苦瓜核糖体失活蛋白的纯化及其诱导HL-60细胞凋亡的研究

EXTRACTION AND PURIFICATION OF BITTER GOURD RIBOSOMAL INACTIVATING PROTEIN AND ANALYSIS OF ITS APOPTOSIS INDUCING ACTIVITY ON HL-60 CELLS

目的: 探讨运用假配基亲和层析和凝胶过滤层析相结合的手段纯化苦瓜核糖体失活蛋白(RIP)的可行性,同时观察其抑制HL-60细胞增殖并诱导其发生凋亡的生物学活性。方法: 利用假配基亲和层析法、凝胶过滤层析法提取纯化苦瓜RIP;通过绘制生长曲线和MTT检测观察苦瓜RIP抑制HL-60细胞增殖的活性,运用流式细胞仪检测细胞周期相分布及细胞凋亡率。结果: 运用硫酸铵分级沉淀、Blue Sepharose CL-6B假配基亲和层析、Sephadex-G75凝胶层析等方法提取、纯化得到了高纯度的苦瓜RIP,并且能够获得较高的得率;一定作用剂量的苦瓜RIP不仅能够抑制HL-60细胞的增殖活性,还可以诱导其发生凋亡。结论: 运用假配基亲和层析和凝胶过滤层析相结合的手段提取、纯化苦瓜RIP是可行的;苦瓜RIP可能是通过诱导细胞发生凋亡来发挥其抗肿瘤活性。

Objective: To explore the feasibility of the combination of psuedo-ligand affinitive chromatography and gel chromatography in purifying RIP from bitter gourd and the cytotoxicity of the RIP. Methods: Using ammonium sulfate precipitation, Blue Sepharose CL-6B psuedo-ligand affinitive chromatography and Sephadex G-75 gel chromatography to isolate the RIP,after treatment of HL-60 cells with RIP for 24-72 h, growth curve, MTT test, as well as flow cytometry test were used to explore its cytotoxicity. Results: After using the methods mentioned above, we obtained the typeⅠRIP with high purity. After treatment of HL-60 cells with the typeⅠRIP the cell viability decreased dramatically. At the same time, flow cytometry test showed 40 mg/ml RIP induced apoptosis of HL-60 profoundly. Conclusion: The methods presented in this paper are feasible to isolate and purify the RIP and the RIP extracted from bitter gourd may exert its anti-tumor activity through induction of apoptosis.