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[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。
[Objective] To investigate the inhibitory effect of TRAIL combined with 5-fluorouracil (IL-5) on the growth of HepG2 hepatocarcinoma cells. [Method] The vector pLenti R.ILZ-s TRAIL and its control vector pLenti R were constructed by polymerase chain reaction (PCR), restriction enzyme digestion, ligation and other methods to drive the expression of TNF-related apoptosis-inducing ligand (TRAIL) by CMV promoter. CopGFP, verified by sequencing the correctness. The 293T cells were transfected with pLenti R.ILZ-s TRAIL and pLenti R.CopGFP, respectively, and the supernatants were collected after 48h. The contents of the target proteins were detected by ELISA. The inhibitory effect of ILZ-s TRAIL combined with 5-Fu on the growth of HepG2 hepatocarcinoma cells and the change of the expression of apoptosis protein were detected by CCK8 cell proliferation inhibition assay and Western blot. [Result] The expression vectors pLenti R. ILZ-s TRAIL and pLenti R.CopGFP were successfully constructed and sequenced correctly. ILZ-s TRAIL expression reached (9.475 ± 0.786) ng / ml. In vitro ILZ-s TRAIL combined with 5-Fu can synergistically inhibit the growth of HepG2 hepatocarcinoma cells. The expression of Bcl-2, Bax, Caspase-8, 9, 3 and PARP were cleaved and activated. [Conclusion] The combined application of ILZ-s TRAIL protein and 5-Fu has an inhibitory effect on HepG2 hepatoma cells, which provides a new strategy for the treatment of hepatocellular carcinoma.