定位在线粒体的肝刺激因子对线粒体膜电势的影响  

Effect of HSS on mitochondrial membrane potential

作　　者：张静1 张静2 李胜兰[1] 吴媛[1] 安威[1] 

机构地区：[1]首都医科大学细胞生物学肝脏保护与再生北京重点实验室,北京100069

出　　处：《中国组织化学与细胞化学杂志》2013年第2期89-94,共6页Chinese Journal of Histochemistry and Cytochemistry

基　　金：国家自然科学基金资助(81070352)

摘　　要：目的肝刺激因子(hepatic stimulator substance,HSS)可以保护肝细胞免受各种毒素的影响,但机制尚未清楚,研究探讨肝刺激因子保护肝细胞的可能机制。方法利用稳定转染FLAG-pcDNA 3.0/hHSS的肝癌细胞BEL-7402为模型,使用Alexa Flour 488、Hoechst 33342、MitoTracker 580分别将HSS、细胞核以及线粒体染色,观察HSS在细胞中的定位情况。当野生型7402细胞、转染空载体FLAG-pcDNA 3.0的7402细胞以及转染FLAG-pcDNA 3.0/hHSS的7402细胞受到线粒体膜孔道开放剂羰基氰化间氯苯腙(carbonyl cyanide m-chlorophenylhydrazone,CCCP)的损伤后,用电镜观察线粒体形态、荧光素酶检测ATP、流式细胞仪测定线粒体膜电位(mitochondrial membrane potential,MMP)等,综合观察过表达HSS的肝细胞的抗损伤能力。结果在稳定转染hHSS基因的7402细胞中,大部分HSS与线粒体共定位;在CCCP作用下,对照组野生型7402细胞以及转染空载体的7402细胞MMP下降明显,线粒体肿胀,嵴断裂、消失,ATP下降显著;实验组稳定转染hHSS基因的7402细胞MMP下降幅度较小,线粒体肿胀与嵴形态的改变明显减轻,ATP的含量较对照组高。结论肝刺激因子HSS在细胞中主要定位于线粒体,可以稳定MMP,维持线粒体形态及细胞内ATP的水平,从而增强肝细胞抗损伤的能力。Objective Hepatic stimulator substance （HSS） can protect hepatocytes from virious toxins, but the mechanism remains unknown. Our study focuses on the possible mechanisms. Methods Human liver cancer cell line BEL-7402,which can stably express HSS ,was used to undergo co-immunofluo- reseence experiment, BEL-7402, BEL-7402/peDNA 3.0 and BEL-7402/hHSS were treated with carbonyl cyanide m-chlorophenylhydrazone （CCCP）, a specific agent that leads to depolarization of the mitochondrial transmembrane potential. DiICI（5）, a mitochondria-specific dye~ was used to evaluate the mitochondri- al membrane potential （MMP）. CellTiter-Glo assay was used to evaluate ATP content, and transmis- sion electronmicroscopy （TEM） was applied to observe the mitochondria structure. Results HSS was mainly localized in the mitochondria of hepatoma cells. In the HSS-transfected cells, loss of MMP was markedly reduced after CCCP treatment as compared with that of the wild-type cells and the vector-transfected cells. The ATP content of the 3 types of cells reduced to a low level, but the amount of ATP in the hHSS-transfected cells was still greater than that in the wild-type cells and the vector-transfected cells. In the wild-type and vector-transfected cells, the mitoehondrial ultrastructures were different from those of HSS-transfected cells, including enlargement in volume and deficient or swollen cristae. Conclusion HSS is mainly localized in the mitochondria of hepatoma cells. In the hHSS-transfected cells, disruption of mito- chondrial membrane potential （MMP） is reduced, and ATP levels are maintained. In addition, HSS expression protects hepatic mitochondrial structure.

关 键 词：肝刺激因子 线粒体 线粒体膜电势转换 ATP 

分 类 号：R735.7[医药卫生—肿瘤]