年龄因素对小鼠肺纤维化模型端粒酶逆转录酶活性影响的研究

摘要

Objective To explore the effect of age factor on the degree of pulmonary fibrosis and on the change in telomerase reverse transcriptase(TERT) activity in mice.Methods A total of 80 healthy male C57BL/6 mice aged 20-weeks were randomized into 4 groups:a young pulmonary fibrosis model group(n=20),a young control group(n=20),a senile pulmonary fibrosis model group(n=20),and an elderly control group (n =20).Two model groups were induced by an intratracheally injected bleomycin in 5 mg/kg,and two control groups by an intratracheally injected normal saline.The elderly were defined as 26 weeks old mice.Five mouse pulmonary fibrosis models and five mouse controls in four groups were randomly selected and killed at 7,14,21,28 days.Lung tissue specimens were stained with hematoxylin eosin (HE)and Masson trichrome (Masson)method,and then pathological changes were observed.In addition,immunohisto-chemical staining was used to observe the expression levels of epithelial cell marker protein E-cadherin(E-cad),stromal cell marker protein Vimentin,and alpha smooth muscle actin(α-SMA).Finally,the expression level of telomerase reverse transcriptase(TERT)protein was detected by Western blotting.Results A bleomycin-induced pulmonary fibrosis mice model was successfully prepared.The degree of pulmonary fibrosis was more severe in old mice than in young mice.Compared with the young pulmonary fibrosis model group,the expression level of E-Cad was decreased in the senile pulmonary fibrosis model group(P ＜0.05).Compared with the young control group and the elderly control group,the expression levels of E-Cad in the young pulmonary fibrosis model group and the senile pulmonary fibrosis model group were decreased (P ＜ 0.05),and decreased along with the prolongation of the modeling time.Compared with the young pulmonary fibrosis model group,the expression levels of alpha-SMA and vimentin were increased in the senile pulmonary fibrosis model group(P ＜ 0.05).The expression levels of alpha-SMA and vimentin were increased in the young pulmonary fibrosis model group and the senile pulmonary fibrosis model group,as compared to the young control group and the elderly control group(P＜0.05),and increased along with the prolongation of the modeling time.The activity of TERT in lung tissue of mice was increased at first and then decreased.Compared with the young pulmonary fibrosis model group,the activity of TERT in the senile pulmonary fibrosis model group significantly fluctuated(P＜0.05).Conclusions Age factor can affect the severity of pulmonary fibrosis by affecting the activity of telomerase reverse transcriptase in mouse models of pulmonary fibrosis.%目的 研究年龄因素对小鼠肺纤维化程度及端粒酶逆转录酶(TERT)活性变化的影响. 方法 20周龄健康雄性C57BL/6小鼠共80只随机分为4组,青年模型组,青年对照组,老年模型组,老年对照组,每组20只.青年对照组气管内注入生理盐水;青年模型组向气管内注射博来霉素(BLM,5mg/kg),老年对照组和老年模型组将小鼠养到26周龄后分别气管内注入生理盐水和BLM(5 mg/kg).各组小鼠于造模后的第7、14、21、28天分别随机抽取5只处死,留取肺组织,HE染色法和Masson染色法观察肺组织病理变化.免疫组化染色观察肺组织上皮细胞标志蛋白E-钙黏蛋白(E-Cad)及间质细胞标志蛋白波形蛋白(Vimentin)、α-平滑肌肌动蛋白(α-SMA)表达.免疫印迹法(Western-bloting)检测各组小鼠TERT蛋白的表达. 结果 气管注入BLM诱导小鼠肺纤维化模型成功.老年小鼠肺纤维化病变程度较青年小鼠严重.相对于青年模型组,E-Cad在老年模型组中表达降低(P＜0.05),相对于青年对照组与老年对照组,E-Cad在青年模型组与老年模型组表达降低(P＜0.05),且随造模时间延长而降低.相对于青年模型组,a-SMA、Vimentin在老年模型组中表达增高(P＜0.05),相对于青年对照组与老年对照组,α-SMA、Vimentin在青年模型组与老年模型组表达增高(P＜0.05),且随造模时间延长而增高.小鼠肺组织TERT的活性先升高再降低.相对于青年模型组小鼠,老年模型组小鼠肺组织TERT变化波动大(P＜0.05). 结论 年龄因素可通过影响肺纤维化小鼠的TERT活性进一步影响肺纤维化病变程度.