定位在线粒体的肝刺激因子对线粒体膜电势的影响

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-pcDNA3．0／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-pcDNA3．0的7402细胞以及转染FLAppcDNA3．0／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（mitoehondrial 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。

Effect of HSS on mitochondrial membrane potential

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.