三种方法气管灌注博来霉素诱导小鼠肺纤维化的比较
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摘要
目的利用腰穿针气管插管、留置针气管插管和气管切开三种不同方法对C57BL/6J小鼠进行气管注射博来霉素诱导肺纤维化,探索腰穿针气管插管造模法、留置针气管插管造模法及气管切开造模法这三种方法对小鼠的影响。方法将120只C57BL/6J小鼠随机分为空白组、腰穿针气管插管模型组、留置针气管插管模型组和气管切开模型组,分别利用不同方法气管注射博来霉素,观察小鼠的一般情况(体重变化和存活率)、组织病理学改变(HE和Masson染色)和羟脯氨酸水平。结果腰穿针气管插管模型组、留置针气管插管模型组造模后28 d小鼠体重均高于气管切开模型组,有统计学差异(P<0.01、P<0.001);各组小鼠存活率存在差异,除空白组外,气管切开模型组最低,留置针气管插管模型组最高,空白组和气管切开模型间有统计学差异(P<0.05),其余各组间没有统计学意义;肺组织肺泡炎及肺纤维化程度由高到低依次为气管切开模型组、腰穿针气管插管模型组和留置针气管插管模型组,且造模后21 d最重;气管切开模型组病灶分布均匀,腰穿针气管插管模型组和留置针气管插管模型组分布不均匀,以气管周围肺组织较重;腰穿针气管插管模型组和气管切开模型组造模后28 d羟脯氨酸水平显著高于空白组(P<0.01、P<0.001)。结论综合多种因素,腰穿针气管插管造模法是三种造模方法中的最佳方法。
Objective Lumbar spinal needle, indwelling needle, and tracheotomy were used to establish a bleomycin-induced pulmonary fibrosis model in C57 BL/6 J mice,respectively,and to compare the effects of these three methods on mice. Methods One hundred and twenty C57 BL/6 J mice were randomly divided into blank, lumbar spinal needle model, indwelling needle model, and tracheotomy model groups. Mice from different groups were administered by intratracheal instillation of bleomycin using the different methods, respectively. The general condition of the mice(changes in body weight and survival rate) was observed, and the histopathological changes(HE staining and Masson staining) and hydroxyproline level were examined. Results The body weight of the lumbar spinal needle model group and the indwelling needle model group was significantly higher than that of the tracheotomy model group at 28 days after modeling(P<0.01、P<0.001). There were no significant differences in survival rate among the four groups except between the blank group and the tracheotomy model group(P<0.05). The degrees of alveolitis and pulmonary fibrosis were highest in the tracheotomy model group, followed by the lumbar spinal needle model group and indwelling needle model group. The degree of alveolitis and pulmonary fibrosis was greatest at 21 days after modeling. The lesions were evenly distributed in the tracheotomy model group, and unevenly distributed in the lumbar spinal needle model and indwelling needle model groups. The lesions in the lung tissue around the trachea were more severe than in other areas in the lumbar spinal needle model and indwelling needle model groups. The level of hydroxyproline in the lumbar spinal needle model and tracheotomy model groups was significantly higher than that in the blank group(P<0.01,P<0.001). Conclusions In summary, tracheal intubation by lumbar spinal needle is the best method among the three methods for the establishment of a bleomycin-induced pulmonary fibrosis in mice.
Objective Lumbar spinal needle, indwelling needle, and tracheotomy were used to establish a bleomycin-induced pulmonary fibrosis model in C57 BL/6 J mice,respectively,and to compare the effects of these three methods on mice. Methods One hundred and twenty C57 BL/6 J mice were randomly divided into blank, lumbar spinal needle model, indwelling needle model, and tracheotomy model groups. Mice from different groups were administered by intratracheal instillation of bleomycin using the different methods, respectively. The general condition of the mice(changes in body weight and survival rate) was observed, and the histopathological changes(HE staining and Masson staining) and hydroxyproline level were examined. Results The body weight of the lumbar spinal needle model group and the indwelling needle model group was significantly higher than that of the tracheotomy model group at 28 days after modeling(P<0.01、P<0.001). There were no significant differences in survival rate among the four groups except between the blank group and the tracheotomy model group(P<0.05). The degrees of alveolitis and pulmonary fibrosis were highest in the tracheotomy model group, followed by the lumbar spinal needle model group and indwelling needle model group. The degree of alveolitis and pulmonary fibrosis was greatest at 21 days after modeling. The lesions were evenly distributed in the tracheotomy model group, and unevenly distributed in the lumbar spinal needle model and indwelling needle model groups. The lesions in the lung tissue around the trachea were more severe than in other areas in the lumbar spinal needle model and indwelling needle model groups. The level of hydroxyproline in the lumbar spinal needle model and tracheotomy model groups was significantly higher than that in the blank group(P<0.01,P<0.001). Conclusions In summary, tracheal intubation by lumbar spinal needle is the best method among the three methods for the establishment of a bleomycin-induced pulmonary fibrosis in mice.
