【摘要】 目的观察酸性鞘磷脂酶(ASM)在二氧化硅(SiO2)粉尘致小鼠胚肺成纤维细胞(NIH3T3)纤维化过程中的变化及规律,探讨ASM在硅尘致肺纤维化体外模型中的作用。方法收集SiO2200 mg·L-1刺激小鼠原代肺泡巨噬细胞(PAM)12 h的培养上清再刺激小鼠胚胎成纤维细胞(NIH3T3)6,12,18,24,36和48 h,ELISA法测定PAM培养上清中白细胞介素8(IL-8)、肿瘤坏死因子ɑ(TNF-ɑ)及转化生长因子β1(TGF-β1)的含量;高效液相色谱法测定NIH3T3细胞内的ASM活性;Western蛋白质印迹法测定NIH3T3细胞内Ⅲ型胶原蛋白质的表达。结果 SiO2200 mg·L-1刺激PAM 12 h,培养上清中IL-8,TNF-ɑ及TGF-β1的分泌中明显增加(P<0.01)。PAM上清刺激NIH3T3细胞24,36和48 h,细胞ASM活性明显升高(P<0.05),在36 h达到峰值,加入地昔帕明1.25μmol·L-1细胞ASM活性随着时间延长而显著降低。Western蛋白质印迹法结果显示,PAM上清刺激NIH3T3细胞24,36及48 h,细胞内Ⅲ型胶原蛋白表达明显增加(P<0.05),在36 h达到峰值,加入地昔帕明1.25μmol·L-1组Ⅲ型胶原蛋白表达随着时间延长而降低。结论抑制ASM活性可能对改善肺纤维化有一定的作用。更多还原

【Abstract】 OBJECTIVE To observe the level and change rule of acid sphingomyelinase( ASM) in silica-induced fibrosis in mouse embryonic fibroblast( NIH3T3),and to explore the functional role of ASM in silica-induced pulmonary fibrosis in vitro. METHODS Pulmonary alveolar macrophages( PAM) were treated with SiO2200 mg·L-1for 12 h,and the medium supernatant was collected to stimulate NIH3T3 for 6,12,18,24,36,and 48 h. ELISA assay was applied to determine the contents of interleukin-8( IL-8),tumor necrosis factor( TNF-ɑ) and transforming growth factor-β1( TGF-β1) in the supernatant collected from the culture medium of PAM. ASM activity of NIH3T3 was assayed using high-performance liquid chromatographic( HPLC). The protein expression level of type Ⅲ collagen was determined by western blotting. RESULTS The levels of IL-8,TNF-ɑ and TGF-β1 in PAM exposed to SiO2 200 mg·L-1were obviously increased( P < 0. 01). ASM activity of NIH3T3 was increased at the time point of 24,36 and 48 h after stimulated with the supernantant( P < 0. 05),and the peak was appeared at 36 h. In the group treated with desipramine 1. 25 μmol·L-1,ASM activity was decreased with the prolonged time. Western blotting results revealed that the expression of typeⅢ collagen was up-regulated at the time point of 24,36 and 48 h after stimulation( P < 0. 05),and the peak appeared at 36 h. And the expression of type Ⅲ collagen was down-regulated with the prolonged time in the group treated with desipramine 1. 25 μmol·L-1. CONCLUSION Inhibition of ASM activity may have a certain effect on improving pulmonary fibrosis.更多还原