摘要
目的 研究胰岛素作用下肺泡上皮细胞钠离子通道α亚基（ENaC-α）的变化及其对细胞凋亡的影响.方法 以人肺腺癌细胞株A549为对象,用含30 U/L的胰岛素培养基培养30 min和60 min,分别用蛋白质免疫印迹法（Western blotting）和逆转录-聚合酶链反应（RT-PCR）法测定ENaC-α及血清和糖皮质激素诱导的蛋白激酶1（SGK-1）的蛋白及mRNA表达,并使用白屈菜红碱阻断蛋白激酶C（PKC）抑制SGK-1后再次测定ENaC-α蛋白表达.用原位末端缺刻标记法（TUNEL）测定经胰岛素处理后的A549细胞凋亡率.结果 用胰岛素培养30 min即可明显上调ENaC-α和SGK-1的蛋白及mRNA表达,并随处理时间延长表达量明显增加.若以未添加胰岛素培养A549细胞蛋白表达量为100%,胰岛素作用30 min时ENaC-α、SGK-1蛋白表达量为124%、135%,60 min时ENaC-α、SGK-1蛋白表达量为186%、176%（均P〈0.05）.胰岛素还可以抑制缺氧诱导的A549细胞凋亡（10.3%比21.6%,P〈0.05）.结论 胰岛素可以通过PKC、SGK-1途径上调ENaC-α表达,并抑制缺氧诱导的A549细胞凋亡,从而有利于急性肺损伤/急性呼吸窘迫综合征（ALI/ARDS）的预后.
Objective To observe the effects of insulin on the expression of epithelial sodium channel α subunit （ENaC-α） in human pulmonary adenocarcinoma cell line （A549 cells） and its impact on apoptosis of the cells. Methods A549 cells were cultured with insulin in a concentration of 30 U/L for 30 minutes or 60 minutes. Then ENaC-α, serum and glucocorticoid-inducible kinase-1 （SGK-1） protein and mRNA were determined with Western blotting and reverse transcription-polymerase chain reaction （RT-PCR）. Then chelidonii, a blocking agent of protein kinase C （PKC）, which could inhibit the effect of SGK-1, was added to the cells culture for 60 minutes, and ENaC-α protein was then determined. Cells were cultured in a hypoxia circumstance for 30 minutes, to induce apoptosis, the rate of which was detected with terminal-deoxynucleotidyl transferase mediated dUTP nick end labeling （TUNEL）, in cells cultured with or without insulin. Results Insulin could up-regulate the protein and mRNA expression of ENaC-α and SGK-1 in A549 cells, and there was a significant difference at 30 minutes, and it gradually increased with prolongation of time. When A549 cells were cultured without insulin, the protein expression of A549 cells was 100%. The protein expression of ENaC-α and SGK-1 was 124% and 135% at 30 minutes after being cultured with insulin, and 186% and 176% at 60 minutes, respectively （all P〈0.05）. Insulin also could inhibit apoptosis which was induced by hypoxia （10.3% vs. 21.6%, P〈0.05）. Conclusion Insulin can up-regulate expression of the ENaC-α in A549 cells via PKC and SGK-1 pathways, also insulin can inhibit hypoxia induced apoptosis. So insulin can be beneficial in the prognosis of acute lung injury/acute respiratory distress syndrome （ALI/ARDS）.
出处
《中国危重病急救医学》
CAS
CSCD
北大核心
2010年第7期385-388,I0001,共5页
Chinese Critical Care Medicine
基金
国家自然科学基金项目（30971303）
