肺纤维化大鼠模型造模方法的优化

Optimization of modeling method for pulmonary fibrosis rat model

目的 比较气管内滴注、气管内雾化喷入博来霉素(5 mg/kg)两种给药方式,以及比较腹腔注射3%戊巴比妥钠麻醉、异氟烷呼吸麻醉(与氧气混合吸入时,异氟烷浓度为0.5%)两种麻醉方式对肺纤维化大鼠模型的影响,探讨更优的造模方法。方法 选择雄性SPF级SD大鼠50只,随机分为空白对照组、腹腔麻醉气管内滴注组、腹腔麻醉气管内雾化喷入组、呼吸麻醉气管内滴注组和呼吸麻醉气管内雾化喷入组,每组各10只。观察给药后1、3、7、14、21 d,各组大鼠的生存状况及体重变化;给药3周后处死大鼠,取肺称重,计算肺系数;HE染色观察肺组织炎症变化;Masson染色观察肺组织中胶原增殖;Western Blot检测肺组织中转化生长因子-β1(TGF-β1)蛋白表达量;碱水解法检测肺组织中羟脯氨酸(HYP)的含量。结果 与空白对照组比,4种模型组的大鼠精神状态不佳、体重下降、肺指数上升;肺组织损伤明显,炎症水平增加,胶原增殖显著;肺组织中TGF-β1蛋白表达水平升高(P<0.001);发现仅气管内雾化喷入博来霉素组大鼠肺组织中羟脯氨酸的含量增加(P<0.05)。发现气管内滴注的建模方式其模型大鼠肺部纤维化病灶分布不均匀,纤维化程度不一。麻醉程度均为中麻醉的情况下,异氟烷呼吸麻醉(与氧气混合吸入时,异氟烷浓度为0.5%)的大鼠平均苏醒时间和死亡率均低于腹腔注射3%戊巴比妥钠麻醉方式。结论 异氟烷呼吸麻醉下气管内雾化喷入博来霉素是建立肺纤维化大鼠模型的优选造模方法。

Objective To compare the effects of intra⁃tracheal drip and intratracheal nebulized spray of bleomycin(5 mg/kg)on the rat model of pulmonary fibrosis,and to compare the effects of intraperitoneal 3%pentobarbital sodium anesthesia and isoflurane respiratory anesthesia(isoflurane concentration of 0.5%when mixed with oxygen for inhalation)on the rat model of pulmonary fibrosis,and to explore a more optimal modeling method.Methods Fifty male SPF⁃grade SD rats were randomly divided into blank control group,intraperitoneal anesthetic intratracheal drip group,intraperitoneal anesthetic intratracheal nebulizer spray group,respiratory anesthetic intratracheal drip group and respiratory anesthetic intratracheal nebulizer spray group,10 rats in each group.The survival status and body weight of rats in each group were observed at 1,3,7,14 and 21 d after drug administration;rats were executed 3 weeks after drug administration,and lungs were weighed to calculate lung coefficients;HE staining was performed to observe inflammatory changes in lung tissues;Masson staining was performed to observe collagen proliferation in lung tissues;Western Blot was performed to detect transforming growth factor⁃β1(TGF⁃β1)protein expression in lung tissues;alkali hydrolysis was performed to detect hydroxyproline(HYP)in lung tissues.Results Compared with the blank control group,the rats in the four model groups had poor mental status,decreased body weight,and increased lung index;lung tissue damage was evident,increased inflammation levels,and significant collagen proliferation;increased TGF⁃β1 protein expression levels in lung tissues(P<0.001);and increased hydroxyproline levels in lung tissues of rats in the intratracheal nebulizer sprayed with bleomycin only group were found(P<0.05).The modeling approach of intratracheal drip injection was found to have a heterogeneous distribution of fibrotic lesions in the lungs of its model rats,with varying degrees of fibrosis.The mean time to awakening and mortality rates were lower in rats under isoflurane respiratory anesthesia(0.5%isoflurane concentration when mixed with oxygen for inhalation)than in the intraperitoneal 3%pentobarbital sodium anesthesia modality when both anesthesia levels were medium.Conclusion Intratracheal nebulized spray of bleomycin under isoflurane respiratory anesthesia is the preferred modeling method for establishing a rat model of pulmonary fibrosis.