【作者】 吴波；

【导师】 李薇；

【作者基本信息】 广州中医药大学 ， 中药学， 2010， 博士

【摘要】 参环毛蚓Pheretima aspergillum(E.Perrier)来源于钜蚓科环毛蚓属,是《中华人民共和国药典》(2005版)“地龙”项下收载的原动物之一。因其主产于广东和广西两地,故被称为“广地龙”。广地龙因其疗效确切,药材加工精细讲究而被业内公认为品质最优,所以既是国内药材销售中的抢手货,又是出口创汇的指定品种。目前我国广地龙药材原动物一参环毛蚓,主要仍依靠野生资源,加上不同环境条件以及采收加工技术的影响,造成药材质量极不稳定,其中重金属超标问题一直难以控制。据前期工作发现：广地龙药材重金属超标的重要原因之一是其原动物参环毛蚓的生理性重金属富集作用所致。因此欲从源头上解决这一难题,应首先对其重金属生物富集过程及其机制进行深入研究,为下一步研究参环毛蚓体内重金属解除策略提供依据。随着工农业的迅速发展,重金属引起的土壤污染问题也越来越严重,并且其在土壤中产生的污染具有隐蔽性、长期性和不可逆性,而且还能被生物体吸收,以至于在食物链末端富集浓缩到极高浓度,后一特性更令人担忧。因为这种特性不仅会严重影响动植物的产量和品质,而且还将通过食物链或药物对人类乃至于整个生物界构成严重的威胁。而土壤动物是土壤生态系统的重要组成部分,土壤中的有毒重金属能够通过各种途径富集到土壤动物体内,但目前国内外多数的研究都是从生态毒理学的角度研究不同种系的蚯蚓对土壤重金属的生物特性变化,并着眼于将蚯蚓对重金属富集的特性当作环境污染的指示标志或作为生态修复的工具,较少研究蚯蚓本身的富集机制,从而以改善药用资源为目的,解决药用蚯蚓体内重金属超标相关的研究。本部分在课题组前研究工作的基础上,开展了参环毛蚓对重金属镉离子耐受能力的考察、重金属镉离子胁迫条件下对参环毛蚓胃肠道上皮细胞结构影响的研究、重金属镉离子胁迫条件下对参环毛蚓体细胞溶酶体膜的影响、重金属镉离子胁迫条件下金属硫蛋白在参环毛蚓不同组织中的表达,并取得了相应的研究结果,现分别概括如下：一、文献综述从现有的研究文献分析,目前国内外对蚯蚓富集特性的研究现状主要体现以下三个方面：1、各项研究结果表明,蚯蚓对重金属有一定的耐受和富集能力,对不同重金属的耐受和富集能力均不同。2、蚯蚓富集重金属特性的研究目前主要用于监测和评价环境污染和修复已污染的环境。3、缺乏以改善药用资源为目的,解决药用蚯蚓体内重金属超标相关的研究。二、参环毛蚓对重金属镉离子耐受能力的考察本实验采用重金属诱导的试验方法探讨在两周时间内参环毛蚓对重金属镉的耐受特性,结果表明,参环毛蚓在土壤镉离子浓度12 mg／kg以下的范围内对镉离子的耐受性良好,其生长几乎不受影响；当土壤镉浓度达到18 mg／kg时,体内镉浓度为100mg／kg,死亡率为25%,其生长明显受到抑制,体重急剧下降到养殖前的60%,体形和形态均发生变化,说明参环毛蚓不能耐受此浓度的镉离子。当土壤镉浓度在24mg／kg以上时,参环毛蚓在一周内全部死亡,死亡率为100%,体重、形态、体形均发生较大变化,出现类似急性中毒现象,显示参环毛蚓对此浓度镉离子的耐受性极差。由实验结果还可以做出以下推断：参环毛蚓对土壤中镉离子的最大耐受浓度在12mg／kg附近。通过对参环毛蚓体内镉浓度及其生长情况进行分析发现,当其体内镉浓度累积达到100 mg／kg时,已经接近其对金属镉离子的最大耐受浓度,进一步表明参环毛蚓对重金属镉的耐受性存在体内最大耐受浓度和体外最大耐受浓度的特点。宋玉芳等对赤子爱胜蚓的研究表明其对土壤中镉离子的耐受浓度可达290 mg／kg,该浓度为参环毛蚓对土壤中镉离子耐受浓度的24倍,提示不同种类的蚯蚓对镉离子的耐受特性存在明显差异。对重金属镉离子在参环毛蚓不同部位的分布情况研究结果表明,生长良好的参环毛蚓(1～4组)镉离子含量内脏均高于躯干,内脏镉含量是躯干的1.8～9.8倍,此结果表明,参环毛蚓主要是通过消化系统吸收镉离子,其对重金属镉具有的耐受特性可能与其体内某种解毒机制有关。此外,将参环毛蚓投入高浓度(24 mg／kg以上)镉土壤中它会迅速死亡,而当投入到较低浓度镉土壤中时,参环毛蚓会适应此环境而正常存活,其内脏和全蚓浓度可分别高达90 mg／kg和70 mg／kg以上,该浓度远远超过24mg／kg。此结果提示参环毛蚓在不超过生理极限的情况下,机体会产生某种有效的重金属解毒机制,而表现出机体对镉离子的高耐受性。本研究考察14 d内参环毛蚓对土壤中镉离子的耐受特性和体内镉离子富集情况及体内镉离子耐受情况,今后应扩大考察时间范围,为研究参环毛蚓重金属富集机制提供了进一步的依据,同时,对于今后解除参环毛蚓重金属富集作用的策略制定提供借鉴。三、重金属镉离子胁迫条件下对参环毛蚓胃肠道上皮细胞结构影响的研究1、重金属对参环毛蚓皮肤的影响参环毛蚓在动物分类上属于环节动物门的寡毛纲钜蚓科环毛蚓属动物,是身体分节的高等蠕虫,由于长期生活于土壤中,其皮肤是抵御外界土壤污染的第一屏障,对土壤的毒性将会作出早期、直接的反应。本实验结果表明,在3mg／kg组和6mg／kg组氯化镉污染的参环毛蚓,虽然在其生长性能未发生明显的变化,但其皮肤的结构却发生了明显的改变,皮肤角质层明显增厚。但是在18mg／kg组氯化镉污染的参环毛蚓,其皮肤的防御体系受到损坏,角质层变薄。正常的角质层为非细胞层,无色而透明,由多层胶原纤维构成,可让气体通过,并保护参环毛蚓体内水分的丢失,同时可抵抗外源化学物质的侵蚀。本实验中角质层的增厚是动物对外源化学物质的一种代偿性反应,使机体免受外源物质的毒害作用。以往的研究结果表明,上角质层的主要成分是酸性多糖,它对于保护动物机体抵抗外源物质的入侵是必需的。上角质层变薄说明了其结构受到了破坏,机体的防御机能下降。本实验中氯化镉也影响了参环毛蚓表皮网状粘液细胞的分泌功能,正常情况下,机体通过粘液形式可以排除参环毛蚓体内的大量氮素,保持体内的代谢平衡,同时对外界环境刺激提供一个保护屏障。参环毛蚓暴露于不同剂量的氯化镉时,皮肤角质层和表皮细胞的结构发生了明显变化,反映了参环毛蚓对该重金属的防御性反应。2、重金属对参环毛蚓体壁的影响通过对比参环毛蚓体壁HE染色的观察,发现其肌肉层损伤的程度有较大的差异,处在最外侧的环肌肉层只是有的部位发生裂隙,处于中层的纵肌排列的间隙有所不同,有的纵肌呈较大裂隙腔,而有的排列十分致密,并没有发生断裂。但是内侧的斜纹肌却发生了较严重的损伤,有的形成较大的断隙,有的完全碎裂,清楚的看到其内侧的斜纹肌损伤要明显比体壁外侧的环肌肉层要严重的多。这说明其体壁的损伤来源于其身体的内部,即对其构成损伤的物质来源于肠道的主动吸收,而不是通过其表皮的被动扩散吸收。3、重金属对参环毛蚓胃肠道的粘膜上皮细胞影响胃肠道是主要的消化器官,动物吞食被污染的土壤后,土壤中的毒物直接刺激胃肠道,容易造成胃肠粘膜上皮细胞的病变。本实验中发现,在3mg／kg组和6mg／kg组氯化镉胁迫条件下的参环毛蚓可见肠道粘膜上皮细胞有大量溶酶体增生,微绒毛、纤毛排列不整齐、紊乱。