3T3-L1脂肪细胞胰岛素抵抗模型的建立、优化及分子指标鉴定

Establishment,Optimaztion and Molecular Marker Identification of the Insulin Resistant Model in 3T3-L1 Adipocytes

目的探讨3T3-L1脂肪细胞胰岛素抵抗(Insulin resistance,IR)模型建立的条件、优化及分子指标鉴定。方法采用3-异丁基-1-黄嘌呤(3-isobutyl-1-methylxanthine,IBMX)、地塞米松(Dexamethason,DEX)和胰岛素联合诱导3T3-L1前脂肪细胞分化为成熟脂肪细胞,跟踪观察其诱导分化过程中的形态变化,并采用油红O染色鉴定。采用DEX(1μmol·L^(-1))单独诱导及DEX(1μmol·L^(-1))+罗格列酮(Rosiglitazone,ROS,10μmol·L^(-1))联合诱导建立IR-3T3-L1脂肪细胞。采用葡萄糖氧化酶-过氧化物酶法(Glucose oxidase-peroxidase,GOD-POD)检测不同时间点细胞培养液中葡萄糖含量,确定IR-3T3-L1脂肪细胞模型的最佳诱导时间,考察3T3-L1脂肪细胞模型IR状态的稳定性;采用CCK-8法测定IR-3T3-L1脂肪细胞活性;采用甘油磷酸氧化酶法(glycerol phosphate oxidase,GPO-POD)测定IR-3T3-L1细胞甘油三酯(Triglyceride,TG)含量;采用实时荧光定量PCR(q PCR)和Western Blot法分别检测IR-3T3-L1脂肪细胞的脂联素(Adiponectin,ADPN)及葡萄糖转运蛋白4(Glucose transporter 4,GLUT4)的mRNA、蛋白质表达水平。结果 DEX诱导3T3-L1脂肪细胞96 h,葡萄糖消耗差值比例达最大为46.84%;DEX+ROS诱导72 h,葡萄糖消耗差值比例达最大为22.77%,可作为2种IR模型建立的最佳时间点。与正常组比较,DEX及DEX+ROS诱导建立的2种IR模型葡萄糖消耗量均显著降低(P<0.05,P<0.01),2种方法建立的IR-3T3-L1细胞模型60 h内均保持稳定;IR模型组细胞活性均无明显变化(P>0.05),2种诱导试剂对IR脂肪细胞活性均无明显影响;IR模型组TG含量均显著升高(P<0.01),DEX+ROS组TG含量明显高于DEX组;IR模型组ADPN、GLUT4 mRNA及蛋白表达水平均显著下调(P<0.01),且DEX组下调幅度较DEX+ROS组更加明显。结论 DEX单独诱导及DEX+ROS联合诱导2种方法均可建立稳定的IR-3T3-L1细胞模型,DEX诱导IR-3T3-L1细胞模型优于DEX+ROS,其机制可能与GLUT4及ADPN表达显著降低有关。

Objective To establish and optimize the insulin resistance（IR） model on 3T3-L1 adipocytes and identify the molecular markers. Methods Differentiation of 3T3-L1 preadipocytes into mature adipocytes was induced by 3-isobutyl-methylxanthine（IBMX）,dexamethasone（DEX） and insulin,and then identified by oil red O staining method. The IR-3T3-L1 adipocyte models were established by induction with 1 μmol · L^（-1） DEX（D-IR）and the combination of 1 μmol·L^（-1） DEX with 10 μmol·L^（-1） rosiglitazone（ROS）（DR-IR）. The glucose consumption was detected at different time points by the glucose oxidase-peroxidase method to defined the optimum induction time and IR stability;intracellular triglyceride（TG） content was measured by glycerol phosphate oxidase method,whereas adiponectin（ADPN） and glucose transporter 4（GLUT4） mRNA and protein expression levels were detected by quantitative PCR and Western Blot. Results The maximal difference in glucose consumption of the control group and D-IR model group was observed as 46.84 % at 96 h,whereas the maximal difference of glucose consumption were 22.77 % at 72 h in the DR-IR group. The IR status maintained for 60 h in both IR groups after inducing drugs were removed from cell culture media. Compared with the control groups,ADPN and GLUT4 mRNA and protein expression levels in both IR groups were significantly down-regulated（P〈0.01）,whereas the mRNA and protein expression of ADPN and GLUT4 in the D-IR group decreased in a greater degree than in the DR-IRgroup. Conclusion The IR-3T3-L1 adipocytes were successfully established in vitro by DEX and the combination of DEX with ROS,respectively,resulted in down-regulation of the mRNA and protein expression levels of ADPN and GLUT4. The IR-3T3-L1 model induced by 1 μmol · L^（-1） DEX is more stable may be associated with more significant down-regulation of glucose consumption, which partly due to more effective inhibition of ADPN and GLUT4 expression.