引文
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[13] 杨聪颖,彭雄群,阳惠湘,等.不同剂量博莱霉素诱导小鼠肺纤维化的差异及动态变化 [J].中国实验动物学报,2013,21(2):45-51.
[14] Cao Z,Lis R,Ginsberg M,et al.Targeting of the pulmonary capillary vascular niche promotes lung alveolar repair and ameliorates fibrosis [J].Nat Med,2016,22(2):154-162.
[15] Cao Z,Ye T,Sun Y,et al.Targeting the vascular and perivascular niches as a regenerative therapy for lung and liver fibrosis [J].Sci Transl Med,2017,9(405):eaai8710.
[16] Lee SH,Lee EJ,Lee SY,et al.The effect of adipose stem cell therapy on pulmonary fibrosis induced by repetitive intratracheal bleomycin in mice [J].Exp Lung Res,2014,40(3):117-125.
[17] Rangarajan S,Bone NB,Zmijewska AA,et al.Metformin reverses established lung fibrosis in a bleomycin model [J].Nat Med,2018,24(8):1121-1127.
[18] Tsubouchi K,Araya J,Minagawa S,et al.Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4 [J].Autophagy,2017,13(8):1420-1434.
[19] Al-Tamari HM,Dabral S,Schmall A,et al.FoxO3 an important player in fibrogenesis and therapeutic target for idiopathic pulmonary fibrosis [J].EMBO Mol Med,2018,10(2):276-293.
[20] 李伟峰,胡玉洁,袁伟锋,等.气管内滴入与雾化博莱霉素致小鼠肺纤维化模型的比较研究 [J].南方医科大学学报,2012,32(2):221-225.
[21] 胡静,刘旭凌.经鼻滴入博莱霉素致小鼠肺纤维化模型的建立及其病理演变 [J].中国实验动物学报,2009,17(5):351-353.
[22] 苏敏红,江宁,李洪涛,等.腹腔注射博来霉素诱导小鼠肺纤维化模型的长期稳定性 [J].中国组织工程研究,2017,21(4):512-519.
[23] 肖强,郑晓滨,刘香,等.静脉注射不同剂量博莱霉素诱导小鼠肺纤维化的比较研究 [J].临床肺科杂志,2015,20(2):251-256.
[24] 张晓晔,志村早苗,增田辙,等.博莱霉素静脉注射制备小鼠肺间质纤维化模型 [J].中国实验动物学报,2007,15(4):241-244.
[25] 付钰,吴瑕,陈随清.甘草查尔酮A通过调节TGF-β/Smad信号通路抑制小鼠肺纤维化 [J].中国实验方剂学杂志,2019,25(4):94-100.
[2] Waseda Y,Yasui M,Nishizawa Y,et al.Angiotensin II type 2 receptor antagonist reduces bleomycin-induced pulmonary fibrosis in mice [J].Respir Res,2008,9:43.
[3] Iyer SN,Gurujeyalakshmi G,Giri SN.Effects of pirfenidone on transforming growth factor-β gene expression at the transcriptional level in bleomycin hamster model of lung fibrosis [J].J Pharmacol Exp Ther,1999,291(1):367-373.
[4] Sriram N,Kalayarasan S,Sudhandiran G.Enhancement of antioxidant defense system by epigallocatechin-3-gallate during bleomycin induced experimental pulmonary fibrosis [J].Biol Pharm Bull,2008,31(7):1306-1311.
