单双次气管内滴注博来霉素诱导肺纤维化大鼠模型的比较

Comparison of rat models of pulmonary fibrosis induced by one or two intratracheal bleomycin instillations

目的采用单次及两次气管内滴注博来霉素(bleomycin,BLM)构建肺纤维化大鼠模型,比较2种造模方式的成模率及稳定性。方法150只SPF级SD大鼠随机分成空白对照组(Control组)、单次气管内滴注博来霉素组(BLM-S组)、两次气管内滴注博来霉素组(BLM-M组)。BLM-S组第1天采用无创气管内滴注BLM(3 mg/kg,单次)诱导;BLM-M组第1、14天分别气管内滴注BLM(3、2 mg/kg)诱导,Control组采用无创气管内滴注0.9%氯化钠注射液(1 mL/kg);于造模后第28、42、56、84天分批取材。检测大鼠深吸气量(inspiratory capacity,IC)、肺活量(vital capacity,VC)、静态肺顺应性(static compliance of lung,Cchord)及动态肺顺应性(dynamic lung complication,Cdyn);观察大鼠肺组织病理变化,并对肺泡炎及纤维化程度进行评分;采用免疫组化检测大鼠肺组织Ⅲ型胶原蛋白(collagen-Ⅲ,COL-Ⅲ)表达。结果(1)一般状态及生存情况:Control组、BLM-S组和BLM-M组生存率分别为:100%、80%、66%。第14~42天,BLM-S组与BLM-M组大鼠体重显著低于Control组(P<0.05,P<0.01)。第28~42天,BLM-M组体重显著低于Control组、BLM-S组(P<0.05,P<0.01)。(2)肺功能:与Control组比较,第28天BLM-S组肺功能IC、VC、Cchord、Cdyn均显著下降(P<0.05,P<0.01),BLM-M组IC、VC、Cchord显著下降(P<0.05,P<0.01);第42天BLM-S组大鼠IC、VC、Cchord显著下降(P<0.05,P<0.01);第42~84天BLM-M组大鼠IC、VC、Cchord明显下降(P<0.05,P<0.01)。(3)肺病理:第28~84天,BLM-S组大鼠出现炎性浸润及纤维条索后逐渐减少(P<0.05,P<0.01);BLM-M组大鼠出现纤维化及肺泡炎后较为稳定(P<0.05,P<0.01)。(4)胶原沉积:各时间点BLM-S组、BLM-M组大鼠肺组织内COL-Ⅲ表达显著高于Control组(P<0.05,P<0.01);第42~84天BLM-S组COL-Ⅲ含量显著低于28 d(P<0.05)。结论2种造模方式均可成功建立肺纤维化模型,其中单次法操作简便,大鼠死亡率更低,28 d时纤维化程度明显,但在42 d之后会逐渐恢复;两次法造模成功率更高,模型稳定性更好,至84 d仍有超半数大鼠存在明显纤维化。

Objective A rat model of pulmonary fibrosis was constructed using a single or two intratracheal drops of bleomycin(BLM)and the modeling rate and stability of the two modeling modalities were compared.Methods A total of 150 specific pathogen-free SD rats were divided randomly into blank control(control),single intratracheal drop of bleomycin(BLM-S),and two intratracheal drops of bleomycin(BLM-M)groups.Rats in the BLM-S group received a single dose of 3 mg/kg BLM by noninvasive intratracheal instillation,and rats in BLM-M group received 3 mg/kg BLM on day 1 and BLM 2 mg/kg on day 14.Rats in the control group were given intratracheal instillation of 0.9%sodium chloride(1 mL/kg).The rats were euthanized on days 28,42,56,and 84 after modelling,respectively.Deep inspiratory capacity(IC),vital capacity(VC),static lung compliance(Cchord),and dynamic lung complication(Cdyn)were measured in all rats.Pathological changes in lung tissue were observed,and the extent of alveolitis and fibrosis was graded.Collagen-III(COL-III)expression in rat lung tissue was detected by immunohistochemistry.Results(1)The survival rates in the control,BLM-S,and BLM-M groups were 100%,80%,and 66%,respectively.Rats in the BLM-S and BLM-M groups had significantly lower body weights on days 14~42 compared with the control group(P<0.05,P<0.01),and rats in the BLM-M group had significantly lower body weight on days 28~42 than rats in the control and BLM-S groups(P<0.05,P<0.01).(2)Regarding lung function,IC,VC,Cchord,and Cdyn were all markedly decreased in the BLM-S group compared with the control group(P<0.05,P<0.01)and IC,VC,and Cchord were significantly decreased in the BLM-M group(P<0.05,P<0.01)on day 28.IC,VC,and Cchord were significantly decreased in rats in the BLM-S group on day 42(P<0.05,P<0.01),and were also significantly decreased in rats in the BLM-M group on days 42~84(P<0.05,P<0.01).(3)In terms of lung pathology,inflammatory infiltration and fibrous cords appeared in the BLM-S group from days 28~84 and then gradually decreased(P<0.05,P<0.01),while fibrosis and alveolitis were relatively stable in the BLM-M group(P<0.05,P<0.01).(4)COL-III expression levels in lung tissue were significantly higher in rats in the BLM-S and BLM-M groups compared with the control group(P<0.05,P<0.01),and the COL-Ⅲcontent in the BLM-S group was significantly lower at 42~84 days than at 28 days(P<0.05).Conclusions Both method are capable of effectively creating pulmonary fibrosis models.The single-dose approach is straightforward,has a lower death rate,and the degree of fibrosis is clearly visible by day 28,but progressively recovers after 42 days.In contrast,the two-dose instillation model has a greater success rate and better stability,with over half the rats still exhibiting visible fibrosis on day 84.