中华糖尿病杂志
中華糖尿病雜誌
중화당뇨병잡지
CHINES JOURNAL OF DLABETES MELLITUS
2014年
12期
881-886
,共6页
李雪梅%翟丽东%王鹏华%于佩%于德民
李雪梅%翟麗東%王鵬華%于珮%于德民
리설매%적려동%왕붕화%우패%우덕민
慢病毒%Chemerin%过表达%3T3-L1脂肪细胞%葡萄糖消耗
慢病毒%Chemerin%過錶達%3T3-L1脂肪細胞%葡萄糖消耗
만병독%Chemerin%과표체%3T3-L1지방세포%포도당소모
Lentivirus%Chemerin%Over expression%3T3-L1 adipocytes%Glucose consumption
目的 应用重组慢病毒构建3T3-L1脂肪细胞chemerin过表达模型并进一步探讨其对糖代谢的影响及可能机制.方法 构建鼠chemerin过表达重组慢病毒,并设对照慢病毒,感染3T3-L1细胞,实时定量聚合酶链反应(RT-PCR)法检测转染后chemerin表达水平;应用胰岛素、3-异丁基1-甲基黄嘌呤、地塞米松诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞,油红O染色鉴定;诱导分化第8天加入慢病毒重组体,继续培养5d,葡萄糖氧化酶法检测各组葡萄糖消耗;RT-PCR法检测各组胰岛素受体底物1(IRS1)、胰岛素受体底物2(IRS2)、蛋白激酶B1 (Akt1)、叉头状转录因子O1 (FoxO1)基因表达水平;Western-blotting检测chemerin、丝氨酸/苏氨酸蛋白激酶(Akt)、磷酸化丝氨酸/苏氨酸蛋白激酶(pAkt)、FoxO1、磷酸化叉头状转录因子O1 (pFoxO1)蛋白水平.两组数据比较应用t检验.结果 Chemerin过表达慢病毒感染3T3-L1细胞72 h后细胞中可见红色荧光,RT-PCR结果显示:过表达组与空载对照组相比chemerin基因表达明显增加(分别为3.04±0.19比1.01±0.11,t=15.65,P＜0.05);chemerin过表达组葡萄糖消耗减少[分别为(3.30± 1.44)比(6.07±1.15) mmol/L,t=-0.35,P＜0.05];RT-PCR结果显示:IRS1、IRS2基因水平无明显变化(均P＞0.05),Akt1基因表达下降(分别为0.76±0.08比1.07±0.15,t=-3.11,P＜0.05),FoxO1基因表达上调(分别为1.53±0.30与1.03±0.21,t=2.34,P＜0.05).Western-blotting结果显示:Chemerin过表达后chemerin蛋白水平增加(相对表达量分别为1.08±0.06比0.72±0.03,t=-10.12;P＜0.05);Akt、pAkt蛋白水平均降低(分别为0.74±0.21比1.23±0.20,0.58±0.17比0.92±0.07;t=2.81、3.17,均P＜0.05),FoxO1蛋白水平升高(分别为1.04±0.09比0.76±0.14,t=-2.91,P＜0.05)、pFoxO1蛋白水平降低(分别为0.61±0.13比0.89±0.10,t=2.93,P＜0.05).结论 Chemerin可能通过下调Akt1 mRNA使3T3-L1脂肪细胞葡萄糖消耗减少.
目的 應用重組慢病毒構建3T3-L1脂肪細胞chemerin過錶達模型併進一步探討其對糖代謝的影響及可能機製.方法 構建鼠chemerin過錶達重組慢病毒,併設對照慢病毒,感染3T3-L1細胞,實時定量聚閤酶鏈反應(RT-PCR)法檢測轉染後chemerin錶達水平;應用胰島素、3-異丁基1-甲基黃嘌呤、地塞米鬆誘導3T3-L1前脂肪細胞分化為成熟脂肪細胞,油紅O染色鑒定;誘導分化第8天加入慢病毒重組體,繼續培養5d,葡萄糖氧化酶法檢測各組葡萄糖消耗;RT-PCR法檢測各組胰島素受體底物1(IRS1)、胰島素受體底物2(IRS2)、蛋白激酶B1 (Akt1)、扠頭狀轉錄因子O1 (FoxO1)基因錶達水平;Western-blotting檢測chemerin、絲氨痠/囌氨痠蛋白激酶(Akt)、燐痠化絲氨痠/囌氨痠蛋白激酶(pAkt)、FoxO1、燐痠化扠頭狀轉錄因子O1 (pFoxO1)蛋白水平.兩組數據比較應用t檢驗.結果 Chemerin過錶達慢病毒感染3T3-L1細胞72 h後細胞中可見紅色熒光,RT-PCR結果顯示:過錶達組與空載對照組相比chemerin基因錶達明顯增加(分彆為3.04±0.19比1.01±0.11,t=15.65,P＜0.05);chemerin過錶達組葡萄糖消耗減少[分彆為(3.30± 1.44)比(6.07±1.15) mmol/L,t=-0.35,P＜0.05];RT-PCR結果顯示:IRS1、IRS2基因水平無明顯變化(均P＞0.05),Akt1基因錶達下降(分彆為0.76±0.08比1.07±0.15,t=-3.11,P＜0.05),FoxO1基因錶達上調(分彆為1.53±0.30與1.03±0.21,t=2.34,P＜0.05).Western-blotting結果顯示:Chemerin過錶達後chemerin蛋白水平增加(相對錶達量分彆為1.08±0.06比0.72±0.03,t=-10.12;P＜0.05);Akt、pAkt蛋白水平均降低(分彆為0.74±0.21比1.23±0.20,0.58±0.17比0.92±0.07;t=2.81、3.17,均P＜0.05),FoxO1蛋白水平升高(分彆為1.04±0.09比0.76±0.14,t=-2.91,P＜0.05)、pFoxO1蛋白水平降低(分彆為0.61±0.13比0.89±0.10,t=2.93,P＜0.05).結論 Chemerin可能通過下調Akt1 mRNA使3T3-L1脂肪細胞葡萄糖消耗減少.
