热休克蛋白70对骨骼肌细胞系(C2C12)葡萄糖代谢的影响  

Effects of HSP70 on the glucose metabolism of C2C12 cells

作　　者：王磊[1] 王尊[1] 杨伟伟[1] 

机构地区：[1]南京中医药大学第二临床医学院康复医学系,江苏南京210001

出　　处：《基础医学与临床》2014年第11期1514-1518,共5页Basic and Clinical Medicine

基　　金：江苏省高校自然科学基金(107KJB320010);全国高校博士点基金(20123237120008)

摘　　要：目的探讨高表达热休克蛋白70(HSP70)对骨骼肌细胞(C2C12)葡萄糖代谢的影响。方法将C2C12细胞分为HSP70转染组与正常对照组。转染组细胞通过构建重组p TRE2hyg-HSP70质粒,稳定转染C2C12骨骼肌细胞系,使其高表达HSP70。待转染组及对照组细胞分化后3和7 d,用葡萄糖氧化酶法检测葡萄糖的消耗,用3H-2-脱氧葡萄糖测定葡萄糖摄取能力,用生化仪检测乳酸的产生,用比色法测定磷酸果糖激酶(PFK),乳酸脱氢酶(LDH),己糖激酶(HK),丙酮酸激酶(PK)等葡萄糖酵解关键酶的活性及细胞内ATP水平,用Western blot检测细胞膜葡萄糖转运体4(Glut4)的表达。结果与对照组相比,在细胞分化后3和7 d,过表达HSP70的C2C12细胞葡萄糖的消耗和乳酸的产生(P<0.01),以及葡萄糖的摄取量都明显升高(P<0.05);细胞磷酸果糖激酶(PFK),乳酸脱氢酶(LDH),己糖激酶(HK),丙酮酸激酶(PK)的活性及ATP的水平明显升高(P<0.01);细胞膜Glut4的表达也明显升高(P<0.05)。结论 HSP70通过提高骨骼肌C2C12细胞膜Glut4的表达增加细胞对葡萄糖的摄取,并通过提高糖酵解关键酶的活性促进细胞的葡萄糖酵解。Objective To examine the effects of over-expression HSP70 on the glucose metabolism in C2C12 cells.Methods C2C12 cells were divided into two groups：transfected group and normal control.HSP70 gene was amplified from pAT153 plasmids and then cloned into pTRE2hyg vector.After the transfection of recombinant plasmids of pTRE2hyg-HSP70 into the C2C12 cells,the expression of HSP70 was examined by Western blot and the C2C12 cell line of up-regulated HSP70 was established.Glucose consumption and glucose uptake were measured by the glucose oxidase method and 3H-labeled glucose respectively at 3 and 7 days after cellular differentiation in two groups.Changes in lactate production were determined by biochemical analyzer.In addition,activities of enzymes involved in glycolysis （phosphofructokinase,PFK ; lactate dehydrogenase,LDH ; hexokinase,HK; pyruvate kinase,PK） and the intracellular ATP level were evaluated by colorimetry in C2C12 cells.The expression of plasma membrane Glut4 in protein level was measured by Western blot.Results Compared with controls,the glucose consumption,glucose uptake and lactate excretion were significantly elevated in cells expressing up-regulated HSP70 （P ＜0.01） at 3 and 7 days after cellular differentiation.The expression of plasma membrane Glut4 in protein level increased in transfected C2C12 cells （P ＜ 0.05）.Conclusions HSP70 promotes glucose uptake by increasing the expression of plasma membrane Glut4 in C2C12 cells.HSP70 also increase the glycolytic capacity by stimulating the activities of glycolytic enzymes of C2C12 cells.

关 键 词：热休克蛋白70 骨骼肌细胞 葡萄糖摄取 糖酵解 葡萄糖转运子4 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]