碳酸氢钠诱导肝癌细胞凋亡机制研究

Mechanism research on apoptosis of hepatocellular carcinoma induced by sodium bicarbonate

目的探讨碳酸氢钠(NaHCO_3)对肝癌生长的影响及其机制。方法 5只7周龄裸鼠于无特定病原体(SPF)条件下的层流架中饲养,皮下注射肝癌细胞株HepG2细胞构建肝癌模型,并通过肿块内局部注射5%NaHCO_3,体内研究肝癌肿块生长及组织坏死。Calcein/PI染色和流式细胞技术体外研究不同酸碱环境和NaHCO_3作用下HepG2凋亡情况。蛋白印迹技术检测NaHCO_3作用下HepG2细胞凋亡调控蛋白Bax/bcl2表达水平。结果 5只裸鼠肝癌5%NaHCO_3局部注射4周,3只肿块明显缩小,1只肿块长大,1只小鼠实验过程中死亡。5%NaHCO_33/4、1/2、1/4、1/8、1/16和0(对照)培养基体积占比,Calcein/PI染色显示HepG2细胞凋亡率依次减低。流式细胞分析显示5%NaHCO_31/2、1/4、1/8、1/16体积占比和对照组细胞凋亡率也依次减低。Calcein/PI染色显示培养基pH6.0、pH6.25、pH6.5、pH6.75、pH7.0、pH7.25、pH7.5、pH7.75、pH8.0的HepG2细胞凋亡率在pH7.0~7.25最低,随着酸碱度的增加凋亡率逐渐增加。流式细胞分析培养基pH6.25、pH6.5、pH6.75、pH7.0、pH7.25和pH7.5组细胞凋亡率变化显示类似结果。5%NaHCO_3占1/4培养基体积比条件下HepG2细胞促凋亡的Bax mRNA和蛋白水平与对照相比较无明显变化,而抑凋亡的bcl2 mRNA和蛋白水平明显低于对照组。结论 NaHCO_3可通过改变肝癌组织内环境酸碱度,继而抑制凋亡功能蛋白blc2诱导肝癌细胞凋亡。

Objective To explore the apoptotic mechanism of hepatocellular carcinoma（HCC） induced by sodium bicarbonate（SB）. Methods HepG2 cells were implanted the subcutaneous of 5 athymic mice（7-week age） to form hepatocarcinoma mouse model and the growth and necrosis of hepatocellular carcinoma was observed under the condition of local mass injection of 5% SB. The apoptosis of hepatocellular carcinoma cell line HepG2 was observed by Calcein/PI staining and flow cytometry at SB exposure and different pH gradients. The expression of Bax/bcl2 were also detected in HepG2 cells under SB exposure by real-time PCR and western blotting. Results Animal in vivo studies revealed that 5% SB local injection for 4 weeks could inhibit tumor growth and cause tumor necrosis in 3 of 5 hepatocarcinoma mice. One hepatocarcinoma mice died and another mice had an enlarged tumor. 5% SB was diluted to the volume ratio 3/4, 1/2, 1/4, 1/8, 1/16 and negative control by DMEM and Calcein/PI staining in vitro showed that HepG2 apoptosis mice decreased in turn. Flow cytometry showed the similar results in 5% SB volume ratio： 1/2, 1/4, 1/8 and 1/16. The medium DMEM was titrated to pH6.0, pH6.25, pH6.5, pH6.75, pH7.0, pH7.25, pH7.5, pH7.75 and pH8.0. The lowest rate of HepG2 apoptosis was at pH7.0 and pH7.25. As the increasing of acidity or alkalinity, the apoptosis rate of HepG2 increased gradually. Flow cytometry also showed the similar results. Compared with control, Bax mRNA and protein expression in SB exposed HepG2 cell were not changed, but bcl2 mRNA and protein expression were decreased. Conclusion SB can induce HCC apoptosis by changing the internal environmental pH and inhibiting the expression of apoptotic protein blc2.