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。
OBJECTIVE To observe the level and change rule of acid sphingo myelinase （ASM）in silica-induced fibrosis in mouse e mbryonic fibroblast （NIH3T3 ）,and to explore the functional role of ASM in silica-induced pul monary fibrosis in vitro.METHODS Pul monary alveolar macrophages （PAM） were treated with SiO2 200 mg·L -1 for 12 h,and the mediu m supernatant was collected to sti mulate NIH3T3 for 6,12,18,24,36,and 48 h.ELISA assay was applied to determine the contents of interleu-kin-8 （IL-8）,tu mor necrosis factor （TNF-ɑ）and transforming growth factor-β1 （TGF-β1 ）in the super-natant collected fro m the culture mediu m of PAM.ASM activity of NIH3T3 was assayed using high-per-formance liquid chro matographic （HPLC）.The protein expression level of type Ⅲ collagen was deter-mined by western blotting.RESULTS The levels of IL-8,TNF-ɑand TGF-β1 in PAM exposed to SiO2 200 mg·L -1 were obviously increased （P ＆lt;0.01 ）.ASM activity of NIH3T3 was increased at the ti me point of 24,36 and 48 h after sti mulated with the supernantant（P＆lt;0.05）,and the peak was appeared at 36 h.In the group treated with desipra mine 1 .25 μmol·L -1 ,ASM activity was decreased with the pro-longed ti me.Western blotting results revealed that the expression of typeⅢ collagen was up-regulated at the ti me point of 24,36 and 48 h after sti mulation（P＆lt;0.05）,and the peak appeared at 36 h.And the expression of type Ⅲ collagen was down-regulated with the prolonged ti me in the group treated with desi-pra mine 1 .25 μmol·L -1 .CONCLUSION Inhibition of ASM activity may have a certain effect on i mpro-ving pul monary fibrosis.
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2014年第2期205-209,共5页
Chinese Journal of Pharmacology and Toxicology
基金
中南大学中央高校基本科研业务费专项资金资助(2013zzts288)
中南大学中央高校基本科研业务费专项资金资助(2282013bks067)~~