但是随着暴露剂量的增加和损伤加剧,主要表现为线粒体肿胀,以至于内膜消失,线粒体嵴减少甚至呈空泡变,这主要是因为线粒体是细胞的能源中心、动力站,是细胞中最敏感的细胞器之一,当受到环境不良因素影响时会发生不同的形态变化。同时在高浓度污染组,如30mg／kg组氯化镉污染的参环毛蚓可见肠粘膜上皮细胞微绒毛出现萎缩,纤毛细胞出现溃疡坏死病灶,有明显孔溃烂病变。四、重金属镉离子胁迫条件下对参环毛蚓体细胞溶酶体膜的影响中性红是一种弱碱性染料,通过非离子被动扩散可穿过细胞膜进入细胞的溶酶体内,当受到环境胁迫时,其溶酶体膜的渗透性发生变化,并失去稳定性,溶酶体膜容易破裂,染料就逐步泄露到细胞质中,使细胞染成红色。目前中性红染色作为一种已确定的技术用于评价化合物对细胞的毒性,已经广泛用于化学物质、药剂、表面活性剂、食品添加剂和杀虫剂对于哺乳类动物、人类和鱼类的细胞毒理测试。在本实验中,镉诱导参环毛蚓体腔细胞溶酶体的损害是明显的,中性红在细胞溶酶体的保留时间随着镉离子浓度有增加呈现逐步下降,这与国外相关报道一致,说明中性红在细胞溶酶体的保留时间可用于评价土壤中重金属离子镉对参环毛蚓的毒性效应。比较第2组(氯化镉浓度为3mg／kg)和第3组(氯化镉浓度为6mg／kg)的参环毛蚓染毒7d和15d溶酶体的中性红保留时间,其组间比较无显著性差异(p>0.05),说明在低浓度时参环毛蚓对重金属离子有一定的耐受和富集作用,此时参环毛蚓可通过自身循环系统解除重金属离子对自己的伤害。但是参环毛蚓对重金属的忍耐和富集能力是有限度的,随着重金属离子浓度的增加和染毒时间的加长,重金属对参环毛蚓细胞的毒性具有明显的区别,如第5组(氯化镉浓度为18mg／kg)的参环毛蚓染毒7d和15d溶酶体的中性红保留时间,其组间比较却具有显著性差异(p<0.05)。当参环毛蚓吸收的重金属超过其自身的耐受范围,则土壤中过高的重金属离子会直接毒害蚯蚓,会对其生命活性产生影响,使生长受到抑制,如本实验中第7组和第6组由于镉离子浓度过高,分别饲养至第7天和第9天就已死亡五、重金属镉离子胁迫条件下金属硫蛋白在参环毛蚓不同组织中的表达本实验通过金属硫蛋白与特异性的一抗相结合,产生抗原抗体复合物,再与二抗反应及一系列呈色反应,在显微镜下清晰表达了金属硫蛋白在参环毛蚓体内的分布,免疫组织化学观察清楚的表明,金属硫蛋白主要在参环毛蚓的肠道中表达,虽然在其表皮之中有类似于MT表达的棕黄色颗粒,但是在其空白对照组的实验和HE染色中,均有此类似的棕黄色染色的细胞,说明它不是MT的表达,而是参环毛蚓本身的色素细胞所形成的黑色素,这与A.J.Morgan和S.R.StUrzenbaum的研究结果一致。由于参环毛蚓的表皮的角质层是高渗透性的,可能有少量的重金属通过被动扩散进入体表,但是在其表皮层和肌肉层均未发现明显的MT表达,而是在其肠道的粘膜下腺细胞质均有明显的棕黄色颗粒,且随着镉浓度的增加,MT表达的越来越强,说明参环毛蚓对重金属的富集是通过其粘膜下层肠上皮细胞来完成,这为我们进一步研究参环毛蚓对重金属的转录调控以及其体内重金属的解除策略的研究提供了依据。更多还原

【Abstract】 Pheretima aspergillum(E.Perrier) from Megascolecidae, Pheretima Division, is one kind of the original animals-Pheretima received under "The People’s Republic of China Pharmacopoeia" (2005 edition).Because of Its main products in both Guangdong and Guangxi, it is called " Lumbricus kwangt ". Because of its curative effect, fine medicine processing,it’squality was recognized as the best in the industry, it is a popular commodity in the domestic medicine sales, but also the designated export varieties.At present, the original animal of China pheretima medicine-pheretima asperillum(PA), still rely on wild resources mainly, with different environmental conditions and harvest processing technologies, ingredient quality is unstable, including heavy metals exceeding which is difficult to control. According to preliminary work:One of the important reasons of heavy metal exceeding of pheretima medicine is the physiological heavy metal enrichment of the original animal-PA. Therefore, wishing to solve this problem from the source, It should be the first step to deeply study the process and mechanism of bioaccumulation of heavy metals, which provides evidence for the next step to discharge heavy metal from PA.With the rapid development of industry and agriculture, The problems of heavy metal soil pollution becomes more and more serious, and the pollution in the soil is hidden, long-term and irreversible. The heavy metal in the soil can be absorbed by organisms so that the metal enrichment concentration of the end of the food chain is very high. The latter characteristic is even more worrying. Because of this feature will not only seriously