摘要
目的：气道上皮是肺部抵御外界理化因素刺激的第一道防线。病原微生物、外界有害的微尘颗粒、气体等因素导致气道上皮损伤是慢性阻塞性肺疾病(COPD)的起始环节和病理基础。吸烟是引起COPD的重要危险因素。吸烟与MMP-9、IL-8引起COPD的关系日益受到重视。分泌性白细胞蛋白酶抑制物(SLPI)是气道重要的保护基质。通过本实验来检测香烟烟雾提取物(CSE)对正常人支气管上皮(NHBE)细胞MMP-9和IL-8表达水平的影响；并观测加入SLPI对上述因素的调控作用。探讨SLPI对慢性气道炎症疾病保护作用的部分机制，为COPD的临床治疗寻求更多的实验依据。
方法：实验分四组：对照组，CSE组，SLPI组，SLPI+CSE组。体外培育NHBE细胞分别给予CSE及SLPI干预处理，采用ELISA法检测NHBE细胞上清液IL-8的表达；免疫细胞化学方法(SP法)检测NHBE细胞MMP-9蛋白的表达及RT-PCR法和图像分析检测NHBE细胞MMP-9mRNA的表达。
结果：1对照组NHBE细胞有少量MMP-9、IL-8的表达。2 CSE组MMP-9、IL-8的表达明显高于对照组、SLPI+CSE组、SLPI组。CSE干预NHBE细胞不同时间证实，其诱导MMP-9、IL-8表达的作用在一定范围内与干预时间呈依赖关系，24小时达高峰，随后呈下降趋势(P＜0.05)。3 SLPI组MMP-9、IL-8的表达水平明显低于对照组(P＜0.05)。SLPI抑制MMP-9、IL-8表达的效应在一定范围内与浓度呈依赖关系，以10ug／ml的浓度抑制作用最强(P＜0.05)。4 SLPI+CSE组MMP-9、IL-8表达较对照组上调但低于CSE组的表达水平。
结论：1 CSE诱导NHBE细胞MMP-9、IL-8的表达上调。2SLPI抑制NHBE细胞MMP-9、IL-8的表达。3 SLPI能抑制CSE诱导NHBE细胞中MMP-9、IL-8的表达。
Objective Airway epithelial cells are the first barrier by whichlung resist injuries coming from outer environment. Some physical andchemical factors such as pathogenic microorganism and various particlesin polluted air of outer environment can stimulate airway epithelial cellsand finally result in its functions damages, which are initial pathologicchanges of some chronic airway diseases such as chronic obstructivepulmonary diseases. Smoking is an important dangerous factor of chronicobstructive pulmonary diseases (COPD). Smoking and matrixmetalloproteinase-9 (MMP-9) with IL-8 beget COPD happening.Secretory leukocyte proteinase inhibitor (SLPI) is important to protect thelocal airway. SLPI inhibited the expression of MMP-9 and IL-8 innormal human bronchial epithelial cells induced by cigarette smokeextract (CSE) through the following experiments. Discussing the partmechanism of SLPI how to protect the local airway of chronic airwaydiseases would afford much more experiments basis of COPD clinictreating.
Methods The experiment was divided into four groups, includingcontrol group, CSE group, SLPI group and SLPI+CSE group. NHBEcells were cultured in vitro. It was treated with different time of CSE anddifferent concentration of SLPI. The following assays were carried out.
The expression levels of IL-8 in NHBE cells supernatant was examinedby ELISA. The expression of MMP-9 protein in NHBE cells wasevaluated by using immuno-cytochemical stain method.The content ofMMP-9 mRNA was determined by reverse transcription -polymerasechain reaction(RT-PCR) method and image analysis.
Results 1 A small quantities of MMP-9 and IL-8 expression wasobserved in control group NHBE cells.2 The expression of MMP-9 andIL-8 in NHBE cells of CSE group was highly than that of the other threegroups. After exposure to different time of CSE, The expression levels ofMMP-9 and IL-8 relied directly on CSE exposure time within certainlimits. A climax expression was observed at the time of 24 hours beingexposured with CSE. But subsequently the tendency descended.3 SLPIdecreased the expression levels of MMP-9 and IL-8. The expressionlevels of MMP-9 and IL-8 in SLPI group NHBE cells were lower thanthat of the other three groups. Within certain limits the inhibiting effectdepended on SLPI concentrations directly. When NHBE cells wereexposured at the 10ug/ml SLPI concentration, the inhibiting effect wasstrongest. 4 The expression levels of MMP-9 and IL-8 in SLPI+CSEgroup were higher than that of the control group and SLPI group. But itwere lower than that of CSE group.
Conclusion 1. CSE could increase the expression levels ofMMP-9 and IL-8 in NHBE cells. 2. SLPI could inhibit the expression levels of MMP-9 and IL-8 in NHBE cells.3. SLPI could inhibit theexpression levels of MMP-9 and IL-8 in NHBE cells induced byCigarette smoking extract.
引文
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