苦瓜核糖体失活蛋白诱导HL-60细胞发生凋亡及其分子机制的研究

Apoptosis-inducing effects of RIP extracted from bitter melon on HL-60 cells

目的 探讨苦瓜核糖体失活蛋白 (Ribosomalinactivatingprotein ,RIP)诱导HL 60细胞凋亡的活性 ,以及对HL 60细胞Bcl 2、Bax、caspase 3表达变化的影响及其分子机制。方法 以一定浓度苦瓜RIP处理HL 60细胞 2 4～ 72h ,用TUNEL法检测细胞凋亡。运用RT PCR和Westernblot免疫印记法检测Bcl 2、Bax、caspase 3mRNA和蛋白表达的变化。结果 苦瓜RIP在一定作用浓度下可使HL 60细胞发生凋亡 ,且随着作用时间的延长凋亡细胞比例逐渐升高。在此过程中 ,HL 60细胞的caspase 3mRNA的表达及其胞内活性亚单位水平显著升高 (P <0 .0 5 ) ,Bcl 2mRNA表达变化虽不显著 ,但其蛋白水平明显降低 (P <0 .0 5 ) ,与此同时 ,BaxmRNA和蛋白表达均有所上调。结论 在苦瓜RIP诱导的HL 60细胞凋亡中 ,caspase 3发挥了极为重要的作用 ,而Bcl 2和Bax可能通过其表达量的变化。

Objective To explore the apoptosis inducing activity of RIP from bitter melon seeds and its effect on the expressions of Bcl 2, Bax and caspase 3. Methods After the treatment of HL 60 cells with 40 μg/ml typeⅠRIP, TUNEL test was employed to test the apoptosis. At the same time, mRNA and protein expressions of Bax, Bcl 2, Caspase 3 were determined by RT PCR and Western blotting. Results TUNEL test showed 40 μg/ml typeⅠ RIP induced apoptosis in a time dependent manner. The expression of caspase 3 mRNA and the active subunit level increased significantly. Bcl 2 mRNA was not significant, but the protein expression decreased significantly. Bax mRNA and the protein expression were found to be up regulated. Conclusion TypeⅠRIP is a sort of effective apoptosis inducing agent and the caspase 3 is a vital molecule in the process of apoptosis induced by typeⅠ RIP. At the same time, Bcl 2 and Bax may probably function as a regulator in the activation of apoptosis.