家蝇幼虫抗菌肽对K562细胞膜的质量浓度阈值作用

中国媒介生物学及控制杂志 ›› 2012, Vol. 23 ›› Issue (1): 42-47.

家蝇幼虫抗菌肽对K562细胞膜的质量浓度阈值作用

程璟侠¹, 樊宏英², 赵瑞君³

1. 山西省疾病预防控制中心病媒生物防控科,山西太原 030012;
2. 湖北省荆州市第一人民医院;
3. 山西医科大学寄生虫学教研室,山西太原 030001

收稿日期:2011-08-23 出版日期:2012-02-20 发布日期:2012-02-20
通讯作者: 赵瑞君,Email: 4935186aaa2272@sina.com
作者简介:程璟侠(1963-),女,主任医师,主要从事病媒生物防制及科研工作。Email: chengjingxia007@163.com
基金资助:
山西省高校科技研究开发项目(200613011);山西省科技攻关项目(2006031087-04);太原市科学技术发展计划项目(081049)

Mass concentration threshold of Musca domestica larvae antimicrobial peptides in K562 cell membrane

CHENG Jing-xia¹, FAN Hong-ying², ZHAO Rui-jun³

1. Prevention and Control of Vectors Division, Shanxi Center for Disease Control and Prevention, Taiyuan 030012, Shanxi Province, China;
2. Office of Hospital Infection Control, First People's Hospital of Jingzhou;
3. Institute of Parasitology, Department of Parasitology, Shanxi Medical University, Taiyuan 030001, Shanxi Province, China

Received:2011-08-23 Online:2012-02-20 Published:2012-02-20
Supported by:
Supported by Universities Science and Technology Research and Development Item of Shanxi Province(No. 200613011), Tackle Key Problems in Science and Technology Item of Shanxi Province(No. 2006031087-04)and Plan Item of Science and Technology Development of Taiyuan City(No. 081049)

摘要/Abstract

摘要：

目的探讨家蝇3龄幼虫抗菌肽对K562细胞膜的作用。方法通过针刺感染大肠埃希菌诱导家蝇3龄幼虫大量表达抗菌肽,然后经过研磨、离心、固相萃取、反相高效液相色谱分离纯化家蝇3龄幼虫抗菌肽,采用四甲基偶氮噻唑蓝比色法和光镜观察法筛选对K562有抑制作用的抗菌肽；用不同质量浓度的峰5、峰8处理K562细胞,采用台盼蓝拒染法测定细胞存活率和生长曲线的变化；用光学显微镜和荧光显微镜检测细胞膜形态的变化；用荧光分光光度计检测细胞膜荧光素的渗漏；用激光共聚焦显微镜观察细胞膜的损伤程度。结果低质量浓度(＜50 μg/ml)对细胞存活率无明显影响,细胞整体形态变化不大,细胞荧光素渗漏较少［(18.95±0.05)～(22.49±0.68)］,细胞损伤甚微；高质量浓度(≥50 μg/ml)使细胞存活率明显下降,细胞形态变化逐渐增大,细胞荧光素渗漏较多［(62.77±4.08)～(70.81±0.18)］,甚至大分子荧光素Dextran-FITC可以通过。结论家蝇3龄幼虫抗菌肽对K562细胞膜的作用存在质量浓度阈值,低质量浓度时可引起膜通透性增加,高质量浓度时可引起细胞膜严重损伤。家蝇幼虫抗菌肽可能通过直接杀伤作用抑制K562细胞生长,其作用机制首先是通过对膜作用,然后可能进一步对膜作用从而使膜上孔洞不断扩大或者进入细胞后经过其他途径抑制K562细胞生长。

关键词: 家蝇幼虫, 抗菌肽, 肿瘤细胞, K562, 膜渗漏

Abstract:

