摘要
目的研究博莱霉素(BLM)对肺纤维化大鼠Ⅱ型肺泡上皮细胞(ATⅡ)凋亡的影响及其可能机制。方法32只雄性Sprague-Dawley大鼠随机分为假手术组(n=8)和BLM组(n=24),分别给予BLM组和假手术组大鼠气管内一次性注入5mg/kgBLM和等量生理盐水。于给药后1、3、7d随机取BLM组大鼠8只,给药后7d取假手术组大鼠进行ATⅡ分离和纯化。采用流式细胞术进行细胞周期、细胞凋亡率、细胞内游离Ca2+浓度、线粒体跨膜电位(MMP)检测;免疫细胞化学法检测ATⅡBax、Bcl-2和Fas的表达;比色法测定ATⅡCaspase-3、Caspase-8和Caspase-9的活性。结果BLM组ATⅡS期细胞比率显著低于假手术组(P<0.05)。BLM组1和3d时ATⅡ的凋亡率显著高于假手术组(P<0.01)。BLM组1、3和7d时ATⅡ内游离Ca2+浓度显著高于假手术组(P<0.05),MMP显著低于假手术组(P<0.05)。BLM组1、3和7d时ATⅡBax和Fas染色阳性率和Caspase-3、Caspase-8、Caspase-9的活性均显著高于假手术组(P<0.05,P<0.01)。BLM组1和3d时ATⅡBcl-2染色阳性率显著低于假手术组(P<0.05)。结论BLM在ATⅡ损伤早期可能通过升高细胞内Ca2+浓度,激活Caspase-3、Caspase-8和Caspase-9,降低MMP,诱导Fas、Bax高表达,促使ATⅡ凋亡,引发肺纤维化。
Objective To investigate the effects of bleomycin ( BLM ) on the apoptosis of typeⅡ alveolar epithelial cell ( AT Ⅱ ) in lung fibrotic rats and its possible mechanisms. Methods Totally 32 male Sprague-Dawley rats were randomly divided into sham group ( n = 8 ) and BLM group ( n = 24 ). Rats in sham group or BLM group were intratracheally instillated with saline or 5 mg/kg of bleomycin, respectively. One, three, and seven days after the instillation of bleomycin, 8 rats in BLM group were taken for AT Ⅱ isolation and purification. Rats in sham group were used to isolate and purify AT Ⅱ on 7 days after the instillation of saline. The cell cycle and apoptosis, intracellular free calcium concentration, and mitochondrial membrane potential (MMP) in AT Ⅱ were determined by flow cytometry. Immunohistochemistry was performed to observe the expressions of Bax, Bcl-2, and Fas. Caspase-3, Caspase-8, and Caspase-9 activities were measured by Caspase activity detection kit. Results The ratio of S phase AT Ⅱ in BLM group was significantly lower than in sham group ( P 〈 0.05 ). AT Ⅱ apoptosis rates on day 1 and 3 were significantly higher in BLM group than in sham group ( P 〈 0. 01 ). Intracellular free calcium concentrations in BLM group were significantly higher than in sham group ( P 〈 0.05 ). However, MMP was significantly lower than sham group ( P 〈 0. 05 ). The positive rates of Bax, Fas and Caspase-3, Caspase-8, and Caspase-9 activities of BLM group were significantly higher than those of sham group ( P 〈 0.05, P 〈 0.01 ). The positive rates of Bcl-2 on day 1 and 3 were significantly lower than those of sham group ( P 〈 0.05 ). Conclusion Early AT Ⅱ apoptosis may be induced by bleomycin, which may be explained by the increase of intracellular free calcium concentration, depression of MMP, increased expressions of Fas and Bax, and increase of Caspase-3, Caspase-8, and Caspase-9 activities.
出处
《中国医学科学院学报》
CAS
CSCD
北大核心
2007年第6期782-786,I0005,共6页
Acta Academiae Medicinae Sinicae
基金
国家自然科学基金(30130220)
北京市中医局资助项目(JJ-2006-24)~~
关键词
肺纤维化
博莱霉素
Ⅱ型肺泡上皮细胞
凋亡
pulmonary fibrosis
bleomycin
alveolar epithelial cell type Ⅱ
apoptosis