affect the yield and quality of plants and animals, but also pose a serious threat to the human being and even to the entire biosphere through the food chain or the drugs. Soil animals are important part of the soil ecology system. The toxic heavy metals in soil could be enriched in soil animal bodies by different ways. Much research currently focuses on the effect of different kinds of earthworms on biological characteristics of soil heavy metals from the perspective of ecological toxicology, and on the accumulation of heavy metals in earthworm as signs of environment pollution or as a tool for ecological restoration. less research focuses on the mechanism of heavy metals enrichment in earthworms to improve medical resources in medical earthworm-related research.On the basis of prior research work of our research group, heavy metal-induced experiment was carried out to explore cadmium tolerance of PA, The effect of heavy metal Cadmium ion on the structural of PA gastrointestinal epithelial cells,The effect of heavy metal Cadmium ion on the lysosomal membrane of PA, and immunochemistry to study the metallothionein expression in different tissues of in PA. The corresponding results obtained are summarized as follows respectively:一、Literature ReviewFrom the analysis of existing research literature, at home and abroad on the Study on Characteristics of earthworm enrichment is mainly reflected in the below three aspects:1. The research results indicate that earthworms have a certain heavy metal tolerance and accumulation capacity, and the accumulation and tolerance capacities to different heavy metals were different.2. heavy metal accumulation characteristics of earthworms is mainly used for monitoring and evaluation of current environmental pollution and restoration of the polluted environment.3. It is lacking for The related research to improve medical resources and solve excessive heavy metals in medicinal earthworm.二、Investigation of heavy metal cadmium tolerance of PAIn this study, heavy metal-induced experiment was used to explore the heavy metal cadmium tolerance of PA in two weeks. the results show that PA could tolerate 12 mg/kg cadmium and its growth was hardly affected; when the soil Cd concentration was 18 mg/ kg, the body cadmium concentration was 100 mg/kg, PA mortality rate was 25%, PA growth WAS suppressed significantly, PA body weight sharply decreased to 60%, PA shape and form was changed, indicating PA can’t tolerate this concentration of cadmium. When the soil Cd concentration is 24 mg/kg or more, all of the PA died within one week, mortality was 100%, weight, shape, body shape changed greatly, and appears similar to acute poisoning, indicating PA tolerance very poor to this concentration of cadmium. The results also can make the following inferences:PA can tolerate Soil cd maximum concentration of 12 mg/kg in the vicinity. Through Cd concentration and PAs growth analysis found that when the Cd