转染adiponectin基因对C2C12肌细胞糖原合成和葡萄糖氧化的影响

[目的]研究adiponectin对C2C12肌细胞糖原合成和葡萄糖氧化的影响.[方法]用阳离子脂质体介导转染和随后G418筛选建立稳定转染小鼠adiponectin cDNA真核表达质粒(pcDNA3.0-mad)及空载pcD-NA3.0的C2C12细胞株并鉴定.C2C12肌细胞糖代谢实验分对照组、载体组和pcDNA3.0-mad(mad)组共3组进行,每组又分0,0.5 nmol/L,5 nmol/L,100 nmol/L胰岛素刺激4个亚组.通过液闪测定细胞合成的糖原中14C的放射活性和氧化产生的14CO2分别检测肌细胞的糖原合成和葡萄糖氧化情况.[结果]Western blot和免疫组化检测证实mad组细胞表达adiponectin蛋白.Mad组葡萄糖氧化量随胰岛素浓度增加的速率较其他两组快,对照,载体和mad组线性回归系数分别为23.34,23.23和26.06.mad组C2C12肌细胞基础状态下和胰岛素刺激下的葡萄糖氧化和糖原合成与其它两组无显著性差异(P＞0.05).[结论]转染adiponectin基因对C2C12肌细胞葡萄糖氧化和糖原合成无显著性影响.

Effects of Transfection with Adiponectin cDNA on Glycogen Synthesis and Glucose Oxidation in Myotubes

To study the effects of adiponectin o n glycogen synthesis and glucose oxidation in C2C12myotubes.Plasmid pcDNA3.0-mad with cDNA of mo use adiponectin,and vector pcDNA3.0were transfecte d into C2C12cells by lipofectAMINE 2000reagent respectively.Stably transfected cells were scree ned by 500mg /L G418for 3weeks.Adiponectin protein expression was determined by Western blot analy sis and immunohistochemistry.The e xperiment were divided into control,vector,and pcDNA3.0-mad(mad)group.Each group was further divided into 4subgroups with 0,0.5nmol /L,5nmol /L,100nmol/L insulin(n=6),respectively.Detection of glucose oxidation and glycogen synthesis wa s carried out with 14 C-labeled glucose by counting radio activity of 14 CO 2 or 14 C labeled glycogen with scintillati on,respectively.Colony growth was seen on the second week,while most of C2C12cells were dead on day 10after transfection.Adiponectin protein expression was only detected in the mad group by either Western blot analy sis or immunostaining.The rate of glucose oxidation increased more with the elevation of insulin concentration in the mad group than in the other 2groups.The regression coefficiencies of control,vector,and mad group were23.34,23.23,and 26.06,respectively.No s ignificant difference in either basic or insulin stimulated glucose oxidation and glycogen synt hesis between the mad group and the other two group was observed(P>0.05).[Conclusion]Transfection with adiponectin cDNA has no significant effect on glucose oxidation and glycogen synthesis of C2C12myotubes.