蛇毒抗高凝状态酶对人肝癌细胞及肝细胞作用的研究

Research on function of AHCSE to human hepatocellular carcinoma BEL-7404, SMMC-7721 and normal hepalocellular LO2

目的:研究蛇毒抗高凝状态酶(antihy-percoagulabilitystateenzyme,AHCSE)对人肝癌细胞BEL-7404(简称7404细胞)、SMMC-7721细胞(简称7721细胞)和正常肝细胞LO2(简称LO2细胞)的作用以及探讨其可能的作用机制。方法:应用光学显微镜、透射电镜、MTT法、TUNEL和FCM等方法观察7404细胞、7721细胞和LO2细胞经过AHCSE处理后,细胞形态学和生物化学等方面的变化。结果:7404细胞和7721细胞经过AHCSE处理后,可见悬浮细胞较多,细胞分裂相减少;培养液内细胞碎片明显增多,胞质内部分线粒体空泡化,成簇,胞膜下多见,胞质中髓鞘样结构形成。AHCSE剂量为0·5μg/mL时对7404细胞和7721细胞具有很强的抑制作用,P值分别为0·000和0·025,但对LO2细胞无影响,P=1·000;当剂量增至75μg/mL,AHCSE对7404细胞和7721细胞抑制率上升不明显,P值分别为0·095和0·115,但是出现对LO2细胞的抑制作用,P=0·049。AHCSE作用致7404细胞和7721细胞发生凋亡具有明显的统计学意义,P值均为0·000,凋亡率随AHCSE浓度和作用时间的增加而增加,P值均为0·000。结论:AHCSE对7404细胞、7721细胞具有很强的抑制作用,对LO2细胞影响较小,AHCSE对肝癌细胞作用安全有效,除直接杀伤作用外,诱导肿瘤细胞凋亡是其作用机制之一。

OBJECTIVE： To research the effect of antihypercoagulability state enzyme （AHCSE） on human hepatocellular carcinoma BEL-7404 cells and SMMC-7721 cells, normal Hepalocellular LO2 cells and probe into its function. METHODS： MTT, phasecontrast-microscope, electron-microcopy, terminal deoxynudeotidy transferase dUTP nick and labeling （TUNEL） and flow cytometer （FCM） were conducted to observe the alteration of cell form and biochemistry variety of the AHCSEed cells of BEL-7404 cells, SMMC- 7721 cells and LO2 cells. RESULTS： The AHCSEed cells of BEL- 7404, SMMC-7721 altered in their cells forms. The suspending cells and the cellular debris got more, the process of cell division got less. Part of mitochondrion became hollows and clusters. There were myeline sheath-liked structures in cytoplasm. The 0. 5μg/mL dosage AHCSE had a very strong inhibition to BEL-7404 cells （P=0. 000）. SMMC-7721 cells （P=0. 025）, but nearly no efficacy on LO2 cells （P= 1.000）. With the dosage increased, the dosage AHCSE was 75 μg/mL, the rate of inhibition to BEL-7404 cells and SMMC-7721 cells did not rise significantly, P=0. 095, 0. 115, but an inhibition to LO2 cells appeared （P=0. 049）. After the action of AHCSE apop- tosis took place in BEL-7404 cells （P=1. 000） and SMMC-7721 cells （P= 1.000）, and the apoptosis index increased with the increase of the dosage （P=1. 000） and time （P=1. 000）. CONCLUSIONS： The AHCSE has very strong inhibition to the BEL-7404 cells and SMMC-7721 cells, but little efficacy on LO2 cells. The efficacy of AHCSE on hepatocarcinoma is safe and effective. Besides the direct damaging effect, inducing apoptosis is one of its functions.