化学合成抗菌肽tachyplesin对肿瘤细胞作用机理的初步研究
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摘要
抗菌肽是一类带正电、多具双亲性的小分子多肽，由于其独
特的结构，抗菌肽对多种细菌和癌细胞有强烈的抑制作用，且多
数抗菌肽在发挥以上作用时对正常细胞没有破坏作用。
Tachyplesin-I是鲎(Tachypleus tridentatus)血液中的一类具β结
构的环状抗菌肽，抗菌作用强烈，是进行抗癌机制研究的一个好
的模板。
本课题以人低分化胃腺癌BGC-823细胞株为模型，探讨人工
化学合成抗菌肽tachyplesin-I对癌细胞的生物学效应和可能的作
用机制。为此，我们首先测定了tachyplesin-I对多种肿瘤细胞存
活率的影响及对BGC-823细胞生长的抑制作用，观察了
tachyplesin-I作用下细胞的形态学变化；然后主要从对膜作用角
度入手，检测与观察荧光探针FDA、Dextran-FITC、JC-1、
YO-PRO-1的强度和分布，分析tachyplesin-I作用后细胞膜、细
胞核和线粒体膜系统的变化。
结果显示：BGC-823细胞的存活率及生长动态受到很大影响；
细胞形态也发生变化，细胞膜出现不完整甚至大面积破损崩溃现
象，线粒体肿胀，次级溶酶体增生，部分细胞核膜崩溃；荧光分
光光度计检测到tachyplesin-I 2小时处理后各组的相对荧光
强度增加，说明荧光均有外泄，细胞膜均受到损伤，低于50 μg／mL
的实验组相对荧光强度在15． 14-17． 84之间，而50 μg／ml以上的
实验组相对荧光强度在60． 54-67． 57之间，存在阈值作用；激光
扫描共聚焦显微镜下观察到Dextran-FITC经2小时处理未能进入
细胞内部，24小时处理后，细胞允许40KD的大分子探针进入，
说明经长时间作用细胞膜受到较大的损伤；正常人红细胞在
tachyplesin-I作用下发生溶血作用，溶血率在26． 66％-44． 39％
之间；激光扫描共聚焦显微镜观察到JC-1染色由强红色变弱，大
多数呈绿色，流式细胞仪检测到绿色荧光强度有所升高，红色荧
光强度大幅降低，说明线粒体膜电位去极化；流式细胞仪检测到
YO干 1绿色荧光增强，说明核膜通透性增加。
结论如下：人工合成抗菌肽tachaplesin刁通过细胞杀伤的途
径抑制肿瘤细胞；作用机制首先是通过对膜作用，其后tachyplesin－
I可能进一步通过对膜作用而使膜上孔洞不断扩大或迅速进入细
胞内部作用在核与线粒体上达到对肿瘤细胞的杀伤低浓度和高
浓度tachyplesin－I对膜有不同的作用方式，但只有高浓度的
tachyplesin刁最终造成细胞死亡。
Antimicrobial peptides, the majority of which are cationic and amphipathic, are small peptides. They can strongly act on many kinds of bacterial and cancer cells, while at the same concentrations they act weakly on normal cells. The ability is due to their unique structures. This selective toxicity has attracted much attention. Now some progress has made on the linear antimicrobial peptides in inhibiting bacterial and cancer cells but not in the case of cyclic peptides. Tachyplesin- I , a small antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus ) , which has a cyclic anti-parallel 3 -sheet structure held together by two disulfide bonds, permeabilizes both bacterial and artificial lipid membranes at low concentrations. It suggests that tachyplesin-1 is also a potent anticancer agent.
In this experiment, we aim at exploring whether, how and to what extent the synthetical antimicrobial peptide tachyplesin inhibits human gastric adenocarcinoma BGC-823 cell. Firstly, we test the effect of tachyplesin- I on cell proliferation, livability and morphology , and then we test the integrity of vital membranes via the change of intensity and distribution of FDA Dextran-FITC JC-1 and YO-PRO-1 probes.
The results show that tachyplesin-1 inhibited the proliferation of BGC-823 cell and reduced the survival ratio of cancer cells. Observation from transmission electron microscope shows tachyplesin- I broke cell membranes and nucleus membranes,
induced mitochondria swelling and increased the number of secondary lysosome. Staining with fluorescent probes of FDA Dextran-FITC JC-1 and YO-PRO-1 indicated that tachyplesin- I could permeabilize cell membranes, induce potential shift of mitochondria membrane and damage on nucleus membranes. Hemolytic test indicated tachyplesin- I could damage on human erymrocyte membranes at low level.
Based on what we have found, we can give a general idea about the anticancer mechanism of tachyplesin- I : 1 ) Synthetical antimicrobial peptide tachyplesin-1 inhibits the BGC-823 cell via the pass way of cytotoxicity; 2) Interaction of the peptide with membrane is the first step, and then possibly there are two ways, the big pores form, or the peptide enters the cell to reach an alternative target, including mitochondria and nucleus; 3 ) Tachyplesin- I interacts with membrane via two ways depending on its concentrations.
