机械拉伸对γ-干扰素诱导肺泡上皮细胞人β-防御素-3表达的影响

Effects of mechanical stretch on the expression of hmnan beta-defensin-3 in alveolar epithelial cells elicited by interferon-gamma

目的研究机械拉伸对γ-干扰素（IFN-γ）诱导肺泡上皮细胞（A549细胞）人β防御素-3（HBD-3）表达的影响并探讨HBD-3在呼吸机相关性肺炎（VAP）发病机制中的作用。方法体外培养A549细胞，分别给予机械拉伸（S组），10g／mlI FN-γ（I组）和10ng／ml IFN-γ＋机械拉伸（IS组）3种处理，未处理的细胞为对照组（C组），处理的时间为2h,4h和6h。拉伸由细胞Flexercell-4000T^TM拉伸系统提供，拉伸的幅度为20％，速率为30次／min。处理后实时定量逆转录-多聚酶链反应（RT-PCR）检测HBD-3 mRNA的表达；处理6h的细胞继续培养至24h，激光扫描共聚焦显微镜检测HBD-3的表达。采用单因素方差分析和q检验对实验数据进行统计学分析。结果单独的机械拉伸不能使HBD-3 mRNA的表达发生显著变化。10g／ml IFN-γ处理2h,4h和6h后，HBD-3 mRNA表达量较之C组分别增加了（2．63±0．60），（8．02±1．63）和（12．21±2．35）倍。10g／ml IFN-γ＋机械拉伸处理2h,4h和6h后，HBD-3 mRNA表达量较之C组分别增加了（1．54±0．16），（4．37±0．87）和（6．99±1．32）倍，但均显著低于I组（P〈0．01）。6h机械拉伸后，IS组HBD-3的表达显著少于I组（P〈0．01），同C组差异无统计学意义。结论机械拉伸能显著抑制IFN-γ诱导肺泡上皮细胞HBD-3表达的上调，这可能是机械通气（MV）的患者易于发生VAP的原因之一。

Objective To study the effect of mechanical stretch on the expression of human beta-defensin-3 （HBD-3） in alveolar epithelial cells（AS49 cells） elicited by interferon-gamma（IFN-γ） and to investigate the role of HBD-3 in the pathogenesis of ventilator-associated pneumonia（VAP）. Method A549 cells cultured in vitro were treated with mechanical stretch （group S）, 10 ng/ml IFN-γ（group I）,and 10 ng/ml IFN-γ with mechanical stretch （group IS）, respectively. Cells without treatment served as controls （group C）. Cells were stretched by 20% amphtude of stretch at 30 cycles/min by Flexercell-4000T^[TM] Unit for 2 h, 4 h, and 6 hours. The HBD-3 mRNA expression was determined by real-time RT-PCR after treatment. After 6 hours, treatment, cells were cultured for 24 hours and the expression of HBD-3 was examined by laser scanning confocal microscope. The experimental data were statistically analyzed by using one-way ANOVA analysis and q-test. Results The expression of HBD-3 mRNA in A549 cells could not significantly be changed by mechanical stretch alone. Compared with group C,the HBD-3 mRNA expression after treatment with 10 ng/ml IFN-γ for 2 hours,4 hours and 6 hours increased significantly by （2.63 ± 0.60）, （8.02 ± 1.63 ） and （ 12.21 ± 2.35 ） times, respectively. Compared with group C,the HBD-3 mRNA expression after treatment with 10 ng/ml IFN-γ and mechanical stretch for 2 hours,4 hours and 6 hours increased by （ 1.54 ± 0.16）, （ 4.37 ± 0.87） and （6.99 ±1.32） times, respectively, however, they were significantly lower than those in group I （P 〈 0.01 ）. The HBD-3 expression in group IS after mechanical stretch for 6 significantly different from than in group C. Conclusions Mechanical stretch can significantly suppress the up-regulation of HBD-3 in alveolar epithelial cells elicited by IFN-γ, and this may be one of the explainations that patients under mechanical ventilation（MV） have a higher risk of VAP.