金纳米棒联合^(125)I粒子诱导肝癌HepG_2细胞凋亡的实验研究

Apoptosis of hepatoma cell line HepG_2 induced by the combination use of GNRs@SiO_2-FA and ^(125)I seeds:an experimental study

目的探讨叶酸偶联二氧化硅包覆的金纳米棒(GNRs@Si O2-FA)联合125I粒子诱导肝癌Hep G2细胞凋亡及其可能的机制,为临床提高肝癌125I粒子组织间植入疗效提供理论依据。方法将体外培养的肝癌Hep G2细胞随机分成空白对照组,单纯金纳米棒组,单纯125I粒子照射组及金纳米棒联合125I粒子照射组,采用流式细胞仪检测细胞凋亡情况,PT-PCR检测bax m RNA、bcl-2 m RNA表达水平,免疫细胞化学检测凋亡相关基因bax、bcl-2表达水平及肿瘤细胞核增殖性抗原Ki67表达情况。结果流式细胞仪检测4组肝癌Hep G2细胞凋亡率,单纯金纳米棒组、单纯125I粒子照射组较空白对照组均有升高(P<0.05);联合组较单纯金纳米棒组和单纯125I粒子照射组明显升高,差异有统计学意义(P<0.05)。联合组bax m RNA表达明显高于单纯金纳米棒组与单纯125I粒子照射组,bcl-2 m RNA表达明显低于单纯金纳米棒组与单纯125I粒子照射组。肝癌Hep G2细胞bax表达于胞质,bcl-2表达于胞质和胞膜,Ki67表达于胞核,均表现为棕黄色细颗粒状沉淀。4组肝癌Hep G2细胞凋亡相关基因bax、bcl-2及增殖核抗原Ki67蛋白表达量比较,差异均有统计学意义(P<0.05);联合组较单纯金纳米棒组和单纯125I粒子照射组Bax蛋白阳性表达率明显升高,Bcl-2、Ki67蛋白阳性表达率明显降低,差异有统计学意义(P<0.05)。结论金纳米棒与125I粒子联合应用能更有效增加肝癌细胞凋亡率,其机制可能是通过增加Bax蛋白的表达,抑制Bcl-2蛋白的表达来实现。

Objective To explore the possible mechanism of the apoptosis of hepatoma cell line Hep G2 induced by the combination use of GNRs@Si O2-FA and^125I seeds and to discuss its relationship with Bcl-2 and Bax protein expressions so as to provide theoretical basis for clinical treatment of hepatic cancer with interstitial brachytherapy by using^125I seeds.Methods In vitro cultured Hep G2 cells were randomly divided into 4 groups：blank control group（not treated）,simple GNRs@SiO2-FA group,simple^125I seeds group,and combination group（GNRs@Si O2-FA plus^125I seeds）.The apoptosis of Hep G2 cells was determined by flow cytometry.The expression of Bax mRNA and Bcl-2 mRNA of Hep G2 cells were tested by RT-PCR.The apoptosis-related genes（Bax and Bcl-2） and the tumor proliferation cell nuclear antigen（Ki67） proteins expression on Hep G2 cells were examined with immunohistochemistry method.Results The flow cytometry examination showed that the apoptosis rate of Hep G2 cells in the simple GNRs@Si O2-FA group and simple^125I seeds group was higher than that in blank control group（P〈0.05）,and the apoptosis rate of the combination group was significantly higher than that of the simple GNRs@Si O2-FA group and the simple^125I seeds group（P〈0.05）.The expression level of Bax mRNA in the combination group was higher than that in the simple GNRs@Si O2-FA group and simple^125I seeds group,while the expression level of Bcl-2 mRNA in the combination group was obviously lower than that in the simple GNRs@Si O2-FA group and simple^125I seeds group.Bax protein was expressed on cytoplasm,Bcl-2 protein was expressed on cytoplasm and cell membrane,while Ki67 protein was expressed on nucleus.All of them presented as brown finely granular precipitations.Statistically significant differences in the amount of Bax,Bcl-2 and Ki67 protein expression existed between each other among the four groups（P〈0.05）.The positive expression rate of Bax protein in the combination group was significantly higher than that of the simple GNRs@Si O2-FA group and the simple^125I seeds group,while the positive expression rate of Bcl-2 and Ki67 protein was significantly lower than that of the simple GNRs@Si O2-FA group and the simple^125I seeds group,and the differences were statistically significant（P〈0.05）.Conclusion The combination use of GNRs@Si O2-FA and^125I seeds can more effectively induce the apoptosis of Hep G2 cells.This effect may be accomplished through increasing the expression of Bax protein and inhibiting the expression of Bcl-2 and Ki67 proteins.