过表达FoxO1对高糖诱导下大鼠肾小球系膜细胞细胞外基质分泌的影响  

Effects of over-expressed forkhead transcription factor O1 on high glucose induced extracellular matrix secretion in rat mesangial cells

作　　者：郭丰[1] 王庆祝[1] 秦贵军[1] 周英旎 吴丽娜[1] 刘飞[1] 

机构地区：[1]郑州大学第一附属医院内分泌科河南省高等学校临床医学重点学科开放实验室,450052

出　　处：《中华医学杂志》2014年第24期1899-1904,共6页National Medical Journal of China

摘　　要：目的 研究叉头转录因子O1（FoxO1）过表达对高糖培养下大鼠肾小球系膜细胞（MCs）细胞外基质（ECM）蛋白分泌的影响及其作用机制.方法 以含组成性激活突变型（CA）FoxO1编码序列的慢病毒载体（LV-CA-FoxO1）及空慢病毒载体（LV-NC-GFP）转染MCs.实验分4组：正常糖（5.6 mmol/L）组（NG组）,高糖（25 mmol/L）组（HG组）、高糖＋LV-CA-FoxO1组（LV-CA组）、高糖＋空病毒载体组（LV-NC组）.各组培养72 h后,实时荧光定量PCR（Real-timePCR）检测FoxO1、纤维连接蛋白（FN）、Ⅰ型胶原蛋白（Col Ⅰ）、Ⅳ型胶原（ColⅣ）、转化生长因子β1（TGF-β1）和TGF-β Ⅰ型及Ⅱ型受体（TGF-βR Ⅰ/Ⅱ）mRNA的表达水平;Western印迹法检测FoxO1、磷酸化FoxO1（p-FoxO1）、TGF-β1、TGF-βRⅠ/Ⅱ的蛋白表达;细胞免疫荧光化学检测TGF-βR Ⅰ在细胞内表达分布.结果 （1） FoxO1表达及活性改变：HG组FoxO1 mRNA及蛋白表达与NG组相比差异均无统计学意义（均P ＞0.05）,p-FoxO1蛋白表达高于NG组（P＜0.05）;与HG组相比,LV-CA组FoxO1mRNA与蛋白表达水平均高于HG组（mRNA：为17.36±1.13比1.01 ±0.12,蛋白：4.38±0.09比0.93 ±0.10,均P＜0.05）,p-FoxO1/FoxO1低于HG组（P＜0.05）,FoxO1表达增加,转录活性增强.（2） ECM蛋白分泌改变：HG组FN、Col Ⅰ及ColⅣmRNA表达高于NG组（均P＜0.05）,LV-CA组FN、Col Ⅰ及ColⅣ表达低于HG组（均P＜0.05）.（3） TGF-β传导通路激活改变：HG组TGF-β1、TGF-βRⅠ/Ⅱ表达均高于NG组（均P＜0.05）,而LV-CA组上述指标均低于HG组（均P＜0.05）;细胞免疫荧光化学结果提示,LV-CA组TGF-βR Ⅰ在细胞内分布与HG组相比减少.LV-NC组上述指标与HG组相比差异均无统计学意义（均P＞0.05）.结论 FoxO1能够抑制TGF-β传导通路的激活,从而减少MCs在高糖诱导下过度分泌ECM蛋白.Objective To explore the role and molecular mechanism of forkhead transcription factor O1 （FoxO1） for extracellular matrix （ECM） protein accumulation in rat mesangial cells （MCs） cultured under high-glucose conditions.Methods The MCs were transfected with either lentiviral vectors containing the sequences of constitutively active FoxO1 （LV-CA-FoxO1） or empty vectors （LV-NC-GFP）.The MCs were divided into 4 groups of normal glucose （5.6 mmol/L） （NG）,high glucose （25 mmol/L） （HG）,high glucose plus LV-CA-FoxO1 （LV-CA） and high-glucose plus empty lentiviral vectors （LV-NC）.After 72 h treatment,the mRNA levels of MCs were measured through real-time polymerase chain reaction （PCR） in each group,including FoxO1,fibronectin （FN）,collagen Ⅰ （Col Ⅰ）,collagen Ⅳ （Col Ⅳ）,transforming growth factor-β1 （TGF-β1）,TGF-β type Ⅰ and type Ⅱ receptor （TGF-βR Ⅰ / Ⅱ）.The protein levels of FoxO1,phosphorylation FoxO1 （p-FoxO1）,TGF-β1 and TGF-βR Ⅰ / Ⅱ were assessed by Western blot.And the distributions of TGF-βR Ⅰ in MCs were detected by immunofluorescence.Results Expression and bioactivity changes of FoxO1：Compared with NG group,there was no significant difference in either mRNA or protein levels of FoxO1 in HG group （P 〉0.05） whereas the protein levels of p-FoxO1 markedly increased （P 〈0.05）.The levels of FoxO1 mRNA and protein increased in LV-CA group versus HG group （mRNA：17.36 ± 1.13 vs 1.01 ±0.12,protein：4.38 ±0.09 vs 0.93 ±0.10,both P 〈0.05）.And there was a significant decrease of p-FoxO1/FoxO1 ratio （P 〈 0.05）.Thus it suggested an enhancement in FoxO1 expression and transcriptional activity.Changes of ECM protein expression.The mRNA expression levels of FN,Col Ⅰ and Col Ⅳ in HG group were elevated compared with NG group （all P 〈 0.05） while the expressions of these indices in LV-CA group became attenuated compared with HG group （all P 〈 0.05）.Changes of TGF-β pathway activation：th

关 键 词：叉头转录因子类 肾小球系膜细胞 细胞外基质 转化生长因子β1 慢病毒载体 

分 类 号：R363[医药卫生—病理学]