concentration in PA body accumulated to 100 mg/kg, it is already close to cd maximum tolerance concentration, and further indicating that PA Cd tolerance is of in vivo maximum tolerance concentration and in vitro the maximum tolerance concentration. Yu-Fang Song show that Eiseniafoetidasavigny could tolerate up to 290 mg/kg soil cadmium, which is higher 24 times than PA’s, suggesting the tolerance characteristics of different types of earthworms significantly different.The distribution of the heavy metal cadmium in different parts of PA showed that the cadmium content of internal organs of well-growthed PA(group 1～4) were 1.8～9.8 times higher than the trunk,. This result shows that PA absorbed Cd mainly through the digestive system, with its tolerance to cadmium may be associated with the body detoxification mechanism. In addition, when PA was put into the soil with high concentration (24 mg/ kg or more) of cadmium, it will quickly die, however, when put into the soil with lower Cd concentration, it will adapt to this environment and survive normally, with its Cd concentration of internal organs and the whole earthworm were as high as 90 mg/kg and 70 mg/kg or more respectly, far more than the concentration of 24 mg/kg. The results suggest that PA was of effective heavy metal detoxification mechanisms with the limit of no more than physiological circumstances, and showed the body’s high tolerance to cadmium.The study examined PA’s cadmium ions tolerance and in vivo cadmium ions enrichment. The future study should expand the scope of study time to provide a further basis for studying PA metal enrichment mechanism and for the strategy laying down to lift Cd enrichment.三、The effect of heavy metal Cadmium ion on the structural of PA gastrointestinal epithelial cells1. The effect of cadmium on the skin of PAIn animal classification, PA is belong to Oligochaeta huge Lumbricidae Earthworm. due to the long life in the soil, the skin is the first barrier to resist external soil pollution and make an early, direct response to the toxicity of the soil. Our results show that the structure of the skin of the PA contaminated with 3-6mg/kg cadmium chliride has undergone a significant change although its growth performance in no obvious change, with stratum corneum thickening. the skin defense system of the PA contaminated with 3-6mg/kg cadmium chliride was damaged and the stratum corneum was thinned. Normal Stratum corneum is non-cell layer, colorless and transparent, multi-layer collagen fibers, allowing air through, and to protect the loss of PA body water, at the same time, it can resist the erosion of exogenous chemicals. Thickening of the stratum corneum is a compensatory response to exogenous chemical substances to avoid the toxic effects of exogenous substances. Previous results show that the main component of the cuticle is acidic polysaccharides, which is required for the protection of animals body against the invasion of exogenous