摘要
目的观察半边旗二萜类化合物5F(5F)对体外培养的人翼状胬肉成纤维细胞(HPF)的致凋亡作用及其对凋亡相关的半胱氨酸蛋白酶3(caspase-3)表达的影响。方法采用5F不同药物浓度组(0,8,32,128 mg·L-1)作用不同时间处理细胞。噻唑蓝比色法检测5F对细胞增殖的影响;Hoechst33258荧光染色法观察细胞凋亡;流式细胞术检测细胞周期及凋亡;RT-PCR检测细胞内凋亡相关基因caspase-3的转录水平;Western blot检测细胞caspase-3蛋白表达;化学比色法检测细胞凋亡过程中caspase-3活性的变化。结果给药组细胞的生长与对照组相比均受到显著的抑制(P<0.01),细胞增殖抑制率呈剂量和时间的依赖关系;Hoechst33258荧光染色显示,给药组部分细胞呈典型的凋亡表现;流式细胞术检测在32 mg·L-1浓度作用下,12 h即出现“凋亡峰”,其峰值达(12.48±1.29)%;RT-PCR检测发现细胞内凋亡相关基因caspase-3 mRNA表达上调;Western blot检测细胞caspase-3蛋白表达水平升高;caspase-3活性检测显示,其活性呈剂量和时间依赖性增高,5F浓度达到128 mg·L-1时,其活性为对照组的3.52倍。结论5F可显著地抑制HPF增殖、诱导细胞凋亡、升高caspase-3活性,并呈明显的量效与时效关系;其诱导细胞凋亡的作用机制可能与升高caspase-3活性,促进caspase-3基因转录和蛋白翻译,使细胞内caspase-3增加从而促进细胞凋亡有关。
Abstract
OBJECTIVE To observe the induced apoptosis effect of 5F on HPF cultured in vitro and the expression of caspase-3.METHODS Cell were treated with different concentration of 5F(0,8,32 and 128 mg·L-1)with different time.The effect of 5F on cell proliferation was detected by thiazole blue chromatomentry.Cell apoptosis was observed by Hoechst33258 fluorescent staining;flow cell cycle and apoptosis were investigated by flow cytometry.The transcription level of caspase-3 were determined by RT-PCR;The expression of caspase-3 protein was detected by Western blot;The change of caspase-3′s activity during the process of cell apoptosis was valued by chemistry chromatometry.RESULTS Compared with the control group,the cell growth in experimental group was inhibited significantly(P<0.01),and the cell proliferation inhibition rate showed a dose-and time-dependent manner.Hoechst33258 fluorescent staining displayed that cells in experimental group partly showed typical apoptosis.Under the effect of 5F with the concentration of 32.18 mg·L-1,the peak of apoptosis was reached at 12 h and was(12.48±1.29)%;RT-PCR showed that the expression of caspase-3 mRNA in cells was increased.Western blot showed that the expression of caspase-3 protein was increased.The caspase-3 activity was increased dose-and time-dependerthy.The activity was 3.52 times as control group when the concentration of 5F was 128 mg·L-1.CONCLUSION 5F can inhibit the proliferation of PHF,induce cell apoptosis,increases the activity of caspase-3,and show a dose-and time-dependent manner.The mechanism of 5F inducing cell apoptosis may relate with the increasing of caspase-3 activity,facilitating of transcription of caspase-3 and protein translation,increasing of casepase-3 in order to accelerate the cell apoptosis.
关键词
半边旗 /
二萜类 /
翼状胬肉 /
成纤维细胞 /
凋亡 /
半胱氨酸蛋白酶3
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Key words
Peris semipinnata L /
diterpeniod /
pterygium /
fibroblasts /
apoptosis /
caspase-3
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林意玲;吴平;廖海兰;张培华c;何惠娟;江黎明.
半边旗二萜类化合物5F诱导人翼状胬肉成纤维细胞凋亡及对caspase-3表达的研究[J]. 中国药学杂志, 2006, 41(13): 993-997
LIN Yi-ling;WU Ping;LIO Hi-ln;ZHNG Pei-huc;HE Hui-jun;JING Li-ming.
Experimental Research of Apoptosis in Pterygium Fibroblasts Induced by 5F and Effect on the Expression of Caspase-3 [J]. Chinese Pharmaceutical Journal, 2006, 41(13): 993-997
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参考文献
[1] SOLOMON A，LI D Q，LEE S B，et al. Regulation of collagenase，stromelysin，and urokinase-type plasminogen activator in primary pterygium body fibroblasts by inflammatory cytokines[J] ．Invest Ophthalmol Vis Sci，2000，41（8）：2154-2163．
[2] ZHANG X，CUI L，NOBUTOSHI T，et al．The active constituents antitumor action of Pteris semipinnata[J] ．Chin Pharm J(中国药学杂志)，1997，32（1）：37-38．
[3] LI J H，LIANG N C，MO L E，et al．Comparison of the cytotoxicity of five constituents from pteris se mipinnatl. In vitro and the analysis of their structure activity relationships[J] ．Acta Pharm Sin（药学学报），1998，33（9）：641-644．
[4] WANG J B，LIANG N C，MO L E．The effects of a diterpenoid compound 5F isolated from Pteris semipinnata L．On the expressions of several oncogenes of K562 cells[J] ．Chin Pharmacol Bull(中国药理学通报)，2002，18（4）：418-421．
[5] LI J H，HE C W，LIANG N C，et al. Effects of antitumor compounds isolated from Pteris semipinnata L on DNA topoisomerases and cell cycle of HL-60 cells[J] ．Acta Pharmacol Sin(中国药理学报)，1999，20(6)：541-545．
[6] LI C L，CUI Z Y，LI S Z．A modified chromatometry method and its application for MTT[J] ．Shanghai J Immunol(上海免疫学杂志)，1996，16（5）：306-307．
[7] WINTER R N，KRAMER A，BORKOWSKI A，et al. Loss of caspase-1 and caspase-3 protein expression in human prostate cancer[J] ．Cancer Res，2001，61(3)：1227-1232．
[8] RAMACHANDRAN A，MOELLERING D R，CEASER E，et al. Inhibition of mitochondrial protein synthesis results in increased endothelial cell susceptibility to nitric oxide-induced apoptosis[J] ．Proc Natl Acad Sci，2002 ，99(10)：6643-6648．
[9] DING H S.Chinese Pharmacon Spore-Producing Plant(中国药用孢子植物)[M] ．Shanghai：Shanghai Science Technology Publishing Company，1971：104．
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脚注
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基金
国家自然科学基金资助项目(No39870900);广东省科委重大科技攻关项目(9622007002)
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