干扰素-γ、肿瘤坏死因子-α和白细胞介素-1β在体外对人气管平滑肌细胞凋亡的作用

Effects of interferon-γ, tumor necrosis factor-α and interleukin-1β on apoptosis of human airway smooth muscle cells in vitro

目的 :研究是否干扰素 -γ(IFN -γ)、肿瘤坏死因子 -α(TNF -α)、白细胞介素 - 1β(IL - 1β)在体外能够诱导人气管平滑肌细胞 (ASMCs)凋亡。方法 :分离人ASMCs并在含有 10 %胎牛血清的DMEM培养基中培养。用 4- 6代的细胞作实验。IFN -γ、TNF -α和IL - 1β ,每种细胞因子单独或一起用于处理人ASMCs。用MTT法在 0、2 4、4 8、72h检测IFN -γ、TNFα和IL - 1β对细胞生长的作用。用光镜和电镜观察形态学的改变。用琼脂糖电泳分析DNA片断。用SP -免疫组化染色方法检测p5 3、bcl- 2、bax基因表达的改变。通过碎片DNA原位末端标记技术(TUNEL)检测凋亡细胞的百分率。结果①IFN -γ以时间依赖的方式单独或 /和TNF -α和IL - 1β一起减低存活的细胞数 ;②光镜和电镜检查显示人ASMCs细胞皱缩、膜出泡 ,核缩小 ,染色质浓聚和核破碎 ;③琼脂糖电泳显示在用上述细胞因子联合处理的人ASMCs ,有代表寡核苷酸片断整倍数的特征性的DNA梯状条带 (大约 180 - 2 0 0bp) ;④在细胞因子联合处理组p5 3和bax基因表达显著高于对照组 ,但bcl- 2基因表达低于对照组 (P <0 0 1) ;⑤用IFN -γ( 4× 10 5U/L)、TNF -α( 4× 10 5U/L)和 /或IL - 1β( 10× 10 4U/L)同时刺激诱导人ASMCs凋亡。在用细胞因子联合处理组的人ASMCs的?

AIM:To clarify if interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)can induce apoptosis of human airway smooth muscle cells (ASMCs) in vitro. METHODS: Human ASMCs were isolated and cultured in DMEM containing 10% fetal bovine serum. Passage 4-6 cell was used in the experiment. IFN-γ,TNF-α and IL-1β, were used separately or together in the treatment of human ASMCs. The effects of IFN-γ,TNF-α and IL-1β on the growth of the cells was detected by MTT method at the hour 0,24,48 and 72. Light microscopy and electron microscopy were used to examine the morphological change. DNA fragmentation was analyzed by agarose gel electrophoresis. SP immunohistological staing method was performed to detect the change of expressions of p53 , bcl-2 and bax gene. The apoptosis cell percentage were detected by in situ end labeling technique (TUNEL)of fragmental DNA. RESULTS: (1)IFN-γ or IFN-γ together with TNF-α and IL-1β decreased the number of viable cells in a time dependent manner. (2) Light and electron microscopic examination showed cell shrinkage, membrane blebbing, nuclear contraction, chromatin condensation and nuclear fragmentation in human ASMCs. (3) Agarose gel electrophoresis showed a characteristic'ladder'of DNA bands representing integer multiples of the internucleosomal fragments (about 180-200 bp) in cytokine cotreated human ASMCs. (4)The expression of p53 and bax gene in cytokine cotreated group was significantly higher than in control group, but the expression of bcl-2 gene was lower than in control group. (5)Stimultaneous treatment with IFN-γ(4×10 5 U/L),TNF-α(4×10 5 U/L)and /or IL-1β (10×10 4 U/L) induced apoptosis of human ASMCs. Apoptotic index of human ASMCs in cytokine co-treated group was significantly higher than in control group ( P< 0.01). CONCLUSION: Stimultaneous treatment with IFN-γ,TNF-α and /or IL-1β induced apoptosis of human ASMCs. These immune cytokines may play an important role in airway remodeling of asthma and of chronic obstructive pulmonary disease.