Objective To investigate the effect of Musca domestica larva antimicrobial peptides on the cell membrane of tumor cells K562. Methods Antimicrobial peptides of M. domestica larvae with inhibition activity to K562 were sieved, through some processes that induced by acupuncture with Escherichia coli induction, isolated and purified by trituration, centrifuga1ization, solid phase extraction (SPE) and reversed-phase high-performance liquid chromatography (RP-HPLC), detected activity by MTT colorimetric method and observed light microscope. After treatment with different concentrations of peaks 5 and 8, the viability, morphological changes, morphological changes, membrane fluorescein leakage and destruction of K562 cells were detected by trypan blue excluding test, optical microscope and fluorescence microscope, spectrofluorophotometer, laser confocal microscope (LSCM), respectively. Results Low concentrations (＜50 μ g/ml) of M. domestica antimicrobial peptides resulted in intact viability, little change of cellular structure, a little of fluorescein leakage [(18.95±0.05)-(22.49±0.68)] and few destruction in K562 cells; however, high concentrations (≥50 μg/ml) of M. domestica antimicrobial peptides induced lower viability, cellular form change increased gradually, a great deal of fluoresceinleakage [(62.77±4.08)-(70.81±0.18)], even macromolecular fluorescent element Dextran-FITC could leak through. Conclusion There is a concentration threshold of M. domestica larvae antimicrobial peptides on K562 cell membrane. Low concentrations of M. domestica larvae antimicrobial peptides can cause the membrane leakage increasing of cell membrane, high concentrations can lead to severe damage of cell membrane, respectively. The antimicrobial peptides inhibit the growth of K562 cells through directly killing the cells, and the mechanism is that firstly damage the membrane function, then may expand the holes or inhibit the growth by other ways after entering the cells.

Key words: Musca domestica larvae, Antimicrobial peptides, Tumor cells, K562, Membrane leakage

中图分类号:

R384.2

程璟侠, 樊宏英, 赵瑞君. 家蝇幼虫抗菌肽对K562细胞膜的质量浓度阈值作用[J]. 中国媒介生物学及控制杂志, 2012, 23(1): 42-47.

CHENG Jing-xia, FAN Hong-ying, ZHAO Rui-jun. Mass concentration threshold of Musca domestica larvae antimicrobial peptides in K562 cell membrane[J]. Chines Journal of Vector Biology and Control, 2012, 23(1): 42-47.

参考文献

[1] Rodrigues EG,DobroffA S,Taborda CP,et al. Antifungal andantitumor models of bioactive protective peptides
[J]. An Acad BrasCienc,2009,81(3):503-520.

[2] Suttmann H,Retz M,Paulsen F,et al. Antimicrobial peptides ofthe Cecropin-family show potent antitumor activity against bladdercancer cells
[J]. BMC Urol,2008,8:5.

[3] Mader JS,Hoskin DW. Cationic antimicrobial peptides as novelcytotoxic agents for cancer treatment
[J]. Expert Opin InvestigDrugs,2006,15(8):933-946.

[4] Lohner K. New strategies for novel antibiotics:peptides targetingbacterial cell membranes
[J]. Gen Physiol Biophys,2009,28(2):105-116.

[5] Silvestro L,Gupta K,Weiser JN,et al. The concentration-dependentmembrane activity of cecropin A
[J]. Biochemistry,1997,36(38):11452-11460.

[6] Heller WT,Waring AJ,Lehrer RI,et al. Membrane thinning effectof the beta-sheet antimicrobial protegrin
[J]. Biochemistry,2000,39(1):139-145.

[7] 王远程,左晓锋,孙东旭,等. 家蝇幼虫抗菌物质组成及其理化性质
[J]. 微生物学报,1997,37(2):148-153.

[8] 王继恒,曲传智. 昆虫抗菌肽的研究进展
[J]. 中国媒介生物学及控制杂志,2000,11(5):392-395.

[9] 周义文,姬尚义,申萍,等. 家蝇抗菌肽的免疫诱导及抗菌活性研究
[J]. 临床检验杂志,2008,26(1):24-26.

[10] 赵瑞君,程璟侠,代培芳,等. 家蝇蛹抗菌肽对肿瘤细胞膜破坏作用
[J]. 中国公共卫生,2007,23(11):1399-1400.

[11] 马卫列,丁航,符伟玉,等. 三羟异黄酮对高转移卵巢癌细胞株HO-8910PM的侵袭、转移及MTA1、nm23H_1基因表达的影响
[J]. 广东医学院学报,2006,24(3):230-232.

[12] Cotter TG,Lennon SV,Glynn JG,et al. Cell death via apoptosis andits relationship to growth,development and differentiation of bothtumor and normal cells
[J]. Anticancer Res,1990,10(SA):1153-1159.

[13] Degos L. A new concept:treatment using differentiation ofmalignant cellinman
[J]. Bull Aead Natl Med,1995,179(8):1689-1700.

[14] 张双全,贾红武,戴祝英. 抗菌肽CM4抗K562癌细胞的超微结构研究
[J]. 生物化学与生物物理进展,1997,24(2):159-163.

[15] 许玉澄,张双全,戴祝英. 家蚕抗菌肽的抗癌作用
[J]. 动物学研究,1998,19(4):263-268.

[16] 郭玉梅,戴祝英,胡云龙. 家蚕抗菌肽的一些性质及抗肿瘤活性
[J]. 南京师范大学学报:自然科学版,1995,18(1):62-67.