中华糖尿病杂志
中華糖尿病雜誌
중화당뇨병잡지
CHINES JOURNAL OF DLABETES MELLITUS
2012年
7期
425-429
,共5页
张鹏宇%秦贵军%李雪峰%任高飞%刘会苗
張鵬宇%秦貴軍%李雪峰%任高飛%劉會苗
장붕우%진귀군%리설봉%임고비%류회묘
叉头转录因子类%游离脂肪酸%固醇调节元件结合蛋白质1
扠頭轉錄因子類%遊離脂肪痠%固醇調節元件結閤蛋白質1
차두전록인자류%유리지방산%고순조절원건결합단백질1
Forkhead transcription factor O1%Free fatty acid%Sterol regulatory element-binding proteins 1c
目的 研究叉头状转录因子O1(FoxO1)对游离脂肪酸介导的人肝癌细胞株(HepG-2)胰岛素抵抗和脂质堆积的作用以及对固醇调节元件结合蛋白-1c( SREBP-1c) mRNA和蛋白表达的影响.方法 将HepG-2细胞培养后用普通培养基培养为对照组,用含5.0×10-4 mol/L软脂酸的培养基诱导为软脂酸组,诱导后转染空白质粒为空白质粒组,诱导后转染FoxO1 siRNA质粒载体为FoxO1 siRNA载体组.运用实时定量聚合酶链反应(RT-PCR)方法检测FoxO1 mRNA表达,噻唑蓝(MTT)比色法检测细胞增殖,葡萄糖氧化酶法检测培养基中葡萄糖消耗量,油红O染色观察细胞脂质堆积；RT-PCR和Western blot技术分别检测SREBP-1c mRNA表达量以及蛋白的表达量.各组间均值比较采用单因素方差分析,样本间比较采用t检验.结果 软脂酸组较对照组细胞葡萄糖消耗量减少(1.17±0.56 vs 4.31±0.21,t=10.587,P＜0.01)、细胞中的脂质堆积增多、FoxO1 mRNA升高(0.78±0.10 vs0.51±0.12,t =3.629,P ＜0.05)、SREBP-1c mRNA升高(0.71±0.17 vs 0.25±0.08,t =6.290,P＜0.05)、SREBP-1c蛋白升高(0.69±0.10 vs0.41±0.07,t=4.797,P＜0.01).转染FoxO1siRNA质粒载体后葡萄糖消耗量较软脂酸组增加(2.26±0.41 vs 1.17±0.56,t =3.144,P＜0.05),FoxO1 mRNA、SREBP-1c mRNA、SREBP-1c蛋白的表达较软脂酸组均减少且接近于对照组(分别为0.38±0.06 vs 0.78±0.10,t=7.164,P＜0.01;0.45 ±0.13 vs 0.71 ±0.17,t =2.479,P＜0.05;0.41 ±0.06 vs 0.69±0.10,=4.797,P ＜0.01),细胞中的脂质堆积也较软脂酸组减少.结论 抑制FoxO1的表达,可改善游离脂肪酸诱导的细胞胰岛素抵抗、减少肝脏细胞内脂肪变性,其机制可能是通过下调SREBP-1c的表达.
目的 研究扠頭狀轉錄因子O1(FoxO1)對遊離脂肪痠介導的人肝癌細胞株(HepG-2)胰島素牴抗和脂質堆積的作用以及對固醇調節元件結閤蛋白-1c( SREBP-1c) mRNA和蛋白錶達的影響.方法 將HepG-2細胞培養後用普通培養基培養為對照組,用含5.0×10-4 mol/L軟脂痠的培養基誘導為軟脂痠組,誘導後轉染空白質粒為空白質粒組,誘導後轉染FoxO1 siRNA質粒載體為FoxO1 siRNA載體組.運用實時定量聚閤酶鏈反應(RT-PCR)方法檢測FoxO1 mRNA錶達,噻唑藍(MTT)比色法檢測細胞增殖,葡萄糖氧化酶法檢測培養基中葡萄糖消耗量,油紅O染色觀察細胞脂質堆積；RT-PCR和Western blot技術分彆檢測SREBP-1c mRNA錶達量以及蛋白的錶達量.各組間均值比較採用單因素方差分析,樣本間比較採用t檢驗.結果 軟脂痠組較對照組細胞葡萄糖消耗量減少(1.17±0.56 vs 4.31±0.21,t=10.587,P＜0.01)、細胞中的脂質堆積增多、FoxO1 mRNA升高(0.78±0.10 vs0.51±0.12,t =3.629,P ＜0.05)、SREBP-1c mRNA升高(0.71±0.17 vs 0.25±0.08,t =6.290,P＜0.05)、SREBP-1c蛋白升高(0.69±0.10 vs0.41±0.07,t=4.797,P＜0.01).轉染FoxO1siRNA質粒載體後葡萄糖消耗量較軟脂痠組增加(2.26±0.41 vs 1.17±0.56,t =3.144,P＜0.05),FoxO1 mRNA、SREBP-1c mRNA、SREBP-1c蛋白的錶達較軟脂痠組均減少且接近于對照組(分彆為0.38±0.06 vs 0.78±0.10,t=7.164,P＜0.01;0.45 ±0.13 vs 0.71 ±0.17,t =2.479,P＜0.05;0.41 ±0.06 vs 0.69±0.10,=4.797,P ＜0.01),細胞中的脂質堆積也較軟脂痠組減少.結論 抑製FoxO1的錶達,可改善遊離脂肪痠誘導的細胞胰島素牴抗、減少肝髒細胞內脂肪變性,其機製可能是通過下調SREBP-1c的錶達.
