不同剂量博莱霉素致小鼠肺纤维化模型的比较

Comparison of mouse pulmonary fibrosis models induced by bleomycin at different doses

背景:经气管注射博莱霉素是制作动物肺纤维化模型最常用的方法,但博莱霉素最佳造模剂量目前国内外尚无统一定论。目的:对比观察不同剂量的博莱霉素对小鼠肺纤维化模型的效果,寻找一种较为合理可靠的小鼠造模剂量。设计、时间及地点:随机对照动物实验,于2007-09/2008-04在上海市发育生物学重点实验室完成。材料:SPF级健康雌性C57BL/6J小鼠60只,体质量18～22g,用于制备小鼠肺纤维化模型。方法:60只小鼠随机数字表法分为博莱霉素组(n=45)和对照组(n=15),博莱霉素组各选15只动物分别气管内注射不同剂量(2,3.5,5mg/kg)的博莱霉素,对照组气管内注射相同体积的生理盐水,所有小鼠于造模后第28天处死并取其左肺叶制作病理切片,右肺叶作羟脯氨酸含量测定。主要观察指标:小鼠28d内的存活情况;光镜观察肺部炎症和纤维化程度;取右肺叶做羟脯氨酸测定。结果:①实验开始后前7d,博莱霉素组和对照组均无小鼠死亡,第8天博莱霉素组小鼠死亡率高于对照组(P<0.05);博莱霉素3个剂量组之间死亡率由高到低分别为5,3.5,2mg/kg博莱霉素组,但差异无显著性意义(P>0.05)。②光镜下可见对照组肺泡形态正常,间质中无炎症细胞浸润;博莱霉素组可见肺实变和胶原沉积,剂量越高,肺纤维化程度越重,以5mg/kg博莱霉素组最明显。③各组小鼠肺组织中羟脯氨酸含量从高到低依次为5mg/kg博莱霉素组、3.5mg/kg博莱霉素组、2mg/kg博莱霉素组和对照组。结论:经气管注射博莱霉素制作小鼠肺纤维化模型应兼顾造模效果和动物的存活率,3.5mg/kg是较为理想的小鼠造模剂量。

BACKGROUND： Intratracheal injection of bleomycin is the most common method to establish pulmonary fibrosis models in mice. However, it still remains to be determined how much bleomycin should be used to establish the pulmonary fibrosis models is better. OBJECTIVE： To comparatively observe the effect of different doses of bleomycin to establish pulmonary fibrosis models in mice, and search for a reasonable and reliable dosage for the mouse pulmonary fibrosis model. DESIGN, TIME AND SETTING： A randomized controlled animal experiment was performed at Shanghai Biology Development Key Laboratory from July 2007 to April 2008. MATERIALS： Sixty healthy female C57BL/6J mice, of SPF grade, weighing 18-20 g, were enrolled. METHODS： All mice were randomly divided into bleomycin groups （n=-45） and control group （n=15）. 2, 3.5 and 5.0 mg/kg bleomycin were given intratracheally to every 15 mice in the bleomycin group, respectively. The mice in the control group were administrated the same volume of normal saline. Twenty-eight days after model establishment, all mice were sacrificed. Their left pulmonary lobe tissues were removed from mice and used for pathological section observation, and the right pulmonary lobe tissues were used for the hydroxyproline contents measurement. MAIN OUTCOME MEASURES： The survival condition of mice in 28 days. The inflammation and fibrosis degree in the left pulmonary lobe were observed by light microscope; Hydroxyproline contents in right pulmonary lobe were measured. RESULTS：（1）At the beginning seven days of the experiment, no mice died in both the bleomycin and control groups. However, the mortality of mice was higher in the bleomycin group than that in the control group on the 8th day （P〈 0.05）. With the increasing of the bleomycin dosage administrated to the mice, the mortality of mice was increased, but there was no significant difference （P 〉 0.05）. （2）The morphology of alveolus was normal and few inflammatory cells infiltrated in the control group. The pulmonary consolidation and collagen deposition appeared in the bleomycin group. The more bleomycin dosage was used, the more severe pulmonary fibrosis would be. （3）Hydroxyproline contents in pulmonary tissue from high to low were mice injected 5, 3.5, 2.0 mg/kg bleomycin, and normal saline. CONCLUSION： Both effect and survival rate should be considered to establish the pulmonary fibrosis model in mice. The ideal dose of bleomycin for inducing mouse pulmonary fibrosis by endotracheal injection is 3.5 mg/kg.