목적 응용중조만병독구건3T3-L1지방세포chemerin과표체모형병진일보탐토기대당대사적영향급가능궤제.방법 구건서chemerin과표체중조만병독,병설대조만병독,감염3T3-L1세포,실시정량취합매련반응(RT-PCR)법검측전염후chemerin표체수평;응용이도소、3-이정기1-갑기황표령、지새미송유도3T3-L1전지방세포분화위성숙지방세포,유홍O염색감정;유도분화제8천가입만병독중조체,계속배양5d,포도당양화매법검측각조포도당소모;RT-PCR법검측각조이도소수체저물1(IRS1)、이도소수체저물2(IRS2)、단백격매B1 (Akt1)、차두상전록인자O1 (FoxO1)기인표체수평;Western-blotting검측chemerin、사안산/소안산단백격매(Akt)、린산화사안산/소안산단백격매(pAkt)、FoxO1、린산화차두상전록인자O1 (pFoxO1)단백수평.량조수거비교응용t검험.결과 Chemerin과표체만병독감염3T3-L1세포72 h후세포중가견홍색형광,RT-PCR결과현시:과표체조여공재대조조상비chemerin기인표체명현증가(분별위3.04±0.19비1.01±0.11,t=15.65,P＜0.05);chemerin과표체조포도당소모감소[분별위(3.30± 1.44)비(6.07±1.15) mmol/L,t=-0.35,P＜0.05];RT-PCR결과현시:IRS1、IRS2기인수평무명현변화(균P＞0.05),Akt1기인표체하강(분별위0.76±0.08비1.07±0.15,t=-3.11,P＜0.05),FoxO1기인표체상조(분별위1.53±0.30여1.03±0.21,t=2.34,P＜0.05).Western-blotting결과현시:Chemerin과표체후chemerin단백수평증가(상대표체량분별위1.08±0.06비0.72±0.03,t=-10.12;P＜0.05);Akt、pAkt단백수평균강저(분별위0.74±0.21비1.23±0.20,0.58±0.17비0.92±0.07;t=2.81、3.17,균P＜0.05),FoxO1단백수평승고(분별위1.04±0.09비0.76±0.14,t=-2.91,P＜0.05)、pFoxO1단백수평강저(분별위0.61±0.13비0.89±0.10,t=2.93,P＜0.05).결론 Chemerin가능통과하조Akt1 mRNA사3T3-L1지방세포포도당소모감소.
Objective To investigate the influence of overexpression of Chemerin on 3T3-L1 adipocyte constructed by recombinant lentiviral on glycometabolism and the potential mechanism.Methods Rat Chemerin expression of recombinant lentivirus was constructed.This recombinant lentivirus and control lentivirus transfected 3T3-L1 cells respectively.The expression of chemerin in 3T3-L1 cells was detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR).3T3-L1 preadipocytes were induced into mature adipocyte by insulin,IBMX and dexamethasone.Oil red O was used to identify the mature 3T3-L1 adipocytes.Recombination lentiviruses were added in culture medium at the eighth day of differentiation.The cells were continuously cultured for five days and then were harvested for series of determinations.Glucose oxidase method was used to test the glucose consumption.The mRNA expression of IRS1,IRS2,Akt1 and FoxO1 were detected by RT-PCR.The protein expression of chemerin,Akt,pAkt,FoxO1 and pFoxO1 were determined by western blotting.The measurement data were measured by mean ± standard deviation ((x) ± s).Student' s t-test was used for comparison of two groups.Results Red fluorescence can be observed in 3T3-L1 cells after being infected with recombination lentivirus for 72 h.The expression of chemerin mRNA significantly increased (relative expression:(3.04±0.19)vs(1.01 ± 0.11),t=15.65,P＜0.05) and glucose consumption decreased ((3.30± 1.44)vs(6.07±1.15) mmol/L,t=-0.35,P＜0.05) in over expression chemerin group comparing with empty-vector control group.There were no difference of IRS1and IRS2 mRNA between the two groups (all P＞0.05).Compared to the empty-vector control group,the over expression chemerin group have higher FoxO1 mRNA and protein expression (relative expression:(1.53±0.30) vs (1.03±0.21),t=2.34,P＜0.05 ; (1.04±0.09)vs(0.76±0.14),t=-2.91,P＜0.05),and lower Akt1 mRNA expression (relative expression:0.76±0.08 vs 1.07±0.15,t=-3.11,P＜0.05),as well as lower Akt,pAkt and pFoxO1 protein expression(relative expression:(0.74±0.21) vs (1.23±0.20),(0.58±0.17) vs (0.92± 0.07),(0.61±0.13)vs(0.89±0.10),t=2.81,3.17,2.93,all P＜0.05).Conclusions Chemerin may reduce glucose consumption in 3T3-L1 by down regulation Akt1 mRNA expression.
