Rottlerin treatment groups had no change. Conclusion Hypertonic saline induces hypersecretion of MUC5AC in human airway epithelial cells. HSP70-2 plays an important role in this process through the μ subtype of PKC.