[关键词]
[摘要]
目的 分离纯化制备羊栖菜褐藻糖胶(CSFP-1),并对其抗肿瘤活性及可能作用机制进行初步研究。方法 通过冷水浸提法制备羊栖菜褐藻糖胶(CSFP),经超滤系统分离获得CSFP-1,并采用苯酚-硫酸比色法、PMP衍生化、高效液相色谱法、红外光谱分析等多种方法对其理化性质进行分析;采用MTT法评价CSFP-1(0、10、100、300 μg/mL)对宫颈癌细胞(HeLa)、肺癌细胞(A549)和肝癌细胞(Hep3B)生存能力的影响;应用流式细胞术评价CSFP-1对HeLa细胞细胞周期的影响;应用Western blotting技术评价CSFP-1对HeLa细胞凋亡和自噬相关蛋白表达的影响。结果 CSFP-1的糖含量为84.18%,糖醛酸含量为14.28%,硫酸根含量为10.02%,平均相对分子质量为5.6×104,主要由半乳糖、岩藻糖、甘露糖、葡萄糖醛酸和木糖组成。CSFP-1具有选择性细胞毒性,对HeLa细胞生长发挥显著抑制作用(P<0.05、0.001),对A549和Hep3B细胞存活率没有明显影响。与对照组比较,HeLa细胞经CSFP-1处理24 h后,G1期细胞比例明显增加(P<0.001);经CSFP-1处理48 h后,S期细胞比例显著增加(P<0.05、0.01、0.001),G2期细胞比例显著减少(P<0.01、0.001),均呈现剂量相关性。与对照组比较,CSFP-1 100、300 μg/mL剂量组处理24 h,300 μg/mL剂量组处理48 h Bcl-2蛋白表达显著减少(P<0.05);100、300 μg/mL剂量组处理24、48 h Beclin-1蛋白表达显著增加(P<0.05、0.01),且存在时间和剂量相关性。结论 CSFP-1具有选择性抑制HeLa细胞增殖作用,作用机制可能与阻滞细胞周期、诱导细胞凋亡相关。
[Key word]
[Abstract]
Objective To extract, purify, and prepare the fucoidin of Sargassum fusiforme (CSFP-1), and preliminary investigate its anti-tumor activity and underlying mechanism. Methods CSFP was extracted by cold water and CSFP-1was obtained by ultrafiltration system. Phenol-sulfuric acid colorimetry, PMP derivatization, high performance liquid chromatography, infrared spectroscopy, and other methods were used to analyze the physical and chemical properties. The effects of CSFP-1 (0, 10, 100, and 300 μg/mL) on the viability of cervical cancer cells (HeLa), lung cancer cells (A549), and hepatoma cells (Hep3B) were evaluated by MTT assay, the effects of CSFP-1 on the cell cycle of HeLa were evaluated by flow cytometry, and the effects of CSFP-1 on apoptosis and autophagy related protein expression in HeLa cells were evaluated by Western blotting. Results CSFP-1 was mainly composed of galactose, fucose, mannose, glucuronic acid, and xylose with the sugar content of 84.18%, glucuronic acid content of 14.28%, sulfate content of 10.02% and average molecular weight of 56 kDa. CSFP-1 had selective cytotoxicity and significantly inhibited the growth of HeLa cells (P < 0.05, 0.001), but had no significant effect on the survival rate of A549 and Hep3B cells. Compared with the control group, HeLa cells treated with CSFP-1 for 24 h increased the proportion of G1 phase cells significantly (P < 0.001); After treated with CSFP-1 for 48 h, the proportion of S phase cells increased significantly (P < 0.05, 0.01, 0.001), and the proportion of G2 phase cells decreased significantly (P < 0.01, 0.001). Compared with control group, the expression of Bcl-2 protein in 100 and 300 μg/mL CSFP-1 groups decreased significantly at 24 h, and that in 300 μg/mL group decreased significantly at 48 h (P < 0.05); The expression of Beclin-1 protein in 100 and 300 μg/mL group increased significantly at 24 and 48 h (P < 0.05 and 0.01), and there was a time-dose correlation. Conclusion CSFP-1 has selective inhibited effect on the proliferation of HeLa cells and its anti-tumor activity mechanism may be related with blocking cell cycle and inducing apoptosis.
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[基金项目]
国家自然科学基金资助项目(314070430);浙江省教育厅科研项目(Y201737374);浙江省基础公益研究计划(LGN18C020004);浙江省科技计划项目(2017C32103);浙江省自然科学基金(LY18C020006)