文蛤体液抗肿瘤蛋白的分离纯化与作用方式研究

IOCAS-IR > 海洋环流与波动重点实验室

	文蛤体液抗肿瘤蛋白的分离纯化与作用方式研究
其他题名	Purification and characterization of a potent anti-tumor protein from Meretrix Meretrix Linnaeus
	宁璇璇
学位类型	博士
	2008-06-11
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	文蛤抗肿瘤蛋白分离纯化 Mtt比色法作用方式
摘要	恶性肿瘤是严重威胁人类健康的疾病，目前的抗肿瘤药物虽然具有一定疗效，但存在选择性差、毒副作用大等缺点，药物治疗期待着新的突破。因此，寻找高效低毒的抗肿瘤药物已经成为当前肿瘤研究的重要内容。传统抗肿瘤天然药物的来源局限在陆地，近年来在海洋来源的具有抗肿瘤活性物质的开发上取得了很多成果，获得了多种高活性、毒副作用小、作用机制新颖的活性化合物，为新型抗肿瘤药物的研发提供了新思路。本研究采用硫酸铵分级沉淀、弱阳离子交换层析、疏水层析、强阴离子交换层析等分离技术，结合MTT活性追踪方法，从海洋软体动物文蛤体液中纯化得到单一蛋白组份MML，SDS-PAGE检测其分子量约为40kD。该蛋白在体外具有显著的抗人肝癌细胞BEL-7402的活性，其IC50达到52µg/mL。相差显微镜下观察发现，MML起效较快，细胞处理半小时后，形态开始发生改变，细胞膜逐渐与胞内物质分离，最终形成明显的空泡状结构。为进一步研究MML的作用方式，分别从细胞膜毒性、细胞周期阻滞及诱导肿瘤细胞凋亡的角度开展了相关研究。在细胞膜毒性方面，采用扫描电镜观察、Hoechst33342/ PI双染色和乳酸脱氢酶漏出率检测的方法，初步证明了MML对BEL-7402具有细胞膜毒性。结合流式细胞仪、微管蛋白免疫荧光检测技术，研究了MML作用后的细胞周期及微管蛋白的变化情况，结果发现MML的抗肿瘤作用方式可能是通过改变肿瘤细胞膜通透性，进而破坏微管蛋白聚合、阻滞细胞周期，产生抑制肿瘤细胞增殖的活性。DNA Ladder琼脂糖凝胶电泳分析发现，MML作用后的细胞出现一定程度的凋亡，这种凋亡现象的产生是由于MML的直接诱导，还是间接作用产生，尚有待于深入研究。
其他摘要	Most of current anti-tumor chemotherapies acted against normal mammalian cells, consequently causing severe side effects. Marine organisms represented an essentially unexploited reservoir for products of potentially biological and pharmacological interest. So far, literature on natural products derived from marine organisms was characterized by low cytotoxity and new mechanisms. Meretrix Meretrix Linnaeus was a traditional Chinese drug with anti-tumor activities. In the present studies, an anti-tumor protein was purified from the cavity fluid of Meretrix Meretrix, and the possible anticancer pharmacological action modes were also investigated. The purification procedure consisted basically of ammonium sulphate fractionation, two cycles of ion exchange chromatography and hydrophobic interaction chromatography. During the process, MTT colorimetric method was employed to trace the active component. The highly purified protein, designated MML, is a single band of 40 kD revealed by SDS-PAGE. It displayed remarkable in vitro toxicity to the tumor cells BEL-7402, but no toxicity to normal fibroblasts 3T3.The experiment revealed that the IC50 on BEL-7402 was about 52µg/mL. The cell death induced by MML was characteristic of cell morphological change and became apparent at nanomolar concentrations. Multiple approaches, including scanning electron microscopy, Hoechst33342/ PI dual staining, LDH leakage, cell cycle arrest assay, tubulin immunofluorescence and DNA ladder, were employed to investigate the possible pharmacological anti-tumor action modes of MML. The results showed that MML treatment could change the permeability of cell membrane, and the growth inhibition effect was perhaps correlated with the arrest of these cells in G2/M and tubulin depolymerization.
页数	59
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1261
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	宁璇璇. 文蛤体液抗肿瘤蛋白的分离纯化与作用方式研究[D]. 海洋研究所. 中国科学院海洋研究所,2008.

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