苦瓜籽RIP的分离纯化及其对K562细胞作用的研究

Extraction and Purification of Ribosome Inhibiting Protein from Momordica Charantia Seeds and Its Effects of Anti-Tumor Activity on Erythroleukemic K562 Cells

目的:采用强阳离子交换层析柱(SP Sepharose H igh Perform ance)分离纯化苦瓜籽R IP,并观察苦瓜R IP对人红白血病细胞株K562细胞的抗肿瘤活性。方法:采用强阳离子交换层析柱(SP Sepharose H igh Perform-ance)分离纯化苦瓜籽R IP,SDS-PAGE、IEF等实验鉴定苦瓜籽R IP的纯度;以一定浓度梯度的苦瓜籽R IP处理K562细胞72 h,CCK-8检测其对细胞生长的影响;流式细细术(AnnexinⅤ)、细胞形态学(光镜及电镜)等方法检测苦瓜R IP诱导K562细胞凋亡的产生。结果:应用强阳离子交换层析柱(SP Sepharose H igh Perform ance)来分离纯化苦瓜籽R IP,可以快速方便分离得到高纯度的苦瓜R IP,得率也较高;一定作用剂量的苦瓜R IP不仅具有抑制K562细胞生长的活性(9.1μg/mL)的作用,还可诱导K562细胞发生凋亡。结论:运用强阳离子交换层析柱(SPSepharose H igh Perform ance)分离纯化苦瓜R IP是便捷可行的;苦瓜R IP具有抗肿瘤的活性,其作用可能是通过诱导细胞发生凋亡来实现。

Objective： To explore the feasibility of a strong cation exchange chromatographic column （ SP Sepharose High Performance） in purifying ribosome inhibiting protein（RIP） from momordica charantia seeds and observe the cytotoxicity and anti-tmnor activity of RIP in erythroleukemic K562. Methods ： Using gradient acetone precipitation, strong cation exchange chromatographic colmnn （SP Sepharose High Performance） to isolate the RIP from momordica charantia seeds, applying sodium dodecyl sulfate polyacrylamide gel electrophomsis （SDS-PAGE） and isoclectric focusing electrophoresis（IFE） to detect the purity of RIP. Erythroleukemic K562 cells were treated by gradient concentration of RIP for 72 hours and detected its cytostatic activity by CCK-8 kit ; as well as FACScan （ FITC- Annexin V staining）, Light and electron microscopy were used to detect its activity of apoptosis. Results：After using the SP Sepharose High Performance chromatographic column, we obtained the RIP from momordica charantia seeds with high purity and convenience; Not only the RIP had strong cytostafic activity but also could induce K562 ceils to apoptosis understand a appropriated concentration of RIP （9.1 μg/mL）. Conclusion：The methods presented in this paper are feasible to extract and purify the RIP and the RIP extracted from momordica charantia seeds may exert its anti-tumor activity through induction of apoptosis.