목적 연구차두상전록인자O1(FoxO1)대유리지방산개도적인간암세포주(HepG-2)이도소저항화지질퇴적적작용이급대고순조절원건결합단백-1c( SREBP-1c) mRNA화단백표체적영향.방법 장HepG-2세포배양후용보통배양기배양위대조조,용함5.0×10-4 mol/L연지산적배양기유도위연지산조,유도후전염공백질립위공백질립조,유도후전염FoxO1 siRNA질립재체위FoxO1 siRNA재체조.운용실시정량취합매련반응(RT-PCR)방법검측FoxO1 mRNA표체,새서람(MTT)비색법검측세포증식,포도당양화매법검측배양기중포도당소모량,유홍O염색관찰세포지질퇴적；RT-PCR화Western blot기술분별검측SREBP-1c mRNA표체량이급단백적표체량.각조간균치비교채용단인소방차분석,양본간비교채용t검험.결과 연지산조교대조조세포포도당소모량감소(1.17±0.56 vs 4.31±0.21,t=10.587,P＜0.01)、세포중적지질퇴적증다、FoxO1 mRNA승고(0.78±0.10 vs0.51±0.12,t =3.629,P ＜0.05)、SREBP-1c mRNA승고(0.71±0.17 vs 0.25±0.08,t =6.290,P＜0.05)、SREBP-1c단백승고(0.69±0.10 vs0.41±0.07,t=4.797,P＜0.01).전염FoxO1siRNA질립재체후포도당소모량교연지산조증가(2.26±0.41 vs 1.17±0.56,t =3.144,P＜0.05),FoxO1 mRNA、SREBP-1c mRNA、SREBP-1c단백적표체교연지산조균감소차접근우대조조(분별위0.38±0.06 vs 0.78±0.10,t=7.164,P＜0.01;0.45 ±0.13 vs 0.71 ±0.17,t =2.479,P＜0.05;0.41 ±0.06 vs 0.69±0.10,=4.797,P ＜0.01),세포중적지질퇴적야교연지산조감소.결론 억제FoxO1적표체,가개선유리지방산유도적세포이도소저항、감소간장세포내지방변성,기궤제가능시통과하조SREBP-1c적표체.
Objective To study the effects and potential mechanisms of Forkhead transcription factor O1 (FoxO1) on free fatty acid (FFA) induced insulin resistance (IR) and steatosis.Methods HepG-2 cells were induced to a status of insulin resistance by being exposed to 5.0 × 10 4 mmol/L palmitic acid (PA) for 24 hours.HepG-2 cells were devided into 4 groups,the HepG-2 cell group cultured with normal medium,the palmitic acid group,the blank plasmid group,and the siFoxO1 group.PA was added to normal medium to a final concentration of 5.0 × 10 -4 mol/L.The expression levels of FoxO1 in different groups were detected by RT-PCR.The glucose consumption was detected by using glucose oxidase method.MTT method was used to detect the proliferation of HepG-2 cells.Steatosis of HepG-2 cells was observed by Oil Red O staining.The mRNA and protein expression of SREBP-1c were determined by RT-PCR and Western blot.Results Compared with control,the glucose consumption of cells cultured with FFA was significantly reduced( 1.17 ±0.56 vs 4.31 ±0.21,t =10.587,P ＜0.01 ).Cellular lipid accumulation,the expressions of FoxO1 mRNA ( 0.78 ± 0.10 vs 0.51 ± 0.12,t =3.629,P ＜ 0.05 ),SREBP-1c mRNA (0.71 ±0.17 vs 0.25 ±0.08,t =6.290,P ＜0.05),and the protein of SREBP-1c (0.69 ±0.10 vs 0.41 ± 0.07,t =4.797,P ＜0.01 )was increased.After transfected by siFoxO1 plasmids using Lipofectamine 2000,the mRNA expression of FoxO1 (0.38 ± 0.06 vs 0.78 ± 0.1 0,t =7.164,P ＜ 0.01 ),SREB9-1 c (0.45 ±0.13 vs 0.71 ±0.17,t =2.479,P ＜0.05) and the SREBP-1 c protein expression (0.41 ±0.06 vs 0.69 ±0.10,t =4.797,P ＜0.01 ) were significantly decreased after tranfected by siFoxO1 plasminds,whereas the glucose consumption obviously the glucose consumption obviously increased ( 2.26 ± 0.41 vs 1.17 ± 0.56,t=3.144,P ＜0.05).The cellular lipod accumulation was decrease after transfected by siFoxO1.There was no remarkable difference between the palmitic acid group,and the blank plasmid group. Conclusions Inhibiting the expression of FoxO1 could improve FFA-induced IR and steatosis by down-regulating SREBP1c expression.
