可溶性肿瘤坏死因子相关的凋亡诱导配体与5-Fu联用对肝癌细胞的抑制作用

Inhibition of Soluble TNF-related Apoptosis-inducing Ligand Combined with 5-Fu on Human Hepatic Carcinoma Cells

[目的]探讨肿瘤坏死因子(tumor necrosis factor,TNF)相关的凋亡诱导配体ILZs TRAIL与5-氟尿嘧啶(5-fluorouracil,5-Fu)联合应用,对HepG_2肝癌细胞生长的抑制作用。[方法]采用聚合酶链反应(PCR)、酶切、连接等方法构建CMV启动子驱动TNF相关凋亡诱导配体(TRAIL)表达的载体pLenti R.ILZ-s TRAIL及其对照载体pLenti R.CopGFP,经测序验证其正确性。将pLenti R.ILZ-s TRAIL和pLenti R.CopGFP分别转染至293T细胞,48h后收集上清并利用ELISA检测目的蛋白含量。利用CCK8细胞增殖抑制实验、Western blot检测ILZ-s TRAIL与5-Fu联用对HepG_2肝癌细胞生长的抑制作用及凋亡蛋白的表达变化。[结果]成功构建表达载体pLenti R.ILZ-s TRAIL及pLenti R.CopGFP,测序正确。ILZ-s TRAIL的表达量达(9.475±0.786)ng/ml。体外ILZ-s TRAIL与5-Fu联用,可协同抑制HepG_2肝癌细胞的生长。其中凋亡相关蛋白Bcl-2表达降低,Bax表达增加,Caspase-8、9、3及PARP被剪切活化。[结论 ]ILZ-s TRAIL蛋白与5-Fu联合应用对肝癌细胞HepG_2具有抑制作用,为肝癌的治疗提供了一种新策略。

[Purpose]To investigate the inhibition effects of TNF-related apoptosis-inducing ligand （ILZ-sTRAIL） combined with 5-Fu on human hepatic carcinoma HepG2 cells. [Methods] Polymerase chain reaction（PCR）,restriction enzyme cleavage and linkage were used to construct the expression vector pLentiR.ILZ-sTRAIL and its control vector pLentiR.CopGFP. The plasmids were verified by DNA sequencing. pLentiR.ILZ-sTRAIL and pLentiR. CopGFP were transfected to 293T cells,respectively;the supernatants of 293T cells were harvested 48h after transfection and the ILZ-sTRAIL in the supernatants was quantified by ELISA. CCK8 assay was employed to detect the growth inhibition of ILZ-sTRAIL on HepG2 cells when combined with low does of 5-Fu. In addition,the apoptosis-related protein in HepG2 cells were determined by Western blotting. [Results] Plasmids pLentiR.ILZ-sTRAIL and pLentiR.CopGFP were successfully constructed and verified by DNA sequencing. The concentration of ILZ-sTRAIL in supernatants reached（9.475±0.786）ng/ml. When treated with ILZ-sTRAIL and 5-Fu in vitro,the growth of HepG2 cells were inhibited significantly. And the expression of Bcl-2 decreased,Bax increased,and caspase-8,9,3 and PARP were cleaved and activated. [Conclusion] The combining of ILZ-sTRAIL with 5-Fu can effectively inhibit human hepatic carcinoma HepG2 cells in vitro,which may provide a new approach for treatment of hepatic carcinoma.