褐藻多糖硫酸酯和吉马酮对几种癌细胞的作用及机制研究

发布时间：2018-04-11 06:05

本文选题：岩藻多糖 + 吉马酮　；参考：《华中科技大学》2015年博士论文

【摘要】：褐藻多糖硫酸酯是多糖中的主要生物活性成分之一。在多种褐藻中,褐藻多糖硫酸酯大约占总干重的4%。褐藻多糖硫酸酯是一种结构复杂的硫酸化多糖,主要成分包括硫酸酯化L-岩藻糖、半乳糖和微量的木糖以及谷氨酸。褐藻多糖硫酸酯作为一种潜在的抗癌药物正在受到人们的关注,但其作用的分子机理仍不十分清楚。本论文首先针对褐藻多糖硫酸酯对人肝癌细胞HepG2的效应及其可能的作用机制开展了研究。褐藻多糖硫酸酯处理后会引起HepG2细胞周期的阻滞和诱导HepG2细胞药物浓度依赖性地发生凋亡。采用浓度为0、0.2、1、2、4和6mg/ml的褐藻多糖硫酸酯处理HepG2细胞后,通过MTT、流式细胞仪和荧光显微镜检测等方法检测了细胞周期和细胞凋亡的变化。流式细胞仪分析的结果表明,褐藻多糖硫酸酯能够将HepG2细胞周期阻滞在G2/M期。Hoechst33258和Annexin V/PI染色结果显示,随着Bax表达的上调以及Bcl-2和p-STAT3表达的下调,细胞出现药物浓度依赖性的凋亡。此外,褐藻多糖处理后还会引起Cyclin B1和CDK1的下调。褐藻多糖硫酸酯引起的p-STAT3的下调会导致细胞凋亡和细胞内活性氧的增加。因此,我们得出结论：褐藻多糖硫酸酯诱导的HepG2细胞凋亡是通过下调p-STAT3实现的。本论文还初步探讨了褐藻多糖硫酸酯对体外培养的神经母细胞瘤SH-SY5Y增殖的影响,发现低浓度褐藻多糖硫酸酯对神经母细胞瘤SH-SY5Y增殖有促进作用,主要是增加了S期细胞的比例。我们还研究了莪术油的有效成分之一吉马酮对肝癌细胞Be17402增殖的影响,并对其作用机制进行了初步的分析。采用MTT法检测了吉马酮对Be17402和L02细胞增殖的影响；通过流式细胞仪检测了吉马酮诱导细胞凋亡情况和细胞内ROS的变化;采用Western Blotting法检测p53、 Bax和Bcl-2等蛋白的表达情况。在实验所用的浓度范围内,发现吉马酮能够浓度依赖性的抑制肝癌细胞Be17402增殖,同时吉马酮还能促进Be17402细胞发生凋亡和ROS合成的增多；在Be17402细胞中观察到吉马酮处理可引起p53和Bax表达的增加以及Bcl-2蛋白表达的降低。吉马酮能够显著的抑制Be17402细胞显著增殖可能与p53, Bax和Bcl-2和ROS的增加相关。以上研究结果对全面揭示褐藻多糖硫酸酯及吉马酮抗癌作用的分子机理及抗癌药物的开发应用具有参考意义。
[Abstract]:Sulfates from brown algae polysaccharides are one of the main bioactive components in polysaccharides.In many kinds of brown algae, the sulfates of polysaccharides from brown algae account for about 4% of the total dry weight.Fucoidan sulfate is a kind of sulfated polysaccharide with complex structure. The main components include sulfated L-fucose galactose and trace xylose as well as glutamic acid.As a potential anticancer drug, fucoidan sulfate has attracted much attention, but the molecular mechanism of its action is still unclear.In this paper, the effect of polysaccharide sulfate of brown algae on HepG2 of human hepatoma cells and its possible mechanism were studied.HepG2 cell cycle arrest and apoptosis in a concentration-dependent manner were induced by polysaccharides sulfate from brown algae.The cell cycle and apoptosis of HepG2 cells were detected by MTT, flow cytometry and fluorescence microscopy.The results of flow cytometry showed that the cell cycle of HepG2 could be blocked in G _ 2 / M phase. Hoechst33258 and Annexin V/PI staining, with the up-regulation of Bax expression and the down-regulation of Bcl-2 and p-STAT3 expression.Cell apoptosis was concentration-dependent.In addition, Cyclin B1 and CDK1 were down-regulated after the treatment of brown algae polysaccharides.The down-regulation of p-STAT3 induced by polysaccharides sulfate from brown algae may lead to apoptosis and increase of intracellular reactive oxygen species (Ros).Therefore, we concluded that the apoptosis of HepG2 cells induced by polysaccharide sulfate of brown algae was achieved by down-regulation of p-STAT3.The effect of polysaccharide sulfate of brown algae on the proliferation of SH-SY5Y of neuroblastoma in vitro was also studied. It was found that the low concentration of polysaccharide sulfate of brown algae promoted the proliferation of SH-SY5Y in neuroblastoma, mainly by increasing the proportion of S phase cells.We also studied the effect of gemanone, one of the effective components of zedoary turmeric oil, on the proliferation of hepatoma cell line Be17402, and analyzed its mechanism.The effects of gemanone on the proliferation of Be17402 and L02 cells were detected by MTT assay, the apoptosis induced by gemone and the changes of intracellular ROS were detected by flow cytometry, and the expression of p53, Bax and Bcl-2 were detected by Western Blotting assay.In the range of concentration used in the experiment, it was found that gemadone could inhibit the proliferation of Be17402 cells in a concentration-dependent manner, and at the same time, it could promote the apoptosis of Be17402 cells and the increase of ROS synthesis.In Be17402 cells, the expression of p53 and Bax was increased and the expression of Bcl-2 protein was decreased.The inhibitory effect of gemadone on the proliferation of Be17402 cells may be related to the increase of p53, Bax, Bcl-2 and ROS.The above results are helpful to reveal the molecular mechanism of the anticancer effects of fucoidan polysaccharides sulfate and jimarone and the development and application of anticancer drugs.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R730.5

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