【作者】 范冠宇；

【导师】 吴志刚；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生学， 2006， 硕士

【摘要】 几丁聚糖(chitosan),化学名为2-氨基-β-1,4-葡聚糖,分子式为:(C6H11O4N)n,是从几丁动物外壳、低等植物菌类、藻类以及高等动物的细胞壁中提取出来的一种含有大量阳离子的葡萄糖胺多聚体。1811年法国科学家Braconnot首先从蘑菇中提取出几丁质并将其命名为Fungine。1823年,法国科学家欧吉尔在甲壳动物的外壳中也提取出几丁质并命名为chitin。1859年,鲁凯特将几丁质置于氢氧化钠溶液中加热,制得一种可溶解于有机酸的物质;1894年,安波索拉将鲁凯特制备的这种物质命名为几丁聚糖(chitosan)并沿用至今。自发现几丁聚糖以来,对它的研究不断深入。1977年在美国波士顿召开了由Mit Sea Grand Program主办的第一届国际几丁质·几丁聚糖学术会议,首次集中探讨了几丁质的生产、开发及利用状况;1982年,第二届国际几丁质·几丁聚糖会议于日本召开;亚洲也于1994年和1996年先后召开了两届几丁质·几丁聚糖学术会议,并深入探讨了几丁聚糖在医药、生物、食品、纺织工业、农业和环境保护诸领域的应用前景。近年来,随着高分子科学和生物医学工程的发展,几丁聚糖在医学方面的研究日益增多,并因其具有多种生理调节功能而被誉为除糖、蛋白质、脂肪、维生素与矿物质外人体必需之第六生命要素。环境重金属镉污染对人体的影响具有时效长、危害大等特点,其致突变和致癌作用引起了国内外学者的广泛关注,并使得重金属拮抗剂、解毒剂的寻找和开发成为环境卫生领域研究的热点。中国环境卫生学界著名教授陈学敏先生通过深入研究硒和锗拮抗环境重金属镉的作用机制,建立了一整套较为完善的重金属研究方法和学术思想,并发现硒、锗等解毒剂的安全剂量范围很窄,用量稍大即引起毒副作用,从而大大限制了该类解毒剂的应用。随着研究领域的拓宽,人们逐渐发现,几丁聚糖对重金属具有很强的吸附作用并将其用于环境重金属污染的治理;Gyliene和Bhanoori通过研究发现,以几丁聚糖作为镉的生化吸附剂可有效促进镉的代谢和排除。尽管这些研究只是刚刚开始,但已凸显出几丁聚糖作为环境重金属解毒剂的潜在价值。本研究在采用Fenton反应产生羟自由基并证实几丁聚糖对羟自由基清除作用的基础上,以氯化镉染毒HepG2细胞产生氧化损伤模型,检测几丁聚糖对细胞活力、ROS水平的影响,深入探讨几丁聚糖对DNA氧化损伤和损伤后修复的作用,以期初步阐明其作用机制。第一部分几丁聚糖对羟自由基的清除作用采用邻二氮菲-Fe2+氧化法研究几丁聚糖对羟自由基的清除作用。以抗坏血酸作为阳性对照,检测不同浓度几对聚糖对Fenton反应体系中羟自由基的清除。结果发现:在0.04mg/ml-0.32mg/ml浓度范围内,几丁聚糖聚对羟自由基(·OH)的清除作用逐渐增强,最大清除率可达93.67%;几丁聚糖对羟自由基有假阳清除现象,扣除假阳清除率后,仍呈现74.98%-84.64%的清除率。研究表明几丁聚糖具有较强的羟自由基清除作用。第二部分几丁聚糖对氯化镉致HepG2细胞氧化损伤的作用及机制研究1.几丁聚糖对氯化镉致HepG2细胞活力降低的影响采用MTT试验检测氯化镉对HepG2细胞的抑制作用及几丁聚糖对氯化镉致HepG2细胞活力降低的影响。结果显示:采用10μmol/L氯化镉染毒HepG2细胞,其活力在所有染毒时间点(4h、8h、16h和24h)均无明显降低;当氯化镉浓度增加至20μmol/L和30μmol/L时,染毒4h后,细胞活力均有一定程度的降低,但与正常对照组相比无显著性差异;当氯化镉浓度增加至40μmol/L和50μmol/L时,在所有染毒时间点上均可见细胞活力显著降低(P<0.05)固定染毒时间,HepG2细胞活力随氯化镉浓度的增加而降低,并呈现明显的剂量-依赖关系。染毒时间为4h时,氯化镉浓度由10μmol/L增加至50μmol/L,HepG2细胞活力从86.3%降至75.7%,仅40μmol/L和50μmol/L剂量组细胞活力与正常对照组有显著性差异(P<0.05);当染毒时间分别延长至8h、16h和24h,除10μmol/L剂量组细胞活力与正常对照组间无显著性差异外,其余染毒组细胞活力与正常对照组相比均有显著性差异(P<0.05)。20μmol/L氯化镉染毒HepG2细胞24h后,细胞活力降至54.8%;在培养液中加入高浓度的几丁聚糖(0.5mg/ml),细胞活力未发生显著性变化(与正常对照组相比:P>0.05);在氯化镉染毒的同时,于培养液中加入不同浓度的几丁聚糖后,HepG2细胞活力随几丁聚糖浓度的增加而增强,并且最高剂量几丁聚糖(0.5mg/ml)与氯化镉联合作用组细胞活力与氯化镉单独作用组相比有显著性差异(P<0.05)。2.几丁聚糖对氯化镉诱导HepG2细胞产生ROS的清除作用研究不同浓度几丁聚糖对氯化镉诱发HepG2细胞产生ROS的清除作用。用氯化镉诱发HepG2细胞产生ROS,以DCFH-DA作荧光探针,采用流式细胞检测技术检测几丁聚糖与氯化镉联合作用下HepG2细胞内ROS水平。结果发现:20μmol/L氯化镉可使HepG2细胞内ROS水平显著增加(P<0.01);以0.50mg/ml几丁聚糖处理HepG2细胞后,细胞内ROS水平较正常对照组略有降低,但并无显著性差异;与氯化镉染毒组相比,几丁聚糖和氯化镉联合作用组细胞内ROS水平随几丁聚糖浓度的增加逐渐降低,并且在高剂量几丁聚糖(0.5mg/ml)和氯化镉联合作用组呈现极显著性差异(P<0.01)。实验表明,几丁聚糖对HepG2细胞内由氯化镉诱发产生的ROS具有较好的清除作用。3.几丁聚糖对氯化镉致HepG2细胞DNA氧化损伤的影响采用单细胞凝胶电泳技术,以代谢酶较为完整的人肝肿瘤(HepG2)细胞为靶细胞,研究氯化镉致HepG2细胞DNA氧化损伤作用及几丁聚糖对氯化镉致HepG2细胞DNA氧化损伤的影响。研究发现:(1)随氯化镉浓度的增加,HepG2细胞Olive尾矩值逐渐增大,并呈明显的剂量-依赖关系;低浓度组(5μmol/L、10μmol/L)HepG2细胞Olive尾矩值与正常对照组相比无显著性差异,但20μmol/L、40μmol/L和80μmol/L浓度组HepG2细胞Olive尾矩值均较正常对照组显著增高(P<0.01);(2)几丁聚糖对照组HepG2细胞DNA损伤较正常对照组略有减轻,但并无显著性差异;分别以不同浓度几丁聚糖与20μmol/L氯化镉联合作用时,HepG2细胞DNA损伤程度均较20μmol/L氯化镉单独作用组减轻,随几丁聚糖浓度的增加,DNA损伤逐渐减轻,呈现明显的剂量-依赖关系;并且,在高剂量几丁聚糖(0.50mg/ml)与氯化镉联合作用组呈现极显著性差异(P<0.01)。研究结果表明,几丁聚糖对氯化镉致HepG2细胞DNA损伤有一定的保护作用。4.几丁聚糖对镉抑制HepG2细胞DNA修复酶hOGG-1表达的影响在以往开展的硒对镉引发氧自由基清除作用和几丁聚糖对羟自由基清除作用研究的基础上,进一步研究几丁聚糖对氯化镉抑制HepG2细胞DNA氧化损伤修复酶hOGG-1表达的影响。在氯化镉染毒HepG2细胞的同时,于培养液中加入不同浓度的几丁聚糖,采用Westernblotting检测氯化镉单独作用以及几丁聚糖和氯化镉联合作用下HepG2细胞DNA氧化损伤修复酶hOGG-1的表达水平。结果发现:(1)以40μmol/L氯化镉染毒HepG2细胞24小时后,细胞内hOGG-1表达显著下降(P<0.01);几丁聚糖对照组与正常对照组相比,hOGG-1表达无显著性差异;(2)不同浓度几丁聚糖和氯化镉联合作用组HepG2细胞hOGG-1的表达水平均较氯化镉单独作用组高,随着几丁聚糖浓度的增加,hOGG-1表达逐渐增强,并且在高剂量几丁聚糖(0.5mg/ml)和氯化镉联合作用组呈现极显著性差异(P<0.01)。研究表明:几丁聚糖对氯化镉致HepG2细胞hOGG-1表达水平降低有拮抗作用。更多还原

