乳酸堆积和二氯乙酸钠对肝癌细胞凋亡及bax、bcl-2 表达 和caspase-3 活性的影响

目的：探讨乳酸堆积和二氯乙酸钠(DCA)对肝癌细胞(HepG2)凋亡和bax、bcl-2 表达及caspase-3 活性的影响。方法：通过体 外培养HepG2，建立稳定的体外培养模型，配制成终浓度分别为0 mmol/L、1.0 mmol/L、2.0 mmol/L、4.0 mmol/L、8.0 mmol/L的乳 酸培养液以及在不同浓度乳酸组中加入终浓度为10-3mmol/L DCA 培养液与HepG2共同培养，其中以0 mmol/L 乳酸组为对照 组。采用MTT法检测乳酸对HepG2 的抑制率，流式细胞仪检测乳酸和DCA 对HepG2的凋亡百分率，用Real-time PCR法测定 bax 及bcl-2 mRNA的表达，用免疫荧光法检测caspase-3 的活性。结果：乳酸对HepG2 的IC50值为13.6 mol/L，与对照组比较，随 着乳酸浓度的增加，HepG2 凋亡率增加，bax mRNA 表达升高，bcl-2 mRNA 的表达降低，caspase-3活性增加，其中1.0 mmol/L 乳 酸组与对照组比较(P>0.05)，2.0 mmol/L，4.0 mmol/L 和8.0 mmol/L乳酸组与对照组比较差异有统计学意义(P<0.05)。加入DCA 后，HepG2 凋亡减少，2.0 mmol/L 乳酸+DCA 组、4.0 mmol/L乳酸+DCA 组、8.0 mmol/L乳酸+DCA 组与同浓度的乳酸组比较， bax mRNA 表达减少（P<0.05），bcl-2 mRNA 表达增加（P<0.05），caspase-3 活性减低（P<0.05）。结论：乳酸可诱导HepG2凋亡，且随 着乳酸浓度的增高，HepG2 的凋亡率增加，其机制可能是通过对bcl-2 及bax mRNA 表达的改变以及激活caspase-3 活性而实现， DCA可以降低HepG2 凋亡，对乳酸堆积造成的HepG2凋亡有抑制作用。

Effect of Lactate Accumulation and Dichloroacetate on Apoptosis of Hepatoma Cells, Expressions of bax, bcl-2 and Activity of Caspase-3

Objective:To investigate the effect of lactate accumulation and dichloroacetate (DCA) on apoptosis of hepatoma cells (HepG2), and the expressions of bax, bcl-2 and activity of caspase 3.Methods:HepG2 were cultivated stable in vitro. HepG2 were co-cultivated with different final concentrations (0 mmol/L, 1.0 mmol/L, 4.0 mmol/L, 8.0 mmol/L) lactate and join the final concentration of 10-3mmol/L DCA in different concentrations groups of lactate. 0 mmol/L lactate group was control group. MTT method was used to test the lactate inhibition rate of HepG2, flow cytometry was used to test the apoptosis of HepG2, Real-time PCR was used to observe the expressions of bax and bcl-2 mRNA, and immunofluorescence method was used to detect the activity of caspase-3.Results:Lactate half inhibitory concentration (IC50) of HepG2 was 13.6mmol/L. Compared with that in control group, with the increase of lactate concentration, HepG2 apoptosis rate, the expressions of bax mRNA and the activity of caspase-3 in creased, while the expression of bcl-2 mRNA reduced. There was no significant difference of the indexes mentioned between the 1.0 mmol/L lactate group and control group (P>0.05). There were significant differences of the indexes mentioned between 2.0 mmol/L, 4.0 mmol/L and 8.0 mmol/L lactate groups and control group (P<0.05). After joined the DCA, the apoptosis of HepG2 decreased. 2.0mmol / L lactate + DCA group, 4.0mmol / Llactate + DCA group, 8.0 mmol/L lactate + DCA group compared with the same concentration of lactate group, bax mRNA expression was decreased (P<0.05), bcl-2 mRNA expression was increased (P<0.05). The activity of caspase-3 was decreased (P<0.05).Conclusion:Lactate can be induced HepG2 apoptosis. The apoptosis rate of HepG2 increased with the increasing of lactate concentration, whith mechanism may be through to change the espressions of bax mRNA, bcl-2 mRNA and the activaty of caspase-3. DCA can reduced HepG2 apoptosis by lactate accumulation.