中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2014年
24期
1899-1904
,共6页
郭丰%王庆祝%秦贵军%周英旎%吴丽娜%刘飞
郭豐%王慶祝%秦貴軍%週英旎%吳麗娜%劉飛
곽봉%왕경축%진귀군%주영니%오려나%류비
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
扠頭轉錄因子類%腎小毬繫膜細胞%細胞外基質%轉化生長因子β1%慢病毒載體
차두전록인자류%신소구계막세포%세포외기질%전화생장인자β1%만병독재체
Forkhead transcription factors%Mesangial cells%Extracellular matrix%Transforming growth factor beta1%Lentiviral vectors
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.
目的 研究扠頭轉錄因子O1(FoxO1)過錶達對高糖培養下大鼠腎小毬繫膜細胞(MCs)細胞外基質(ECM)蛋白分泌的影響及其作用機製.方法 以含組成性激活突變型(CA)FoxO1編碼序列的慢病毒載體(LV-CA-FoxO1)及空慢病毒載體(LV-NC-GFP)轉染MCs.實驗分4組:正常糖(5.6 mmol/L)組(NG組),高糖(25 mmol/L)組(HG組)、高糖+LV-CA-FoxO1組(LV-CA組)、高糖+空病毒載體組(LV-NC組).各組培養72 h後,實時熒光定量PCR(Real-timePCR)檢測FoxO1、纖維連接蛋白(FN)、Ⅰ型膠原蛋白(Col Ⅰ)、Ⅳ型膠原(ColⅣ)、轉化生長因子β1(TGF-β1)和TGF-β Ⅰ型及Ⅱ型受體(TGF-βR Ⅰ/Ⅱ)mRNA的錶達水平;Western印跡法檢測FoxO1、燐痠化FoxO1(p-FoxO1)、TGF-β1、TGF-βRⅠ/Ⅱ的蛋白錶達;細胞免疫熒光化學檢測TGF-βR Ⅰ在細胞內錶達分佈.結果 (1) FoxO1錶達及活性改變:HG組FoxO1 mRNA及蛋白錶達與NG組相比差異均無統計學意義(均P ＞0.05),p-FoxO1蛋白錶達高于NG組(P＜0.05);與HG組相比,LV-CA組FoxO1mRNA與蛋白錶達水平均高于HG組(mRNA:為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組(P＜0.05),FoxO1錶達增加,轉錄活性增彊.(2) ECM蛋白分泌改變:HG組FN、Col Ⅰ及ColⅣmRNA錶達高于NG組(均P＜0.05),LV-CA組FN、Col Ⅰ及ColⅣ錶達低于HG組(均P＜0.05).(3) TGF-β傳導通路激活改變:HG組TGF-β1、TGF-βRⅠ/Ⅱ錶達均高于NG組(均P＜0.05),而LV-CA組上述指標均低于HG組(均P＜0.05);細胞免疫熒光化學結果提示,LV-CA組TGF-βR Ⅰ在細胞內分佈與HG組相比減少.LV-NC組上述指標與HG組相比差異均無統計學意義(均P＞0.05).結論 FoxO1能夠抑製TGF-β傳導通路的激活,從而減少MCs在高糖誘導下過度分泌ECM蛋白.
목적 연구차두전록인자O1(FoxO1)과표체대고당배양하대서신소구계막세포(MCs)세포외기질(ECM)단백분비적영향급기작용궤제.방법 이함조성성격활돌변형(CA)FoxO1편마서렬적만병독재체(LV-CA-FoxO1)급공만병독재체(LV-NC-GFP)전염MCs.실험분4조:정상당(5.6 mmol/L)조(NG조),고당(25 mmol/L)조(HG조)、고당+LV-CA-FoxO1조(LV-CA조)、고당+공병독재체조(LV-NC조).각조배양72 h후,실시형광정량PCR(Real-timePCR)검측FoxO1、섬유련접단백(FN)、Ⅰ형효원단백(Col Ⅰ)、Ⅳ형효원(ColⅣ)、전화생장인자β1(TGF-β1)화TGF-β Ⅰ형급Ⅱ형수체(TGF-βR Ⅰ/Ⅱ)mRNA적표체수평;Western인적법검측FoxO1、린산화FoxO1(p-FoxO1)、TGF-β1、TGF-βRⅠ/Ⅱ적단백표체;세포면역형광화학검측TGF-βR Ⅰ재세포내표체분포.결과 (1) FoxO1표체급활성개변:HG조FoxO1 mRNA급단백표체여NG조상비차이균무통계학의의(균P ＞0.05),p-FoxO1단백표체고우NG조(P＜0.05);여HG조상비,LV-CA조FoxO1mRNA여단백표체수평균고우HG조(mRNA:위17.36±1.13비1.01 ±0.12,단백:4.38±0.09비0.93 ±0.10,균P＜0.05),p-FoxO1/FoxO1저우HG조(P＜0.05),FoxO1표체증가,전록활성증강.(2) ECM단백분비개변:HG조FN、Col Ⅰ급ColⅣmRNA표체고우NG조(균P＜0.05),LV-CA조FN、Col Ⅰ급ColⅣ표체저우HG조(균P＜0.05).(3) TGF-β전도통로격활개변:HG조TGF-β1、TGF-βRⅠ/Ⅱ표체균고우NG조(균P＜0.05),이LV-CA조상술지표균저우HG조(균P＜0.05);세포면역형광화학결과제시,LV-CA조TGF-βR Ⅰ재세포내분포여HG조상비감소.LV-NC조상술지표여HG조상비차이균무통계학의의(균P＞0.05).결론 FoxO1능구억제TGF-β전도통로적격활,종이감소MCs재고당유도하과도분비ECM단백.
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:the expressions of TGF-β1 and TGF-βR Ⅰ / Ⅱboth increased in HG group (all P ＜ 0.05),but decreased in LV-CA group (all P ＜ 0.05).The results of immunofluorescence suggested that the staining of TGF-βR Ⅰ decreased in MCs in LV-CA group versus HG group.All indices of LV-NC group had no statistical difference with those of HG group (all P ＞ 0.05).Conclusion FoxO1 plays a pivotal role in down-regulating the activation of TGF-β pathway and thereby inhibiting the secretion of ECM.
