地塞米松通过稳定磷脂酰肌醇3－激酶/蛋白激酶B分子信号通路对嘌呤霉素诱导足细胞凋亡的抑制作用  

Inhibitory of Dexamethasone on podocytes apoptosis induced by Puromycin via stabilizing phosphatidylinositol 3 kinase/protein kinase B signaling pathway

作　　者：何绍平[1] 于力[1] 于生友[1] 郝志宏[1] 张瑶[1] 

机构地区：[1]广州医科大学附属广州市第一人民医院儿科,510180

出　　处：《中华实用儿科临床杂志》2017年第9期677-681,共5页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金（81670652,81273205）;广东省科技计划项目（2016A020215010）

摘　　要：目的探讨磷脂酰肌醇3－激酶（PI3K）/蛋白激酶B（Akt）分子信号通路在地塞米松（DEX）抑制嘌呤霉素（PAN）引起足细胞凋亡中的作用。方法体外培养小鼠肾小球足细胞系，并设立对照组、溶媒溶剂二甲基亚砜（DMSO）组、PAN组、DEX组及PI3K特异性抑制剂LY294002组。采用实时荧光定量PCR、间接免疫荧光染色检测CD2相关蛋白（CD2AP）mRNA表达水平及细胞内分布情况，激光共聚焦检测其与p85的共定位分布的情况；Western blot检测Akt、磷酸化Akt（p－Akt）、糖原合成酶激酶－3β（GSK3β）和磷酸化GSK3β（p－GSK3β）的表达。结果PAN组各时间点（8 h、24 h、48 h） CD2AP mRNA （1.11±0.16、0.78±0.09、0.56±0.43）显著低于对照组（1.90±0.26、2.09±0.12、2.28±0.95），差异均有统计学意义（均P〈0.05），CD2AP蛋白呈均匀细丝状分布于胞质足突并在细胞核内转变为不连续粗颗粒状集中分布于核周，CD2AP与p85在细胞核膜、细胞质及细胞膜均匀的重叠转变为核内增多的强荧光信号的重叠；DEX组各时间点（8 h、24 h、48 h） CD2AP mRNA（1.53±0.14、2.15±0.27、2.13±0.15）显著高于PAN组，差异均有统计学意义（均P〈0.05），CD2AP蛋白在细胞膜、胞质的分布密度、分布范围大于PAN组，与p85其重叠荧光信号向核内积聚明显减轻；PAN刺激足细胞15 min后p－Akt蛋白表达水平最低，p－GSK3β蛋白表达水平在30 min最低，p－Akt及p－GSK3β蛋白表达水平呈PAN浓度依赖性下降（P〈0.05），不同浓度DEX干预后p－Akt及p－GSK3β蛋白表达水平均呈DEX浓度依赖性地恢复（P〈0.05），LY294002组p－Akt及p－GSK3β的表达则显著下降（P〈0.01）。结论DEX通过稳定CD2AP的表达水平和分布情况来保护足细胞，抑制足细胞凋亡；PI3K/Akt分子信号通路稳定的表达水平是DEX保护足细胞的关键因素。ObjectiveTo investigate the role of phosphatidylinositol 3 kinase （PI3K）/protein kinase B （Akt） signaling pathway in Dexamethasone （DEX） inhibiting podocytes apoptosis which was induced by Puromycin（PAN）.MethodsMouse glomerular podocytes were cultured in vitro, and were divided into control group, dimethyl sulfoxide（DMSO） group, PAN group, DEX group, and LY294002 （inhibitor of PI3K） group.The mRNA expression of CD2-associated protein （CD2AP） was measured by using real time fluorescent quantitative polymerase chain reaction, and intracellular distribution was detected by using indirect immunofluorescence staining.Co localization of CD2AP and p85 was detected by using confocal fluorescence microscopy.The expressions of Akt, phosphorylated（p）-Akt, glycogen synthase kinase-3β （GSK3β） and phosphorylated（p）-GSK3β were evaluated by using Western blot.ResultsThe expressions of CD2AP mRNA in PAN group at each time point （8 h, 24 h, 48 h） （1.11±0.16, 0.78±0.09, 0.56±0.43） were significantly lower than those in the control group （1.90±0.26, 2.09±0.12, 2.28±0.95）, and the differences were statistically significant （all P〈0.05）; CD2AP distributed in foot process with uniform filament and discontinuous coarse particle around perinuclear; CD2AP and p85 distributed in cell membrane and cytoplasm evenly in control group, but accumulated in nuclei in the PAN group.The expressions of CD2AP mRNA in DEX group at each time point （8 h, 24 h, 48 h） （1.53±0.14, 2.15±0.27, 2.13±0.15） were significantly higher than those in the PAN group, and the differences were also statistically significant （all P〈0.05）; the distribution density and range of CD2AP were greater than those in the PAN group, and the accumulation with p85 in nuclei decreased obviously.The expressions of p-Akt and p-GSK3β were inhibited by PAN in a dose-dependent manner （P〈0.05）. The expressions of p-Akt and p-GSK3β were lowest after PAN stimulated at 15 min and 30 min respectively.Ho

关 键 词：足细胞 CD2相关蛋白 磷脂酰肌醇3激酶/蛋白激酶B 地塞米松 

分 类 号：R726.9[医药卫生—儿科]