Leptin对缺氧诱导胎肺Ⅱ型上皮细胞凋亡的影响及机制

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5 mmol/L连二亚硫酸钠（Na₂S₂O₄）培养液培养AECⅡ细胞12 h建立缺氧诱导细胞凋亡模型，处理组含不同浓度LEP（100-1 600 μg/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caspase 3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；5 mmol/L Na₂S₂O₄能诱导AECⅡ细胞凋亡和caspase 3活化，LEP（100-1 600 μg/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caspase 3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caspase 3活化有关。

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

AIM: To investigate the effects of leptin (LEP) on the alveolar type Ⅱ cells(AECⅡ) apoptosis induced by Na₂S₂O₄ and explore the molecular mechanisms. METHODS: Primary AECⅡ culture was prepared according to a specific immunosorption procedure with slight modification and the cells were identified by transmission electron microscope and immunocytochemistry. AECⅡ 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 (MTT) assays. Cell cycle and apoptosis were analyzed by flow cytometry and the level of caspase-3 was measured by Western blotting. RESULTS: Highly purified AECⅡ, 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 Na₂S₂O₄, 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 AECⅡ 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.