[5] Gunther A,Lubke N,Ermert M,et al.Prevention of bleomycin-induced lung fibrosis by aerosolization of heparin or urokinase in rabbits [J].Am J Respir Crit Care Med,2003,168(11):1358-1365.
[6] Hinz B,Phan SH,Thannickal VJ,et al.Recent developments in myofibroblast biology:paradigms for connective tissue remodeling [J].Am J Pathol,2012,180(4):1340-1355.
[7] 陶章,李惠萍.不同剂量博莱霉素致小鼠肺纤维化模型的比较 [J].中国组织工程研究与临床康复,2009,13(7):1214-1218.
[8] Degryse AL,Tanjore H,Xu XC,et al.Repetitive intratracheal bleomycin models several features of idiopathic pulmonary fibrosis [J].Am J Physiol Lung Cell Mol Physiol,2010,299(4):L442-L452.
[9] 李丽娜,王华,周蕾,等.博莱霉素诱导小鼠肺间质纤维化造模方式的选择 [J].中国免疫学杂志,2010,26(3):254-257.
[10] 张成华,朱庆均,田景振.博莱霉素诱导的肺纤维化动物模型评价及应用研究进展 [J].中南药学,2017,15(4):472-475.
[11] Szapiel SV,Elson NA,Fulmer JD,et al.Bleomycin-induced interstitial pulmonary disease in the nude,athymic mouse [J].Am Rev Respir Dis,1979,120(4):893-899.
[12] Adamson IY,Bowden DH.The pathogenesis of bleomycin-induced pulmonary fibrosis in mice [J].Am J Pathol,1974,77(2):185-197.
[13] 杨聪颖,彭雄群,阳惠湘,等.不同剂量博莱霉素诱导小鼠肺纤维化的差异及动态变化 [J].中国实验动物学报,2013,21(2):45-51.
[14] Cao Z,Lis R,Ginsberg M,et al.Targeting of the pulmonary capillary vascular niche promotes lung alveolar repair and ameliorates fibrosis [J].Nat Med,2016,22(2):154-162.
[15] Cao Z,Ye T,Sun Y,et al.Targeting the vascular and perivascular niches as a regenerative therapy for lung and liver fibrosis [J].Sci Transl Med,2017,9(405):eaai8710.
[16] Lee SH,Lee EJ,Lee SY,et al.The effect of adipose stem cell therapy on pulmonary fibrosis induced by repetitive intratracheal bleomycin in mice [J].Exp Lung Res,2014,40(3):117-125.
[17] Rangarajan S,Bone NB,Zmijewska AA,et al.Metformin reverses established lung fibrosis in a bleomycin model [J].Nat Med,2018,24(8):1121-1127.
[18] Tsubouchi K,Araya J,Minagawa S,et al.Azithromycin attenuates myofibroblast differentiation and lung fibrosis development through proteasomal degradation of NOX4 [J].Autophagy,2017,13(8):1420-1434.
[19] Al-Tamari HM,Dabral S,Schmall A,et al.FoxO3 an important player in fibrogenesis and therapeutic target for idiopathic pulmonary fibrosis [J].EMBO Mol Med,2018,10(2):276-293.
[20] 李伟峰,胡玉洁,袁伟锋,等.气管内滴入与雾化博莱霉素致小鼠肺纤维化模型的比较研究 [J].南方医科大学学报,2012,32(2):221-225.
[21] 胡静,刘旭凌.经鼻滴入博莱霉素致小鼠肺纤维化模型的建立及其病理演变 [J].中国实验动物学报,2009,17(5):351-353.
[22] 苏敏红,江宁,李洪涛,等.腹腔注射博来霉素诱导小鼠肺纤维化模型的长期稳定性 [J].中国组织工程研究,2017,21(4):512-519.
[23] 肖强,郑晓滨,刘香,等.静脉注射不同剂量博莱霉素诱导小鼠肺纤维化的比较研究 [J].临床肺科杂志,2015,20(2):251-256.
[24] 张晓晔,志村早苗,增田辙,等.博莱霉素静脉注射制备小鼠肺间质纤维化模型 [J].中国实验动物学报,2007,15(4):241-244.
[25] 付钰,吴瑕,陈随清.甘草查尔酮A通过调节TGF-β/Smad信号通路抑制小鼠肺纤维化 [J].中国实验方剂学杂志,2019,25(4):94-100.