中文摘要：

目的：研究天花粉蛋白（trichosanthin,TCS）抑制人乳腺癌MDA-MB-231细胞增殖和诱导细胞凋亡过程中脾酪氨酸激酶（spleen tyrosine kinase,syk）基因甲基化状态的改变,以寻找新的去甲基化药物。方法：采用MTT法分析TCS对MDA-MB-231细胞增殖的影响,Giemsa染色法观察TCS作用后细胞形态学的变化,FCM检测不同浓度TCS作用48h后MDA-MB-231细胞凋亡和细胞周期分布情况,甲基化特异性PCR（methylation-specific PCR,MSP）法检测MDA-MB-231细胞经TCS作用前后syk基因甲基化状态的改变,RT-PCR方法分析TCS作用后MDA-MB-231细胞中syk、Dnmts和MBD2 mRNA表达量的变化,比色法检测TCS对MDA-MB-231细胞DNA甲基转移酶活性的影响。结果：TCS体外对MDA-MB-231细胞增殖具有显著抑制作用（P〈0.05）,并呈时间和浓度依赖性;TCS可诱导MDA-MB-231细胞出现空泡样变性、核固缩和核碎裂等细胞凋亡形态学改变,能使MDA-MB-231细胞凋亡并可诱导细胞生长周期阻滞,凋亡率明显高于对照组（P〈0.05）,并呈时间和浓度依赖性。TCS可以逆转MDA-MB-231细胞syk基因启动子区甲基化状况;MDA-MB-231细胞syk mRNA表达阴性,经TCS作用后,syk mRNA表达阳性;MDA-MB-231细胞经TCS作用48h前后Dnmts和MBD2 mRNA表达量没有明显变化（P〉0.05）,但Dnmts活性降低。结论：TCS在抑制MDA-MB-231细胞增殖、诱导细胞凋亡过程中对syk基因具有明显的去甲基化作用,因而可能成为新型的去甲基化药物。

英文摘要：

Objective：To study the methylation status of spleen tyrosine kinase （syk） gene during apoptosis of human breast cancer MDA-MB-231 cells induced by trichosanthin （TCS）,and explore the relationship between the apoptosis of MDA-MB-231 cells and methylation status of syk gene to search for a novel demethylated medicine. Methods：The effects of TCS on proliferation of MDA-MB-231 cells were detected by MTT assay. The change of cell morphology was observed by Giemsa staining. Cell cycle distribution was detected by flow cytometry after treatment with different concentrations of TCS for 48 h. The methylation status of syk was detected by methylation-specific PCR （MSP）. The expression of syk,Dnmts（including dnmt1,dnmt3a,dnmt3b）,and demethylation gene （MBD2） at mRNA levels was assayed using RT-PCR. The effect of TCS on the activity of Dnmts in MDA-MB-231 cells was examined by colorimetric method. Results：TCS significantly inhibited the growth of MDA-MB-231 cells in vitro in a time-and concentration-dependent manner （P〈0.05）. TCS caused typical apoptotic morphological changes in MDA-MB-231 cells such as vacuolar degeneration,pyknosis,and karyorrhexis,induced apoptosis,and arrested cells in S phase. The apoptotic rate of MDA-MB-231 cells was significantly higher than control group （P〈0.05）. All the effects were in a time-and concentration-dependent manner. TCS reversed the methylation of the promoter of syk gene in MDA-MB-231 cells. Syk mRNA had a negative expression in MDA-MB-231 cells but its expression was detected after TCS treatment. No significant difference in expression of Dnmts and MBD2 at mRNA levels was detected in MDA-MB-231 cells before and after TCS treatment （P〉0.05）. However,the activity of Dnmts in MDA-MB-231 cells was decreased. Conclusion：TCS inhibits proliferation of MDA-MB-231 cells and has significant demethylation effects on sky gene,so it may become a novel kind of demethylation drugs.