论文部分内容阅读
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 various toxins, but the mechanism is not yet clear. The possible mechanism of hepatic stimulator in protecting hepatocytes was explored. Methods HSL, nucleus and mitochondria were stained with Alexa Flour 488, Hoechst 33342 and MitoTracker 580 by using BEL-7402 hepatoma cells stably transfected with FLAG-pcDNA 3.0 / hHSS. The localization of HSS in cells was observed. When wild-type 7402 cells, 7402 cells transfected with the empty vector FLAG-pcDNA 3.0 and 7402 cells transfected with FLAG-pcDNA 3.0 / hHSS were stimulated by the mitochondrial membrane pore opener carbonyl cyanide m-chlorophenylhydrazone (CCCP ), The morphology of mitochondria was observed by electron microscope, ATP was detected by luciferase, and mitochondrial membrane potential (MMP) was measured by flow cytometry. The anti-injury ability of hepatocytes over-expressing HSS was observed. Results Most HSS colocalized with mitochondria in 7402 cells stably transfected with hHSS gene. Under the action of CCCP, the MMPs of wild-type 7402 cells in control group and 7402 cells transfected with empty vector were significantly decreased, mitochondria swollen and cristae fractured and disappeared , ATP decreased significantly; experimental group stably transfected hHSS gene 7402 cells MMP decreased less mitochondrial swelling and cristae morphology significantly reduced, ATP content higher than the control group. Conclusions HSS mainly located in mitochondria in cells, which can stabilize MMPs, maintain mitochondrial morphology and intracellular ATP levels, and thus enhance the ability of hepatocytes to resist damage.