CAJ | 학술논문

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。
목적：관찰산성초린지매（ASM）재이양화규（SiO2）분진치소서배폐성섬유세포（NIH3T3）섬유화과정중적변화급규률，탐토 ASM재규진치폐섬유화체외모형중적작용。방법수집 SiO2200 mg·L -1자격소서원대폐포거서세포（PAM）12 h 적배양상청재자격소서배태성섬유세포（NIH3T3）6，12，18，24，36화48 h，ELISA 법측정 PAM배양상청중백세포개소8（IL-8）、종류배사인자ɑ（TNF-ɑ）급전화생장인자β1（TGF-β1）적함량；고효액상색보법측정 NIH3T3세포내적 ASM 활성；Western 단백질인적법측정NIH3T3세포내Ⅲ형효원단백질적표체。결과SiO2200 mg·L -1자격 PAM 12 h，배양상청중 IL-8，TNF-ɑ급 TGF-β1적분비중명현증가（P＜0．01）。PAM상청자격 NIH3T3세포24，36화48 h，세포 ASM활성명현승고（P＜0．05），재36 h 체도봉치，가입지석파명1．25μmol·L -1세포 ASM활성수착시간연장이현저강저。Western 단백질인적법결과현시，PAM상청자격 NIH3T3세포24，36급48 h，세포내Ⅲ형효원단백표체명현증가（P＜0．05），재36 h 체도봉치，가입지석파명1．25μmol·L -1조Ⅲ형효원단백표체수착시간연장이강저。결론억제 ASM활성가능대개선폐섬유화유일정적작용。
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 <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<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<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.