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目的:对蓖麻毒素纯化方法进行改良并探讨其体外抗血管生成的药理作用。方法:通过磷酸盐缓冲液抽提、Sepharose 4B亲和层析与Sephacryl S-100分子筛层析纯化蓖麻毒素,反相疏水高效液相色谱法测定蛋白纯度,免疫印迹法进行鉴定;原代培养人脐静脉内皮细胞,免疫细胞化学法鉴定,MTT法检测蓖麻毒素对细胞增殖的抑制作用,流式细胞术定量分析细胞凋亡率。结果:采用改良法纯化蓖麻毒素明显缩短了纯化周期,纯化的蛋白经免疫印迹技术证实为蓖麻毒素,蛋白纯度高达95.3%;纯化的蓖麻毒素抑制体外培养人脐静脉内皮细胞增殖作用具有明显的时间及剂量依赖效应,并且可明显诱导细胞凋亡,随着药物浓度增加,细胞凋亡率,尤其是晚期凋亡率上升更明显。结论:采用改良方法纯化可获得高纯度的蓖麻毒素,纯化的蓖麻毒素具有明显的抑制血管内皮细胞增殖,诱导细胞凋亡作用。
Objective: To improve the purification method of ricin and investigate its pharmacological effects on anti-angiogenesis in vitro. METHODS: Ricin was purified by phosphate buffer extraction, Sepharose 4B affinity chromatography and Sephacryl S-100 molecular sieve chromatography. Protein purity was determined by reversed-phase hydrophobic high performance liquid chromatography and identified by immunoblotting. Primary culture was performed. Human umbilical vein endothelial cells were identified by immunocytochemistry. MTT assay was used to determine the inhibitory effect of ricin on cell proliferation. Flow cytometry was used to quantitatively analyze the apoptosis rate. RESULTS: Purification of ricin by modified method significantly shortened the purification cycle. The purified protein was confirmed to be ricin by immunoblot technique. The purity of the protein was as high as 95.3%. Purified ricin inhibited the proliferation of human umbilical vein endothelial cells in vitro. Obvious time and dose-dependent effects, and can significantly induce apoptosis, with the increase of drug concentration, apoptosis rate, especially the late apoptosis rate increased more significantly. Conclusion: The purified ricin can be purified by the improved method. The purified ricin can inhibit the proliferation of vascular endothelial cells and induce apoptosis.