CAJ | 학술논문

目的建立博来霉素诱导小鼠肺纤维化模型,观察ROCK抑制剂法舒地尔对肺纤维化的干预作用;同时体外培养NIH3T3成纤维细胞,观察法舒地尔对细胞增殖和迁移能力的影响及其可能的机制.方法 SPF级雄性C57BL/6小鼠36只通过随机数字表法分为对照组(A组)、法舒地尔组(B组)、博来霉素组(C组)、博来霉素+法舒地尔低剂量组(D组)、博来霉素+法舒地尔中剂量组(E组)、博来霉素+法舒地尔高剂量组(F组),每组6只.腹腔内注射法舒地尔,肺组织病理切片Ashcroft评分及羟脯氨酸测定评估小鼠肺纤维化的严重程度.给予法舒地尔对NIH3T3细胞进行干预,MTT法、平板克隆形成实验检测细胞增殖能力;划痕实验、Transwell小室实验检测细胞迁移能力;RT-PCR法和Western blot法测定细胞CyclinD1、MMP2、TIMP1 mRNA和蛋白的表达水平及MYPT1磷酸化水平.结果 A组及B组小鼠第21天的Ashcroft评分分别为0.50±0.22和0.67±0.21,评分无明显差异;C组评分为6.00±0.26,明显高于A组;D组、E组和F组评分分别为5.50±0.22、3.83±0.31和3.33±0.21.A组及B组小鼠肺组织羟脯氨酸含量分别为(36.32±1.08)和(37.25±2.16)μg/ml,含量无明显差异;C组羟脯氨酸含量为(84.79±3.04) μg/ml,明显高于A组;D组、E组和F组评分分别为80.73±2.21、71.80±2.33和62.18±2.75.MTT法、平板克隆形成实验、划痕实验和Transwell小室迁移实验提示法舒地尔可呈时间和浓度依赖性地抑制NIH3T3细胞的增殖能力和迁移能力.结论法舒地尔对肺纤维化具有保护作用,为临床应用法舒地尔治疗IPF提供一定的理论和实验依据.
목적 건립박래매소유도소서폐섬유화모형,관찰ROCK억제제법서지이대폐섬유화적간예작용;동시체외배양NIH3T3성섬유세포,관찰법서지이대세포증식화천이능력적영향급기가능적궤제.방법 SPF급웅성C57BL/6소서36지통과수궤수자표법분위대조조(A조)、법서지이조(B조)、박래매소조(C조)、박래매소+법서지이저제량조(D조)、박래매소+법서지이중제량조(E조)、박래매소+법서지이고제량조(F조),매조6지.복강내주사법서지이,폐조직병리절편Ashcroft평분급간포안산측정평고소서폐섬유화적엄중정도.급여법서지이대NIH3T3세포진행간예,MTT법、평판극륭형성실험검측세포증식능력;화흔실험、Transwell소실실험검측세포천이능력;RT-PCR법화Western blot법측정세포CyclinD1、MMP2、TIMP1 mRNA화단백적표체수평급MYPT1린산화수평.결과 A조급B조소서제21천적Ashcroft평분분별위0.50±0.22화0.67±0.21,평분무명현차이;C조평분위6.00±0.26,명현고우A조;D조、E조화F조평분분별위5.50±0.22、3.83±0.31화3.33±0.21.A조급B조소서폐조직간포안산함량분별위(36.32±1.08)화(37.25±2.16)μg/ml,함량무명현차이;C조간포안산함량위(84.79±3.04) μg/ml,명현고우A조;D조、E조화F조평분분별위80.73±2.21、71.80±2.33화62.18±2.75.MTT법、평판극륭형성실험、화흔실험화Transwell소실천이실험제시법서지이가정시간화농도의뢰성지억제NIH3T3세포적증식능력화천이능력.결론 법서지이대폐섬유화구유보호작용,위림상응용법서지이치료IPF제공일정적이론화실험의거.
Objective To determine the beneficial effects and mechanisms of fasudil,a selective ROCK inhibitor,on bleomycin-induced pulmonary fibrosis in mice and to determine the effects and mechanisms of fasudil on the biological behaviors in NIH3T3 mouse fibroblast cell line.Methods The BPF model was induced by a single dosage of 2.5 mg/kg bleomycin intratracheal injection in mice and fasudil intraperitoneal injection was given to the mice.The fibrosis degree was determined pathologically by using the Ashcroft scoring method and biochemically by hydroxyproline assay in lung tissue.NIH3T3 mouse fibroblast cell line was cultured in vitro and fasudil was given to the cell.The proliferation activity in NIH3T3 cells were detected by MTT assay and fiat colony forming experiment.The migration activity in NIH3T3 cells were detected by scratch test and transwell chamber experiment.The expression of CyclinD1,MMP2 and TIMP1 mRNA in NIH3T3 cells was detected by RT-PCR.The expression of CyclinD1,MMP2 and TIMP1 protein and the level of MYPT1 phosphorylation in NIH3T3 cells was detected by Western blot.Results Compare to the mice administrated by bleomycin,the Ashcroft score and hydroxyproline content were significantly decreased in the mice administered fasudil.Administration of fasudil can reduce the ability of proliferation and migration in a dose-dependent manner in NIH3T3 cells.The effect of fasudil was possibly related to increase the production of TIMP1 and decrease the production of CyclinD1 and MMP2.Conclusions Administration of fasudil can attenuate pulmonary fibrosis both in vivo and in vitro.These findings suggest that fasudil may be a potential therapeutic candidate for the treatment of pulmonary fibrosis.