二氧化硅刺激矽肺肺泡巨噬细胞上清介导的人胚肺成纤维细胞Ⅲ型胶原和Ⅲ型前胶原的表达

Effects of silicotic alveolar macrophages exposed to SiO2 on the expression of type Ⅲ collagen and type Ⅲ procollagen in human lung fibroblasts

目的 探讨经SiO2刺激的肺泡巨噬细胞（AM）通过人胚肺成纤维细胞（HELF）对Ⅲ型胶原（CⅢ）和Ⅲ型前胶原（pCⅢ）表达的影响及抗TGF-β1抗体的干预作用.方法 收集矽肺患者AM,将其分为加入SiO2粉尘悬液的处理组和仅加入无血清培养液的对照组.培养18 h后获取培养上清.将原代培养的HELF分为：（1）处理组：加入经SiO2刺激的AM上清;（2）对照组：加入未经SiO2刺激的AM上清;（3）空白对照组：仅加入含体积分数为3%胎牛血清的DMEM;（4）处理组＋抗转化生长因子β1（TGF-β1）抗体干预组：在处理组的基础上加上抗TGF-β1抗体（10 μg/ml）;（5）对照组＋抗TGF-β1抗体干预组：在对照组的基础上加上抗TGF-β1抗体（10 μg/ml）.前3组分别培养6、12、18、24、36、48 h,后2组分别培养18、24、36 h,用免疫细胞化学法检测HELF中pCⅢ的表达,用免疫印迹（Western blot）法检测HELF条件培养上清中CⅢ的表达.结果 与对照组（0.1212±0.0079、0.1414±0.0058、0.1620±0.0081、0.1965±0.0103、0.1715±0.0116）相比,处理组12、18、24、36和48 h pCⅢ表达（0.1423±0.0107,0.1624±0.0011,0.1925±0.0056,0.2421±0.0097,0.2102±0.0103）明显升高,差异有统计学意义（P＜0.05或P＜0.01）.与对照组（0.2296±0.0121、0.2778±0.0116、0.3367±0.0269、0.3722±0.0214）相比,处理组12、18、24、36、48 hCⅢ含量（0.2559±0.0061、0.3249±0.0110、0.4171±0.0193、0.5441±0.0452、0.4751±0.0252）明显增高,差异均有统计学意义（P＜0.05或P＜0.01）.与同期相对应的非干预组相比,18、24、36 h抗TGF-β1抗体干预组CⅢ和pCⅢ均明显降低,差异均有统计学意义（P＜0.01或P＜0.05）.结论 SiO2经AM的介导,诱导HELF高表达CⅢ和pCⅢ,部分效应是通过TGF-β1起作用.

Objective To study the effects of supernatant of alveolar macrophages （AM） exposed to SiO2 on the expression of type Ⅲ collagen and type Ⅲ procollagen in human lung fibroblasts （HELF） and the intervention effects of anti-TGF-β1 antibody Methods AMs collected from a silicotic by bronchoalveolar lavage were divided into 2 parts, one part was exposed to SiO2 and other part served as control. The supernatant was obtained from AMs cultured for 18 h. HELF were divided into （1） exposure group, which was added with supernatant from AMs exposed to SiO2; （2） control group, which was added with the supernatant from AMs not exposed to SiO2; （3） blank control group, which was added with DMEM; （4） exposure group plus anti-TGF-β1antibody （10μg/ml）; （5） control group plus anti-TGF-β1 antibody （10 μg/ml）. （1）-（3） groups were cultured for 6, 12, 18, 24, 36, 48h, respectively. （4）-（5） groups were cultured for 18, 24, 36, respectively.Immunocytochemical test and Western blot assay were used to detect pC Ⅲ expression levels in HELF and C Ⅲ expression levels in the supernatant of HELF culture, respectively. Results The pC Ⅲ expression levels of exposure group were 0.1423 ±0.0107,0.1624±0.0011,0.1925 ±0.0050,0.2421 ±0.0097 and 0.2103 ±0.0103,respectively, which were significantly higher than those （0.1212±0.0079、0.1414±0.0058、0.1620±0.0081、0.1965±0.0103 、0.1715±0.0116） of control group （P＜0.05 or P＜0.01）. The C Ⅲ levels of exposure group were （0.2559±0.0061、0.3249±0.0110、0.4171±0.0193、0.5441 ±0.0452、0.4751±0.0252）, respectively, which were significantly higher than control group （0.2296±0.0121、0.2778±0.0116、0.3367±0.0269、0.3722±0.0214）. The pC Ⅲ and C Ⅲ expression levels of exposure plus anti-TGF-β1 antibody group were significantly lower than those of control plus anti-TGF-β1 antibody group （P＜0.05 or P＜0.01）.Conclusion AMs exposed to SiO2 can induce the elevated pC Ⅲ and C Ⅲ expression levels in HELF by TGF-β 1 to some extent.