低剂量5-Fu与Ad-IL24联用对肝癌细胞HepG2生长的抑制作用及其机制

Inhibition Effect of Ad-IL24 Combined with Low Dose of 5-Fu on Hepatocarcinoma Cells HepG2 and Its Mechanism

目的探讨低剂量5-Fu与腺病毒表达的IL24联用对HepG2细胞的生长抑制作用及其机制。方法采用PCR、酶切、连接等方法构建pAd-IL24腺病毒表达载体,Western blot检测IL24蛋白水平表达情况。CCK8法确定5-Fu联用剂量,检测与Ad-IL24联用对HepG2肝癌细胞的抑制效果,并计算联合指数。流式细胞术检测不同腺病毒感染复数及不同5-Fu浓度下绿色荧光蛋白(green fluorescent protein,GFP)阳性细胞的比例。流式细胞术检测5-Fu作用前后HepG2细胞表面柯萨奇腺病毒受体(coxsackie adenovirus receptor,CAR)的表达变化。结果成功构建腺病毒表达载体pAd-IL24。确定5-Fu联用剂量为1μg/ml和2μg/ml,Ad-IL24与低剂量5-Fu联用可以协同抑制HepG2肝癌细胞生长,联合指数分别为0.75和0.64。低剂量5-Fu作用于HepG2后使其表面CAR水平显著增加(P<0.01)。结论低剂量5-Fu与Ad-IL24联用对HepG2肝癌细胞系的抑制有协同作用,可能与5-Fu上调HepG2表面CAR水平相关。

Objective To investigate the inhibition effect of Ad-IL24 combined with low dose of 5-Fu on hepatocarcinoma cells HepG2 and its mechanism. Methods PCR, restriction enzyme cleavage and linkage were used to construct the adenovirus expression vector pAd-IL24. The expression of IL24 was detected by Western blot. CCK8 assay was applied to confirm the doses of 5-Fu and to detect the viability of HepG2 cells which were treated by Ad-IL24 with or without 5-Fu. The proportion of GFP positive cells in HepG2 cells treated by different does of 5-Fu and adenovirus was detected by flow cytometry. The expression levels of coxsackie adenovims receptor （CAR） were detected by flow cytometry before and after 5-Fu treatment. Results The adenovirus expression vector pAd-IL24 was successfully constructed. The doses of 5-Fu were confirmed to be l and 2μg/ml by CCK8 assay; Ad-IL24 combined with low doses of 5-Fu synergistically inhibited the growth of HepG2 cells, and the combination index were 0.75 and 0.64 respectively. After treated with low dose of 5-Fu, the level of CAR expression on HepG2 cells was significantly increased（P〈0.01）. Conclusion The combination treatment of Ad-IL24 and low dose of 5-Fu synergistically inhibit the growth of hepatocarcinoma cells HepG2, which maybe related with up-regulation of CAR induced by 5-Fu.