热疗诱导人肝癌细胞HepG2凋亡及机制的研究

目的探讨不同温度热疗对人肝癌细胞Hep G2的凋亡作用。方法人肝癌Hep G2细胞常规方法培养,采用水浴加热法对实验组(41℃,42℃,43℃)和对照组(37℃)细胞进行加温(0.5 h)处理,处理后继续培养24 h。采用流式细胞术检测细胞的凋亡情况,免疫组织化学技术检测细胞凋亡相关蛋白P53的水平,实时荧光定量PCR法检测热疗作用后生存素(Survivin),caspase-9基因mRNA的表达变化。应用SPSS16.0软件进行统计学分析。凋亡率、P53蛋白值、caspase-9 mRNA表达水平等计量资料采用均数±标准差表示,组间比较采用t检验或方差分析。P0.05差异有统计学意义。结果在37℃,41℃,42℃,43℃,细胞凋亡率分别为(9.58±1.22)%、(16.47±3.41)%、(20.07±1.85)%和(23.42±1.93)%;P53蛋白表达OD值分别为(0.083±0.015)、(0.145±0.024)、(0.267±0.031)、(0.389±0.019)。与对照组比较,survivin基因mRNA的表达量下降(P0.05),caspase-9基因mRNA的表达显著增加(P0.05)。不同温度热疗后,随着加热温度的增高,细胞凋亡率增加,促进了基因p53及caspase-9的表达,凋亡抑制基因survivin的表达量下降。结论通过热疗可以增加p53及caspase-9基因的表达,抑制survivin基因的表达,从而诱导肝癌Hep G2细胞凋亡。

Effect of Hyperthermia on inducing Apoptosis of Human Hepatoma Cell Line HepG2 and its relevant mechanism

Objective To study the effect of hyperthermia at different temperature on the apoptosis of human hepatoma cell line HepG 2. Methods HepG2 cells were cultured with the common method and treated with hyperthermia at different temperature (37℃, 41℃, 42℃and 43℃)respectively for 0.5 h, and 37℃-treated cells were served as control .After cell harvesting , apoptosis was observed with flow cytometer , and the expression of the apoptosis-related gene of p53 was detected by immunohistochemistry staining after cultured for 24-hours, The expression of survivin and caspase-9 mRNA was detected by using real-time fluorescence quantitative PCR (qRT-PCR).SPSS16.0 software was used for statistical analysis .Apoptosis rate, P53 protein value, caspase-9 mRNA expression and other measurement data were presented as mean ± standard deviation, and the differences between groups were compared by t test or by variance.P<0.05 was thought to be of statistically significant difference . Results The results showed that the rates of apoptosis after 24 hours at 37℃, 41℃, 42℃ and 43℃ were (9.58 ±1.22)%、 (16.47 ±3.41)%、(20.07 ± 1.85)%and(23.42 ±1.93)% , respectively.The ODs of P53 protein expression at 37℃, 41℃, 42℃and 43℃were (0.083 ±0.015), (0.145 ±0.024), (0.267 ±0.031), and (0.389 ±0.019), respectively .The rate of cell apoptosis and the expression of P 53 protein were increased gradually with the increase of heating temperature, while the mRNA expression of survivin was decreased (P<0.05, 0.01). Caspase-9 mRNA expression was increased with the increase of heating temperature when compared with that in the control group ( P <0.05, 0.01 ). Conclusions Hyperthermia could induce the apoptosis of hepatoma cell line HepG2 of human through increasing the expression of p 53 and caspase-9 gene, inhibiting the expression of survivin gene .