上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

5-氨基乙酰丙酸介导声动力诱导Hep G2细胞体外凋亡的机制研究

李永宁1，熊小婧2，李青松2,刘莹2，胡铮3，曹文武3   

  1. 1.大连医科大学附属第一医院急诊科， 大连 116011； 2.大连医科大学附属第一医院心内科， 大连 116011； 3．哈尔滨工业大学声光诊疗实验室， 哈尔滨  150001
  • 出版日期:2016-05-28 发布日期:2016-05-26
  • 作者简介: 李永宁（1980—）， 男， 副主任医师， 博士生; 电子信箱: lyn1009@126.com。
  • 基金资助:

    国家自然科学基金（81400247）

Study on the mechanism of in vitro apoptosis induced with 5-aminolevulinic acid-mediated sonodynamic therapy in Hep G2 cells

LI Yong-ning1, XIONG Xiao-jing2, LI Qing-song2, LIU Ying2, HU Zheng3, CAO Wen-wu3   

  1. 1.Department Emergency, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China; 2.Cardiovascular Institute, First Affiliated Hospital of Dalian Medical University, Dalian 116011, China; 3.Laboratory of Sono- and Photo-theranostic Technologies, Harbin Institute of Technology, Harbin 150001, China
  • Online:2016-05-28 Published:2016-05-26
  • Supported by:

    National Natural Science Foundation of China ，81400247

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摘要：

目的 研究5-氨基乙酰丙酸（5-ALA）介导的声动力疗法(SDT)对人肝癌Hep G2细胞的促凋亡作用及机制。方法 取对数期生长Hep G2细胞，通过CCK-8法检测5-ALA和超声干预对细胞存活率的影响，优化出声动力治疗最佳5-ALA药物浓度及超声辐照强度。取对照组（未处理细胞）、5-ALA组、超声组和SDT组Hep G2细胞，流式细胞仪检测细胞凋亡率、坏死率及线粒体膜电位（MMP）的改变，荧光探针DCFH-DA检测活性氧类（ROS）的产生，Western blotting检测凋亡蛋白caspase-3、caspase-9和细胞色素C（Cyt-C）的表达，透射电镜观察细胞超微结构的改变。结果 与对照组、5-ALA组和超声组比较，SDT组Hep G2细胞存活率较低，细胞凋亡率升高，ROS的产生增加，MMP降低，caspase-3、caspase-9和Cyt-C高表达。透射电镜观察发现SDT组Hep G2细胞表面微绒毛减少、内质网和线粒体肿胀、凋亡小体产生。结论 5-ALA介导的SDT诱导Hep G2细胞凋亡以内源性线粒体凋亡为主要途径。

关键词: Hep G2细胞, 5-氨基乙酰丙酸, 声动力治疗, 凋亡

Abstract:

Objective To investigate the underlying mechanisms of cell apoptosis induced by 5-aminolevulinic acid-mediated sonodynamic therapy (5-ALA-SDT) in human hepatocellular carcinoma Hep G2 cells.  Methods The CCK-8 assays were used to examine the effects of 5-ALA and ultrasound intervention on the viability of Hep G2 at logarithmic growth phase. The 5-ALA level and intensity of ultrasonic irradiation with best efficacy were identified. The apoptosis rate, mortality rate, and changes of mitochondrial membrane potential (MMP) in Hep G2 cells in the control group (untreated cells), 5-ALA group, ultrasonic group, and SDT group were detected with flow cytometry assays. The production of reactive oxygen species (ROSs) were detected with the fluorescent probes. Western blotting was used to examine expressions of cytochrome C (Cyt-C) and apoptosis-associated proteins caspase-3 and caspase-9. The ultrastructural changes in cells were observed with the transmission electron microscope. Results The SDT group had lower survival rate, higher apoptosis rate, increased ROS production, decreased MMP, and elevated expressions of caspase-3, caspase-9, and Cyt-C as compared with the control group, 5-ALA group, and ultrasonic group. The transmission electron microscope revealed changes such as reduced cell microvilli, swelling endoplasmic reticulum and mitochondria, and the production of apoptotic bodies in Hep G2 cells in the SDT group. Conclusion Endogenous mitochondrial apoptosis is the main mechanism of 5-ALA-SDT-induced cell apoptosis in Hep G2 cells.

Key words: hepatocellular carcinoma, 5-aminolevulinic acid, sonodynamic therapy, apoptosis