鲎血细胞多肽诱导HL-60细胞凋亡

Apoptosis of HL-60 Cells Induced by Peptide from Hemocyte of Horseshoe Crab

目的：研究鲎血细胞多肽（下称鲎血多肽）诱导ＨＬ－６０细胞凋亡的作用。方法：ＭＴＴ法测定鲎血多肽对ＨＬ－６０细胞的毒性和ＨＬ－６０细胞的相对存活率，荧光显微镜观察ＨＬ－６０细胞形态的变化，流式细胞仪鉴定细胞凋亡和分析细胞周期，扫描电镜观察细胞膜的改变。结果：鲎血多肽对ＨＬ－６０细胞有明显的细胞毒性，ＩＣ５０值为２４ μｇ／ｍｌ；荧光显微镜观察到凋亡细胞主要表现为细胞体积缩小，核染色质固缩，荧光染色增强。５０－１００μｇ／ｍｌ的鲎血多肽处理６ｈ， ＨＬ－６０细胞主要表现为细胞凋亡；而鲨血多肽浓度为２００ μｇ／ｍｌ时主要为细胞坏死。５０ μｇ／ｍｌ鲎血多肽处理 ＨＬ－６０细胞０～１２ ｈ，流式细胞仪检测到凋亡细胞亚二倍体峰出现，细胞周期分析 Ｇ１期细胞减少，Ｇ２期细胞增加。鲎血多肽浓度为２５－１００μｇ／ｍｌ时，细胞凋亡率随浓度增加而增加，鲎血多肽为２００ μｇ／ｍｌ时，则主要为细胞坏死，凋亡细胞减少。扫描电镜观察发现，ＨＬ－６０细胞用鲎血多肽处理后，细胞膜损伤，出现空洞。结论：鲎血细胞多肽能诱导ＨＬ－６０细胞凋亡；凋亡的发生较早，与细胞膜受损有一定的关系；Ｇ１期细胞对鲎血多肽更敏感。

Objective: This study was designed to investigate the apoptosis of HL-60 cells induced by the peptide from hemocyte of horseshoe crab (horseshoe crab hemocyte peptide). Methods: Cytotoxicity and cell viability were assayed by MTT method. Morphologic assessment of apoptosis was performed with fluorescence microscope; the apoptosis of the cells was confirmed by flow cytometry; cell cycle was analyzed by flow cytometry, changes of cell membrane were observed by scanning electron microscope. Results: HL-60 cells treated with horseshoe crab hemocyte peptide were showed apparently cytotoxicity with IC50 24 μg/ml. The morphologic changes including reduction in volume, nuclear chromatin condensation, fluorescence strength were observed with fluorescence microscope. Treated with horseshoe crab hemocyte peptide 50 - 100 μg/ml for 6 h, apoptostic HL-60 cells were predominant. However the HL-60 cells were shown necrotic and apoptostic cells were reduced when treatment with horseshoe crab hemocyte peptide of 200 μg/ml. SubG1 peak was detected by flow cytometry. Cell cycle analysis showed Phase G1 cells decreased and Phase G2 cells increased with horseshoe crab hemocyte peptide 50 μg/ml treating for 0 - 12 h. Ratio of apoptosis increase depended on concentrations of horseshoe crab hemocyte peptide in 25 - 100 μg/ml but it reduced in 200 μg/ml, the HL-60 cells were shown necrotic mainly. Scanning electron microscope showed membrane of HL-60 cells was damaged by horseshoe crab hemocyte peptide treatment, cell membrane showed some holes. Conclusion: Peptide from hemocytes of horseshoe crab can induce apoptosis in HL-60 cell, it was early event, and may correlate with membrane damage. The HL-60 cell in Phase G1 seemed to he sensitive to the peptide.