siRNA沉默转酮醇酶样蛋白1基因对人肝癌HepG2细胞生长微环境的影响  

Effects of TKTL1silencing by siRNA on growth microenvironment of human hepatocellular carcinoma HepG2 cells

作　　者：张德太[1] 左曙荣[1] 杨文[1] 张科[1] 宋斌[1] 涂启明[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院检验科,湖北武汉430022

出　　处：《中国病理生理杂志》2012年第8期1383-1387,共5页Chinese Journal of Pathophysiology

摘　　要：目的:通过siRNA技术沉默肝癌细胞系HepG2转酮醇酶样蛋白1(TKTL1)基因后观察其对生长微环境中乳酸生成水平、还原型谷胱甘肽(GSH)水平及NADPH/NADP+比值的影响,旨在探讨肿瘤细胞TKTL1表达与其转移特性的内在联系。方法:用构建的靶向TKTL1基因siRNA干扰质粒载体转染HepG2细胞株,RT-PCR检测TKTL1 mRNA表达并结合转酮醇酶(TKT)活性测定判断其干扰效率;以未转染HepG2细胞为对照,进一步观察TKTL1沉默的癌细胞内乳酸生成水平、GSH含量、NADPH/NADP+比值及葡萄糖-6-磷酸脱氢酶(G-6-PD)活性的变化。结果:siRNA沉默HepG2细胞TKTL1基因后TKTL1 mRNA表达下降、TKT活性显著降低(P<0.01),乳酸生成、GSH水平及NADPH/NADP+比值较对照组细胞均显著降低(P<0.01),但G-6-PD活性无明显变化(P>0.05)。结论:肿瘤可能通过TKTL1高表达调整葡萄糖代谢从而改变乳酸及氧化自由基生成,使细胞生长微环境利于肿瘤转移。To explore the relationship between the expression of transketolase - like 1 protein （ TKTL1 ） and the metastasis of hepatocellular carcinoma by investigating the change of some indicators in growth cluding lactate production, reduced glutathione （GSH） level and ratio of NADPH/NADP ＋ in hepatocellular carcinoma cell line HepG2 after silencing of TKTL! expression by siRNA. METHODS： Specific siRNA expression vector targeting TKTL1 gene was constructed and transfected into HepG2 cell line. The effect of TKTL1 silencing was evaluated by detecting the mRNA level and the activity of transketolase （TKTL1）. The changes of lactate production, GSH level, the ratio of NAD- PH/NADP ~ and glucose- 6 -phosphate dehydrogenase （G -6 -PD） activity were observed in transfected HepG2 cells compared with the untransfected control cells. RESULTS： Compared with the untransfected control cells, the mRNA ex- pression of TKTL1 and the TKT activity decreased significantly （P 〈0.01 ）. Meanwhile, the lactate production, GSH level and ratio of NADPH/NADP ~ also decreased significantly （ P 〈 0.01 ）. However, no change of the G - 6 - PD activity was observed. CONCLUSION： Carcinoma cells switch the glucose metabolism by overexpression of TKTL1 to modify the lac- tate production and the levels of reactive oxygen species, thus changing the growth microenvironment in favor of tumor me- tastasis.

关 键 词：RNA 小干扰 转酮醇酶样蛋白1 葡萄糖-6-磷酸脱氢酶 HEPG2细胞 

分 类 号：R735.3[医药卫生—肿瘤]