【Abstract】 Chitosan, 2-amino-2-deoxy-β-D-glucose, with the formula of (C6H11O4N)n, is naturally abundant in crustaceans, exoskeletons of insects, algaes, fungi and cytoderm of superior animals. In 1811, a French scientist Braconnot isolated a new substance from mushroom and denominated it chitin; Later in 1823, the same substance was also extracted from the shell of crustaceans by another French scientist and was given a new name as Fungine. Then in 1859, Lucheit obtained a new substance by treating chitin with sodium hydroxide at 120℃for 3 hours; Different to chitin, this substance was soluble in organic acids, and finally was denominated as chitosan by Annbosola in 1894.Researches about chitosan were carried out further deeply since chitosan was discovered. In 1977, the 1th International symposium about chitin and chitosan , at which the production、exploitation and utilization of chitin and chitosan was discussed for the first time, was hosted by Mit Sea Grand Program in Boston,US. Then, the 2th was held in Japan,1982; Recently in 1996 and 1999, symposium about chitin and chtosan was held subsepuently in Beijing and Wuhan, which deeply investigated and prospected the applications of chitosan in medicine and pharmacology、biology、food processing、textile industry、agriculture and environmental protection. With the development of science of macromolecule and biomedicine , the applications of chitosan get expanded and chitosan has been honoured the 6th element essential for life after glucose、protein、fat、vitamine and minerals for its extensive physiological functions.Pollution of environmental heavy metal of cadmium was persistent、hazardous and undegradable. The mutagenesis and carcinogenesis of cadmium pollution has provoked worldwide interest, which in turn make exploitation of antagonist and antidote of cadmium the focus of environmental health. Pro.Chen Xuemin , a Chinese specialist in the field of environmental hygiene, has established a series of methodology about heavy metals by a large number of studies such as the hydroxy free radical-scavenging effect of combined selenium and germanium, et al. But as antidotes of cadmium, the application of selenium and germanium was restricted severely because of the rather narrow safety dose range. In recent years, chitosan was found to be effective as adsorbent of heavy metals by Bhanoori and Gyliene, who have reported that chitosan could promote the metabolism and elimination of cadmium significantly. All of these studies have revealed the potential of chitosan as antidote of environmental heavy metals, especially cadmium.The scavenging effect of chitosan on hydroxy free radical produced in Fenton-reaction system has been testified by our former experiment. In this study, we employed cadmium as toxicant to induce oxidative damage of HepG2 cell, then investigated the role and mechanism of chitosan on cell viability、ROS level、oxidative damage of DNA and DNA repair.PartⅠ: The hydroxy free radical-scavenging effect of water-soluble chitosanPhenanthroline-Fe(Ⅱ) oxidation assay was adopted to detect the hydroxy free radical-scavenging effect of water-soluble chitosan in this study. In Fenton reaction system , ascorbic acid ( Vc ) was used as positive control , the hydroxy free radical-scavenging effect of water-soluble