摘要
目的探讨半胱氨酸蛋白酶(caspase)在ω-3脂肪酸联合细胞外源性跨膜型TNF-α诱导MCF-7细胞凋亡过程中的作用。方法带有脂肪酸受体反应元件的跨膜型TNF-α表达载体转染MCF-7细胞,经ω-3脂肪酸处理后,MTT比色法检测细胞增殖能力,DNA梯形带分析ω-3脂肪酸对细胞凋亡的影响,RT-PCR和W estern b lot方法检测细胞caspase-1、8和9基因的表达变化,应用caspase特异性底物检测肿瘤细胞caspase-8活性变化,观察caspase特异性抑制剂Ac-IETD-CHO对ω-3脂肪酸联合细胞外源性跨膜型TNF-α抑制肿瘤细胞增殖、诱导凋亡的拮抗作用。结果与MCF-7细胞相比,MCF-7转染细胞经ω-3脂肪酸处理后,细胞增殖能力减弱,检测到明显的DNA梯形带,caspase-1、8、9基因表达增加,caspase-8活性增高,caspase抑制剂可以部分拮抗ω-3脂肪酸联合跨膜型TNF-α抑制肿瘤细胞增殖和诱导凋亡的发生。结论ω-3脂肪酸联合跨膜型TNF-α可以更有效地诱导MCF-7细胞凋亡,而上调caspase的表达和/或活性可能在此过程中发挥重要作用。
Objective To study the apoptosis and its molecular mechanism of MCF-7 ceils induced by ω- 3 polyunsaturated fatty acid（ ω-3 PUFA）combined with the product of exogenous transmembrane TNF-α （tmTNF-α） gene. Methods The tmTNF-α eukaryotic expression vector containing the PPRE-tk promoter was transfected into the human breast cancer MCF-7 cells. The effects of ω-3 PUFA and/or exogenous tmTNF-α on the cell proliferation and apoptosis were measured by MT7 and DNA ladder assay. The activity of caspase-8 was examined by using special fluorescence substrate, and the expression of caspases （ 1, 8 and 9 ） were analyzed by RT-PCR and Western blotting. The inhibitor of caspases was used to confirm the function of caspase. Resuits In 6.0×10^-5mol/L ω-3 PUFA-treated MCF-7 ceils, only the growth suppression was found. In transfected MCF-7 ceils after treated with ω-3 PUFA, not only the proliferation capacity was decreased but the DNA ladder was detectable. The expression changes of caspases （ 1, 8 and 9） and caspase-8 activity were obvious in MCF-7 transfected ceils treated with ω-3 PUFA. Growth inhibition and apoptosis induced by ω-3 PUFA and tmTNF-α were partly prevented by the special caspase inhibitor. Conclusion These results suggested that upregulated expression and activity of caspase might promote MCF-7 cells apoptosis induced by ω-3 PUFA and exogenous tmTNF-α, indicating that ω-3 PUFA and exogenous tmTNF-α could cooperate in inhibition of the MCF-7 cell growth and induction of apoptosis, caspase network pathway may play a key role in these processes.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第13期1380-1383,共4页
Journal of Third Military Medical University
基金
国家自然科学基金资助项目(30100150)~~
