Leptin对缺氧诱导胎肺Ⅱ型上皮细胞凋亡的影响及机制

Effects of leptin on hypoxia-induced apoptosis in cultured alveolar typeⅡ cells of fetal rat and its mechanism

目的:观察瘦素(LEP)对缺氧诱导胎鼠肺Ⅱ型上皮细胞(AECⅡ)凋亡的拮抗作用,并探讨其机制。方法:采用改良免疫黏附法原代培养胎鼠AECⅡ细胞,并用SP-A免疫细胞化学法和透射电镜进行鉴定;用含5mmol/L连二亚硫酸钠(Na2S2O4)培养液培养AECⅡ细胞12h建立缺氧诱导细胞凋亡模型,处理组含不同浓度LEP(100-1600μg/L);噻唑兰(MTT)比色法检测细胞存活情况;流式细胞术分析细胞凋亡和细胞周期;蛋白质免疫印迹法(Western blotting)检测凋亡蛋白caspase3的表达。结果:采用免疫黏附法获得高纯度原代培养的AECⅡ细胞,免疫细胞化学法示SP-A阳性表达,电镜下可见细胞内特征性板层小体;5mmol/LNa2S2O4能诱导AECⅡ细胞凋亡和caspase3活化,LEP(100-1600μg/L)能减轻Na2S2O4所致的细胞损伤,表现为AECⅡ存活率提高、增殖指数(PI)增高及凋亡峰下降、细胞形态恢复和caspase3活化受抑制。结论:LEP可拮抗缺氧所致AECⅡ细胞凋亡,这可能与其促使细胞周期从G1期进入S期及抑制凋亡蛋白caspase3活化有关。

AIM： To investigate the effects of leptin （LEP） on the alveolar type Ⅱ cells （AEC Ⅱ ） apoptosis induced by Na2 S2O4 and explore the molecular mechanisms, METHODS： Primary AEC Ⅱ culture was prepared according to a specific immunosorption procedure with slight modification and the cells were identified by transmission electron microscope and immunocytochemistry. AEC Ⅱ damage was induced by 5 mmol/L Na2S2O4. LEP group cells were treated with LEP at concentrations from 100 μg/L to 1 600 μg/L. The cell survival rate was evaluated by 3 - （4,5 - dimethylthiazol - 2 -yl） -2,5 -diphenyltetrazolium bromide （MTY） assays. Cell cycle and apoptosis were analyzed by flow cytometry and the level of caspase - 3 was measured by Western blotting. RESULTS ： Highly purified AEC Ⅱ , obtained by the method of modified immunosorption, were identified with the positive expression of SP - A and intracellular lamellarbodies were found under electron micrography. The cells, exposed to 5 mmol/L Na2 S2O4, showed characteristic changes of apoptosis and activation of caspase 3. These damages were relieved by the treatment of LEP （ 100 - 1 600 μg/L）, with survival increasing, apoptosis peak decreasing, cell morphology restoring and caspase 3 activation inhibiting. CONCLUSION： Leptin prevents AEC Ⅱ from apoptosis induced by Na2S2O4 or hypoxia. The potential mechanism of its action may be related to promoting cell cycle from G1 phase to S phase and inhibiting the activating of caspase 3.