chitosan with different concentration was tested. It showed that: (1) the rate of hydroxy free radical-scavenging of water-soluble chitosan improved from 14.68% to 93.67% when the concentration of chitosan increased from 0.04mg/ml to 0.32 mg/ml; (2) The pseudo-positive scavenging effect on hydroxy free radical of water-soluble chitosan with the highest value of 9.03% was tested specially in the experiment. The rate of hydroxy free radical-scavenging of the chitosan was still up to 74.98%-84.64% when the rate of pseudo-positive scavenging was calibrated. The results demonstrated that water-soluble chitosan is an appropriate kind of hydroxy free radical-scavenging agent.PartⅡ: Mechanism of chitosan on the damage of HepG2 cell induced by cadmium chloride1. Effect of chitosan on the decrease of HepG2 cell viability induced by cadmium chloride .MTT Assay was employed to investigate the cytotoxic effect of cadmium chloride on human hepatocyte carcinoma cell (HepG2 cell) and antagonistic effect of chitosan on the decrease of HepG2 cell viability induced by cadmium chloride. The results showed that:(1) Cadmium caused decrease in cell viability in a time-dependent manner. Compared to normal control, cell viability decreased in 10μmol/L cadmium chloride group after exposured for 4, 8, 16 and 24h,respectively, but no significant difference was observed. In 20μmol/L and 30μmol/L cadmium chloride group, cell viability decreased significantly after exposured for 8,16 and 24h, respectively. In comparison with normal control, cell viability decreased with significant difference in 40μmol/L and 50μmol/L after exposured for 4, 8, 16 and 24h,respectively(P<0.05). (2) Compared to normal control, cell viability decreased in dose-dependent fashion with concentration of cadmium chloride. After exposured to cadmium chloride for 4h, cell viability decreased from 86.3% to 75.7% when concectration of cadmium chloride increased from 10μmol/L to 50μmol/L,but significant difference was only be observed in 40μmol/L and 50μmol/L group(P<0.05).In comparison with normal control,significant decrease of cell viability was observed in all but 10μmol/L cadmium treated group when exposure time was extended to 8, 16 and 24h, respectively(P<0.05).(3) Compared to normal control,cell viability decreased from 100% to 54.8% after exposured to 20μmol/L cadmium chloride for 24h.There is on significant difference between the cell viability of 0.50mg/ml chitsoan group and that of normal control. In experimental groups treated with chitosan and 20μmol/L cadmium chloride simultaneously, cell viability increased from 59.8% to 81.5% when chitosan concentratioin improved from 0.10mg/ml to 0.50 mg/ml. In comparison with 20μmol/L cadmium chloride group, significant increase of cell viability was observed in experimental group exposured to 0.50mg/ml chitosan and 20μmol/L cadmium chloride simultaneously.It has demonstrated that high dose of chitosan has no significant effect on viability of HepG2 cell, but has protect effect on HepG2 cell damage induced by cadmium chloride.2. The scavenging effect of chitosan on ROS in HepG2 cell induced by cadmium chloride.HepG2 cell was exposured to chitosan and cadmium chloride simultaneously for 4h.Two groups treated with cadmium chloride and chitosan respectively were used as control. The fluorescence of 