特的结构，抗菌肽对多种细菌和癌细胞有强烈的抑制作用，且多
数抗菌肽在发挥以上作用时对正常细胞没有破坏作用。
Tachyplesin-I是鲎(Tachypleus tridentatus)血液中的一类具β结
构的环状抗菌肽，抗菌作用强烈，是进行抗癌机制研究的一个好
的模板。
本课题以人低分化胃腺癌BGC-823细胞株为模型，探讨人工
化学合成抗菌肽tachyplesin-I对癌细胞的生物学效应和可能的作
用机制。为此，我们首先测定了tachyplesin-I对多种肿瘤细胞存
活率的影响及对BGC-823细胞生长的抑制作用，观察了
tachyplesin-I作用下细胞的形态学变化；然后主要从对膜作用角
度入手，检测与观察荧光探针FDA、Dextran-FITC、JC-1、
YO-PRO-1的强度和分布，分析tachyplesin-I作用后细胞膜、细
胞核和线粒体膜系统的变化。
结果显示：BGC-823细胞的存活率及生长动态受到很大影响；
细胞形态也发生变化，细胞膜出现不完整甚至大面积破损崩溃现
象，线粒体肿胀，次级溶酶体增生，部分细胞核膜崩溃；荧光分
光光度计检测到tachyplesin-I 2小时处理后各组的相对荧光
强度增加，说明荧光均有外泄，细胞膜均受到损伤，低于50 μg／mL
的实验组相对荧光强度在15． 14-17． 84之间，而50 μg／ml以上的
实验组相对荧光强度在60． 54-67． 57之间，存在阈值作用；激光
扫描共聚焦显微镜下观察到Dextran-FITC经2小时处理未能进入
细胞内部，24小时处理后，细胞允许40KD的大分子探针进入，
说明经长时间作用细胞膜受到较大的损伤；正常人红细胞在
tachyplesin-I作用下发生溶血作用，溶血率在26． 66％-44． 39％
之间；激光扫描共聚焦显微镜观察到JC-1染色由强红色变弱，大
多数呈绿色，流式细胞仪检测到绿色荧光强度有所升高，红色荧
光强度大幅降低，说明线粒体膜电位去极化；流式细胞仪检测到
YO干 1绿色荧光增强，说明核膜通透性增加。
结论如下：人工合成抗菌肽tachaplesin刁通过细胞杀伤的途
径抑制肿瘤细胞；作用机制首先是通过对膜作用，其后tachyplesin－
I可能进一步通过对膜作用而使膜上孔洞不断扩大或迅速进入细
胞内部作用在核与线粒体上达到对肿瘤细胞的杀伤低浓度和高
浓度tachyplesin－I对膜有不同的作用方式，但只有高浓度的
tachyplesin刁最终造成细胞死亡。
Antimicrobial peptides, the majority of which are cationic and amphipathic, are small peptides. They can strongly act on many kinds of bacterial and cancer cells, while at the same concentrations they act weakly on normal cells. The ability is due to their unique structures. This selective toxicity has attracted much attention. Now some progress has made on the linear antimicrobial peptides in inhibiting bacterial and cancer cells but not in the case of cyclic peptides. Tachyplesin- I , a small antimicrobial peptide from the hemocytes of the horseshoe crab (Tachypleus tridentatus ) , which has a cyclic anti-parallel 3 -sheet structure held together by two disulfide bonds, permeabilizes both bacterial and artificial lipid membranes at low concentrations. It suggests that tachyplesin-1 is also a potent anticancer agent.
In this experiment, we aim at exploring whether, how and to what extent the synthetical antimicrobial peptide tachyplesin inhibits human gastric adenocarcinoma BGC-823 cell. Firstly, we test the effect of tachyplesin- I on cell proliferation, livability and morphology , and then we test the integrity of vital membranes via the change of intensity and distribution of FDA Dextran-FITC JC-1 and YO-PRO-1 probes.
The results show that tachyplesin-1 inhibited the proliferation of BGC-823 cell and reduced the survival ratio of cancer cells. Observation from transmission electron microscope shows tachyplesin- I broke cell membranes and nucleus membranes,
induced mitochondria swelling and increased the number of secondary lysosome. Staining with fluorescent probes of FDA Dextran-FITC JC-1 and YO-PRO-1 indicated that tachyplesin- I could permeabilize cell membranes, induce potential shift of mitochondria membrane and damage on nucleus membranes. Hemolytic test indicated tachyplesin- I could damage on human erymrocyte membranes at low level.
Based on what we have found, we can give a general idea about the anticancer mechanism of tachyplesin- I : 1 ) Synthetical antimicrobial peptide tachyplesin-1 inhibits the BGC-823 cell via the pass way of cytotoxicity; 2) Interaction of the peptide with membrane is the first step, and then possibly there are two ways, the big pores form, or the peptide enters the cell to reach an alternative target, including mitochondria and nucleus; 3 ) Tachyplesin- I interacts with membrane via two ways depending on its concentrations.
引文
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