substances. The thinning of the stratum corneum Notes the destruction of its structure, the body’s defense function declines. In this study, cadmium chloride also affected the secretion of PA reticular epidermal mucous cell. under normal circumstances, the body can rule out a large of nitrogen to maintain the body’s metabolic balance and to provide a protective barrier against the external environmental stimulation. When PA was exposed to different doses of cadmium chloride, the structure of the stratum corneum and epidermal cells changed significantly, reflecting the early defense response of the PA to heavy metals.2. The effect of heavy metal on the body wall of PABy comparing the HE staining of the body wall of PA, we found that the damage degree of different muscle layers is different. The fracture of the outermost ring muscle layer occurred only somewhere, the longitudinal muscle in the middle space is different, some longitudinal muscle showed large crack chamber, and some arranged very compact, no fracture. But the inside drill muscle underwent a more serious injury, some formed large fractures, and some completely fragmented, it is clear to seethat the inside drill muscle injury is significantly seriouser than the outer ring muscles layer of the body wall. This shows that the damage of the body wall is from his body interior, showing that the active substances derived from intestinal positive absorption, rather than passive diffusion through the skin absorption.3. The effect of heavy metal on PA gastrointestinal mucosal epithelial cellsGastrointestinal tract is the main digestive organs, animals eat contaminated soil, the poison in the soil directly stimulate the gastrointestinal tract, likely to cause the lesion of gastrointestinal epithelial cells. This study found that in the cadmium chloride 3mg/kg group and 6mg/kg group, a large number of lysosomal hyperplasia could be seen in the PA intestinal epithelial cells, microvilli, cilia arranged in irregular, disordered. However, with increasing exposure dose, damage increased, mainly mitochondrial swelling, endometrial disappear, mitochondria showed vacuolization. This is mainly because mitochondria is cell’s energy center, power station, and one of the most sensitive cell organelles, when it is impacted by environmental adverse factors, different morphological changes occur. While in the group contaminated with the high concentration cadmium chloride, such as 30mg/kg, the microvilli of the PA intestinal epithelial cells contracted, necrotic ulcer lesions occurred in the ciliated cells.