Troglitazone对小鼠Ⅱ型葡萄糖载体及Ⅰ型葡萄糖载体mRNA表达影.docVIP

Troglitazone对小鼠Ⅱ型葡萄糖载体及Ⅰ型葡萄糖载体mRNA表达影 　【摘要】 　　目的探讨Troglitazone对小鼠Ⅱ型葡萄糖载体（mGLUT2）和I型葡萄糖载体（mGLUT1）mRNA表达的影响。方法采用分子克隆技术，将过氧化物酶体增殖物受体γ(PPARγ)和维甲酸类受体X(RXRα)及mGLUT2和mGLUT1cDNA分别克隆到表达载体pCMX和pGL3b上。PPARγ与RXRα及mGLUT2与mGLUT1克隆载体转染NIH 3T3细胞，处理或不处理Troglitazone，应用荧光素酶活性测定法及RNA印迹等方法测定Troglitazone对mGLUT2和mGLUT1重组体荧光素酶活性调节及对mRNA表达的影响。结果Troglitazone可激活mGLUT2和mGLUT1重组体荧光素酶的活性，并且可增加mRNA的表达水平。结论Troglitazone可增强mGLUT2和mGLUT1的表达，可能参与PPARγ对mGLUT2和mGLUT1的调节过程。 【关键词】 Troglitazone 小鼠Ⅰ型葡萄糖载体 Ⅱ型葡萄糖载体 mRNA表达 　　AbstractObjectiveTo understand the possibility of the effect of Troglitazone on mGLUT2 and mGLUT1 mRNA expression. Methods PPARγ , RXRα and mGLUT2 and mGLUT1 cDNA were cloned into pCMX and pGL3b expression vector. The recombinant DNA transfected into NIH 3T3 cells, treated Trogiltazone or not. The mGLUT2 and mGLUT1 activity and mRNA expression were determined by the Trogiltazone through PPARγ activation with luciferase assay and Northern blot.ResultsTroglitazone activated the mGLUT2 and mGLUT1 activity and increased the mGLUT2 and mGLUT1 mRNA expression through PPARγ activation. ConclusionTroglitazone perhaps binds to the regulations on mGLUT2 and mGLUT1 by PPARγ. 　　Key words Troglitazone; mGLUT; mRNA expression 　　mGLUT2在胰岛、肝脏等组织中表达，mGLUT1在脂肪、肌肉等多种组织中表达，参与各组织吸收葡萄糖的过程。据报道，GLUT2和GLUT1在多种病理状态下有较高水平的表达，但机理不详。Troglitazone作用靶点是一种核转录因子，即过氧化物酶体增殖物激活受体（PPAR）γ，PPARγ和配体结合而激活后，与维甲酸类受体X(RXRα)形成二聚体，再结合于PPARs反应元件(PPRE)上而发挥转录调控作用。PPRE存在于许多编码与糖代谢与脂类代谢相关的蛋白质的DNA上游序列。为了探讨Troglitazone 是否通过激活PPARγ参与mGLUT2和mGLUT1的表达调节，进而参与组织吸收葡萄糖的过程,达到降血糖的目的,本实验利用分子克隆技术构建PPARγ、RXRα和mGLUT2和mGLUT1的表达载体系统，将其转染NIH 3T3细胞后处理Troglitazone，通过荧光素酶活性测定和Northern blot等实验方法观察Troglitazone对mGLUT2和mGLUT1的活性调节及对mRNA 表达调节。 　　1 材料与方法 　　1.1 材料 　　NIH 3T3 细胞株购自 ATCC；pCRⅡ-TOPO 载体(Life Technologies, Gaithersburg, MD, USA)；Dulbecco`s modified Eagle`s 培养液 (DMEM, Life Technologies, Gaithersburg, MD,USA)；层析柱(Qiagen,Hilden,Germany)；LipofectAMINE PLUS试剂 (Life Technologies, Gaithersburg, MD, USA)；OPTI-MEN I(Life Technologies, Gaithersburg,MD, USA)；裂解缓冲液 (Pro