摘要
目的研究中性粒细胞弹力蛋白酶(NE)通过表皮生长因子受体(EGFR)引起黏蛋白(MUC)5AC 高表达的上游信号通路。方法培养人气道 BEAS-2B 上皮细胞,给予 NE 刺激,以肿瘤坏死因子转换酶抑制剂-1(TAPI-1)及活性氧(ROS)清除剂 DMTU 为干预因素。RT-PCR 检测干预前后细胞中 MUC5AC mRNA 含量;酶联免疫吸附测定法(ELISA)检测培养上清中 MUC5AC、可溶性转化生长因子(TGF)-α蛋白含量;Western 印迹分析磷酸化表皮生长因子受体(p-EGFR)蛋白水平。结果NE 刺激后引起 MUC5AC 基因转录和蛋白表达水平明显高于未给予 NE 刺激的对照组(P<0.01),同时伴随可溶性 TGF-α及 p-EGFR 蛋白水平的增高,与对照组相比,差异有统计学意义(均 P<0.01);加入浓度为20μmol/L 的肿瘤坏死因子转换酶抑制剂 TAPI-1后可明显下调上述指标的表达水平(均 P<0.01),DMTU 也同样降低了 MUC5AC 的基因转录、产物水平以及可溶性 TGF-α蛋白相对含量,与单纯 NE 刺激组比较差异均有统计学意义(P<0.01),但对单纯外源性 TGF-α刺激组引起的MUC5AC 基因转录和蛋白合成增加无明显作用(均 P>0.05)。结论 NE 能刺激细胞产生 ROS,再活化肿瘤坏死因子转换酶(TACE)引起黏蛋白产生的增多,组成 EGFR 通路上游的主要信号分子,故ROS/TACE/TGF-α前体/ECFR 转导途经是 NE 引起气道黏液高分泌的重要机制。
Objective To investigate the upstream signal transduction pathway of neutrophil elastase （NE） -induced mucous hypersecretion via epidermal growth factor receptor（EGFR）. Methods The airway epithelial cells of the line BEAS-2B were cultured and divided into 6 groups： control group, cultured in serum-frec DMEM/Ham＇s F12 mixture; NE group, incubated with NE alone, transforming growth factor （TGF）-α protease inhibitor（TAPI）-1 ＋ NE group, incubated with NE and TAP I-1; 1,3-dimethyl-2- thiourea（DMTU） ＋ NE group, preincubated with DMTU, a reactive oxygen species（ROS） scavenger, and then incubated with NE; TGF-α group, incubated with TGF-α; and DMTU ＋ TGF-α group, incubated with DMTU and TGF-α. The mucin （MUC）5AC mRNA level was analyzed by RT-PCR, MUCSAC protein and soluble TGF-et were detected by enzyme-linked immunosorbent assay, and the pbospborylated EGFR （p- EGFR） protein production was assayed by Western blotting. Results The MUCSAC mRNA expression and protein expression of the NE group were 0. 81 ± 0.13 and 0. 78 ± 0. 06 respectively, both significantly higher than those of the control group （ 0. 32 ± 0.07 and 0. 24 3= 0. 08 both P 〈 0. 01. The protein expression of soluble TGF-α and the protein expression of p-EGFR protein of the NE group were 0. 83 3= 0. 14 and 0. 88 3= 0. 07 respectively, both significantly higher than these of the control group （0. 25 3=0. 13 and 0. 28 3=0. 11 respectively, both P 〈 0. 01 ）. The expression rates of MUCSAC mRNA, MUCSAC protein, soluble TGF-α, and p-EGFR of the TAPI-1 group were 0.37 ± 0.04,0.30 ± 0.11,0.32 ± 0.07, and 0.43 3=0.09 respectively, all significantly lower than these of the NE group （ all P 〈 0. 01 ）. The level of MUCSAC mRNA,MUCSAC mucin protein ,and soluble TGF-α of the DMTU ＋ NE group were 0. 36 3=0. 03,0. 39 3= 0. 12, and 0. 43 ± 0. 05 respectively, all significantly lower than those of the NE group （ all P 〈 0. 01 ）, however, the MUCSAC mRNA expression and MUC5AC protein expression of the DMTU ＋ TGF-α group were 0. 58 3=0. 13 and 0. 55 3= 0. 11 respectively, both not significantly different from these of the TGF-α group （0. 56 ± 0.08 and 0. 57 ±0. 13 respectively, both P 〉 0. 05 ）. Conclusion NE induces MUC5AC expression via ROS/TACE/pro-TGF-α/EGFR in the airway epithelial cells.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2007年第5期348-352,共5页
National Medical Journal of China