四、The effect of heavy metal Cadmium ion on the lysosomal membrane of PANeutral red is a weak basic dye, it can go across the cell membrane into cell lysosomes by non-ionic passive diffusion. as environmental stresses, cell lysosomal membrane permeability changes and loses its stability, enzyme membrane becomes easy to break, the dye gradually leakes to the cytoplasm, the cells will be stained red. Neutral red staining as the current already established technique for the evaluation of the toxicity of compounds on cells, has been widely used in chemicals, pharmaceuticals, surfactants, food additives and pesticides for mammalian animal, human and fish cell toxicity test. In this experiment, cadmium induced PA coelomocytes lysosomal damage is obvious, neutral red retention time of lysosomes in the cells showed an increase with the cadmium concentration gradually decreased, which is consistent with reports of foreign-related, indicating lysosomal neutral red retention time in the cells can be used for evaluation the toxic effects of heavy metal Cd in soil on PA.Compared Group 2 (cadmium chloride concentration of 3mg/kg) with Group 3 (cadmium chloride concentration 6mg/kg) (Cd exposure 7 d and 15 d), lysosomal neutral red retention time is of no significant difference (p> 0.05), shows PA has some tolerance and accumulation at low concentrations of heavy metal ions Cd, indicating that PA can lift heavy metal Cd through their circulatory system. But PA’s heavy metal tolerance and capacity is limited, with the heavy metal concentration and exposure time longer, heavy metals cytotoxicity on PA is of obvious differences, such as the group 5 (cadmium chloride concentration of 18mg/kg) (Cd exposure 7 d and 15d), lysosomal neutral red retention time is of significant difference (p<0.05). When the heavy metals uptake of PA is more than their own tolerance range, high heavy metals in soil will be directly toxic to earthworm activity and impact on their lives, so that PA growth is inhibited, such as the experimental group 7 and Group 6, because the concentration of cadmium ions is too high, all of the PAs died when raised to 7 days and 9 days.五、The expression of Metallothionein in different tissues of PAIn this study, immunohistochemistry was used to explore the expression of Metallothionein in different tissues of PA. immunohistochemistry clearly shows that PA metallothionein was mainly expressed in intestine, although there was positive result in the epidermis similar to their epidermal MT expression, among the control group and HE staining there was similar positive results, indicating that it is not the expression of MT, but formed by PA pigment cells, which is consistent to A,J.Morgan and S.R.StUrzenbaum’s research results. Although PA epidermis cuticle is of high permeability, there may be a small amount of heavy metals by passive diffusion into the surface, MT expression were not found in its muscle layer and the epidermis. However, there were visible brown spots between the intestinal epithelial cells and basement membrane, and with the increase of Cd concentration, MT expression was growing, indicating heavy metal accumulation of PA is by intestinal epithelial cells. This study provides a basis for our further study about the MT transcriptional regulation of PA and the lifting of heavy metal.更多还原