2′,7′-dichlorofluorescin(DCF) was measured (by flowcytometry) as a mean of estimating the formation of reactive oxygen species (ROS).In comparison with normal control ,the DCF intensity of chitosan group only exhibited somewhat decrease(P>0.05) and a significant increase(P<0.01) was observed in cadmium chloride group(20μmol/L, 4h). The DCF intensity decreased in all three groups treated with cadmium chloride and chitosan(0.02, 0.10 and 0.50mg/ml, respectively) simultaneously are all higher than that of cadmium chloride-treated group, and when the concentration of chitosan improved from 0.01mg/ml to 0.50mg/ml, the DCF intensity decreased from 275.59 to 174.59 and significant decrease was observed at concentration of 0.50mg/ml. These results suggested that chitosan could antagonized the increase of ROS in HepG2 cell induced by cadmium chloride.3. The effect of chitosan on cadmium-induced DNA damage in HepG2 cell.In this part of study, we used single cell gel electrophoresis to measure DNA damaging effect caused by cadmium chloride in HepG2 cell and the effect of chitosan on DNA damage in HepG2 cell induced by cadmium chloride. The results showed that (1) cadmium chloride caused increase in Olive tail moment in a dose-dependent fashion. Statistical significant increases of Olive tail moment were observed in HepG2 cell treated with 20, 40 and 80μmol/L cadmium chloride for 4 hours. (2) The Olive tail moment of 0.50mg/ml chitosan group exhibited somewhat decrease in comparison with that of normal control, but no significant difference was observed. In the three groups treated with 20μmol/L cadmium chloride and chitosan(0.02, 0.10 and 0.50mg/ml,respectively) simultaneously, the Olive tail moment was 5.29, 5.11 and 1.61,respectively,which were all lower than that of 20μmol/L cadmium chloride-treated group, and significant decrease was observed in the group treated with cadmium and 0.50mg/ml chitosan simultaneously. These results demonstrated that chitosan was effective in protect HepG2 cell from DNA damage caused by cadmium chloride.4. The effect of chitosan on expression of hOGG-1 in oxidative damaged HepG2 cell induced by cadmium chloride.To make a further investigation on the effect of chitsoan on expression of hOGG-1 in cadmium chloride-induced HepG2 cell based on the studies on scavenging effect of cadmium-induced free radical with selenium and the hydroxy free radical- scavenging effect of water-soluble chitosan. HepG2 cell was treated with 40μmol/L cadmium chloride and chitosan simultaneously for 24h. 40μmol/L cadmium chloride and 0.50mg/ml were used as control,respectively. The expression of hOGG-1 was semi-quantitated by westernblotting. Results showed that: In comparison with normal control, the hOGG-1 content in 40μmol/L cadmium chloride group decreased significantly(P < 0.01). Compared to cadmium chloride-treated group, the hOGG-1 content were much higher in the groups treated with cadmium chloride(40μmol/L) and chitosan(0.02, 0.10 and 0.50mg/ml, respectively) simultaneously, and when the concentration of chtosan improved from 0.02mg/ml to 0.50 mg/ml, the hOGG-1 content increased and significant difference(p<0.01)was observed at the concentration of 0.50mg/ml .This study suggested that the decrease of hOGG-1 content in HepG2 cell induced by cadmium chloride could be antagonized effectively by chitosan .更多还原