MMP-9在LPS诱导的支气管上皮细胞MUC5AC合成中的作用及其 调控机制的研究 第一部分 MMP-9在LPS诱导的NCI-H292细胞MUC5AC合成中的作用 【摘要】目的 旨在探讨基质金属蛋白酶9 (MMP-9)在脂多糖(LPS)诱导的NCI-H292细胞MUC5AC合成中的作用。方法用LPS刺激NCI-H292细胞， Realtime-PCR(RT-PCR)检测 MMP-9 和 MUC5AC mRNA水平的变化，western blotting、ELISA法检测MMP-9和MUC5AC蛋白的变化，明胶酶谱法检测MMP-9活性的变化。应用MMP-9抑制剂doxycycline (DOX)和MMP-9 siRNA观察MMP-9在NCI-H292细胞MUC5AC合成中的作用。结果LPS刺激后NCI-H292细胞MMP-9和MUC5AC的mRNA和蛋白表达增高，MMP-9的活性增强，DOX、 MMP9 siRNA抑制MMP-9和MUC5AC的表达。结论MMP-9参与了 LPS诱导的NCI-H292细胞MUC5AC的合成。 【关键词】MMP-9；MUC5AC；LPS；支气管上皮细胞 第二部分 MMP-9在LPS诱导的支气管上皮细胞MUC5AC合成中的调控机制 【摘要】目的 探讨MMP-9在LPS诱导的支气管上皮细胞MUC5AC合成中的调控机制。方法LPS刺激NCI-H292细胞前用MMP-9抑制剂DOX、EGFR抑制剂AG1478及MAPK抑制剂(ERK抑制剂PD98059、p38 MAPK抑制剂ML3404、JNK抑制剂SP600125)干预，然后用RT-PCR检测MMP-9 mRNA的表达、ELISA法检测MUC5AC、western blotting检测信号传导蛋白的磷酸化。结果MMP-9抑制剂DOX和EGFR抑制剂AG1478能抑制LPS诱导的NCI-H292细胞MUC5AC的合成。 AG1478、ML3404、SP600125能抑制NCI-H292细胞MMP-9 mRNA 的表达。LPS诱导的p38、JNK的磷酸化能被AG1478所抑制。结论MMP-9是通过EGFR信号传导通路介导LPS刺激所致的MUC5AC的产生。LPS刺激NCI-H292细胞分泌MMP-9是通过EGFR-p38/JNK信号传导通路。 【关键词】MMP-9； MUC5AC；酪氨酸激酶磷酸化；支气管上皮细胞 第三部分 MMP-9对人原代支气管上皮细胞MUC5AC合成的影响 【摘要】目的 探讨MMP-9对LPS诱导的人原代培养的支气管上皮细胞MUC5AC合成的影响。方法应用Transwell建立气液平面培养原代人支气管上皮细胞。用LPS刺激原代人支气管上皮细胞，用RT-PCR检测MMP-9和MUC5AC mRNA 水平的变化，用 western blotting、ELISA 法检测 MMP-9 和 MUC5AC 蛋白的变化，用明胶酶谱法检测MMP-9活性的变化。并用MMP-9抑制剂DOX和EGFR抑制剂AG1478干预观察MMP-9和EGFR在原代支气管上皮细胞MUC5AC合成中的作用。结果LPS刺激后原代支气管上皮细胞MMP-9和MUC5AC的mRNA和蛋白表达增高，MMP-9的活性增强，DOX、AG1478能抑制MMP-9和MUC5AC的表达。结论MMP-9通过EGFR通路介导了 LPS诱导的原代人支气管上皮细胞MUC5AC的合成。 【关键词】MMP-9； MUC5AC； LPS；支气管上皮细胞 第四部分 MMP-9对LPS诱导的大鼠气道杯状细胞增生的影响 【摘要】目的 探讨MMP-9对LPS诱导的大鼠气道杯状细胞增生的影响及其机制。方法 SD大鼠气道内注入LPS建立气道粘液高分泌模型，用MMP-9抑制剂DOX干预观察对杯状细胞增生的影响。AB-PAS染色检测大鼠气道杯状细胞的增生、免疫组化检测大鼠气道MMP-9、muc5ac的表达，western blotting检测EGFR的表达。计数气道中性粒细胞的浸润和检测支气管肺泡灌洗液(BALF)中TNF-α、 IL-1β、IL-8的变化。结果LPS刺激后大鼠气道上皮AB-PAS和muc5ac、MMP-9的阳性染色明显增多，EGFR的表达增高，气道中性粒细胞的浸润和BALF中TNF-α、IL-1β， IL-8含量增高。除TNF-α外，这些变化均能被DOX所抑制。结论MMP-9参与了LPS诱导的大鼠气道杯状细胞的增生，DOX能通过抑制MMP-9的表达、中性粒细胞的浸润和IL-1β、IL-8的表达而对气道粘液高分泌起治疗作用。 【关键词】MMP-9；杯状细胞；EGFR；细胞因子

 Part Ⅰ Role of MMP-9 in LPS induced MUC5AC mucin production in NCI-H292 cells Abstract Objective In this study, we explored the role of matrix metalloproteinasen -9 (MMP-9 ) in lipopolysaccharide (LPS ) induced MUC5AC mucin production in NCI-H292 cells. Methods After NCI-H292 cells were stimulated with LPS, MMP-9 and MUC5AC mRNA were determined by real-time PCR (RT-PCR) , MMP-9 and MUC5AC protein were measured by western blotting and ELISA, and MMP-9 activity was detected by gelatin zymography. To examine the role of MMP-9 in the production of MUC5AC, we treated NCI-H292 cells with MMP-9 inhibitor doxycycline (DOX) and small interfering RNA (siRNA) for MMP-9. Results LPS significantly increased MMP-9 and MUC5AC mRNA expression and protein production, and also enhanced the MMP-9 activity. These effects were inhibited by treatment with DOX and MMP-9 siRNA. Conclusions Our results indicate that MMP-9 plays a critical role in LPS induced MUC5AC mucin production by NCI-H292 cells. [Key words] MMP-9; MUC5AC; LPS; bronchial epithelial cells Part Ⅱ Regulation of LPS induced MUC5AC mucin production in human airway epithelial cells by MMP-9 Abstract Objective To investigate the regulation of LPS induced MUC5AC mucin production in human airway epithelial cells by MMP-9. Methods NCI-H292 cells were pretreated with EGFR inhibitor AG1478, MMP-9 inhibitor DOX and MAPK inhibitors (ERK inhibitor PD98059, p38 MAPK inhibitor ML3404, JNK inhibitor SP600125) before LPS stimulation. MMP-9 mRNA was determined by RT-PCR, MUC5AC protein were measured by ELISA and phosphorylation of signaling proteins was assessed by western blotting. Results LPS induced MUC5AC mucin production in NCI-H292 cells was inhibited by MMP-9 inhibitor DOX and EGFR inhibitor AG1478. MMP-9 transcript level in NCI-H292 cells was significantly diminished by treatment with AG1478, ML3404 and SP600125. It was also found that the phosphorylation of p38 MAPK or JNK induced by LPS was inhibited by AG1478. Conclusions LPS induced MUC5AC production was mediated by MMP-9 through EGFR activation. MMP-9 mRNA expression induced by LPS in NCI-H292 cells was through EGFR- p38/JNK signaling pathway. [Key words] MMP-9; MUC5AC;tyrosine kinase phosphorylation; bronchial epithelial cells Part Ⅲ Role of MMP-9 in LPS induced MUC5AC mucin production in human primary cultured bronchial epithelial cells. Abstract Objective To investigate the role of MMP-9 in LPS induced MUC5AC mucin production in human primary cultured bronchial epithelial cells. Methods Human primary cultured bronchial epithelial cells were cultured in Transwell chamber and grown on an air-liquid interface (ALI). After cells were stimulated with LPS, MMP-9 and MUC5AC mRNA were determined by RT-PCR, MMP-9 and MUC5AC protein were measured by western blotting and ELISA, and MMP-9 activity was detected by gelatin zymography. To examine the role of MMP-9 and EGFR in the production of MUC5 AC, we treated human primary cultured bronchial epithelial cells with MMP-9 inhibitor DOX and EGFR inhibitor AG1478. Results LPS significantly increased MMP-9 and MUC5AC mRNA expression and protein production, and also enhanced the MMP-9 activity. These effects were inhibited by treatment with DOX and AG1478. Conclusions MMP-9 mediated LPS induced MUC5AC mucin production through EGFR signaling pathway in human primary cultured bronchial epithelial cells. [Key words] MMP-9; MUC5AC; LPS; bronchial epithelial cells. Part Ⅳ Effects of MMP-9 on LPS induced goblet cell metaplasia in rats Abstract Objective In this study, we investigated the effects and the mechanism of MMP-9 on LPS induced goblet cell metaplasia in rats. Methods LPS were instilled into the tracheas of Sprague-Dawley rats to establish airway mucous hypersecretion model. To determine the role of MMP-9 in goblet cell metaplasia, we treated rats with DOX before and after LPS instillation. Alcian blue/periodic acid-schiff (AB-PAS) staining for mucin glycoprotein, immunohistochemical staining for muc5ac and MMP-9, and western blotting for EGFR were performed. Airway neutrophilic infiltration and TNF-α, IL-1β、IL-8 levels in bronchoalveolar lavage fluid (BALF) were also measured. Results LPS instillation not only increased AB-PAS, muc5ac and MMP-9 staining in airway epithelium, but also increased EGFR expression, airway neutrophil infiltration, and TNF-α、 IL-1β、 IL-8 levels in BALF, and DOX was found to significantly prevent goblet cell metaplasia, neutrophil recruitment, EGFR and MMP-9 expression and IL-1β、 IL-8 production. DOX had no inhibitory effect on TNF-α level. Conclusions This study demonstrated that MMP-9 contributed to the LPS induced goblet cell metaplasia, and MMP-9 inhibitor DOX has potential therapeutic role in mucus hypersecretion by inhibiting MMP-9 expression, neutrophil infiltration and IL-1β、 IL-8 production. [Key words] MMP-9; goblet cell; EGFR; cytokine