【作者】 郭文韬；

【导师】 余龙江； 敖明章；

【作者基本信息】 华中科技大学 ， 生物制药工程， 2013， 博士

【摘要】 大鲵作为中国特有濒临灭绝的二级保护动物,国家已经建立16个专业大鲵保护区及一些自然保护区(大鲵列为保护名录),对大鲵的适生环境和人工繁殖技术开展研究,逐步建立起大鲵人工繁育体系,有效地保护该物种。目前大鲵保护存在一些不足：(1)大鲵种质资源混乱,近亲繁殖,导致大鲵种质的退化,不利于物种的长期生存,有必要对人工养殖大鲵的亲本进行遗传多样性及亲缘关系分析,辨别其混乱的亲本,提供合理的保护策略。(2)缺乏可持续保护方法,大鲵皮肤附属物分泌物和蜕皮是大鲵自然生理特征,可以多次产生,属于可以利用的可再生物质,对这部分加以研究,利用好这部分物质,更适合大鲵资源的长远保护。本课题以大鲵皮肤附属物分泌物和蜕皮为研究对象,建立了非伤害取样技术提取基因组DNA,筛选SSR标记位点,分析了大鲵亲本的遗传多样性和亲缘关系,为制定大鲵长远保护策略提供依据。对其组成及理化性质进行分析,并结合动物模型评价其具体功效,为充分合理利用大鲵皮肤附属物提供支持：(1)大鲵亲本的遗传多样性分析：建立了非损伤大鲵样本组织替代技术,从大鲵分泌物和蜕皮中提取大鲵基因组DNA。通过扩增线粒体12S序列证实该方法获得的DNA可以用于遗传结构分析。从这种组织样本获得的基因组DNA扩增出34个微卫星序列,其中10个微卫星序列具有良好的多态性,用于大鲵资源遗传多样性分析。张家界大鲵核心保护区人工养殖大鲵亲本具有丰富的遗传多样性,聚类分析属于4个种群。(2)分泌物的理化性质及活性分析：为高度含水的凝胶样物质,主要成分为蛋白质。仅溶于5%(V/V)醋酸溶液和蛋白酶溶液,具有很强的粘性,其平均分子量为3.61×106。分泌物可溶组分含有蛋白水解酶活性和磷脂酶A2活性。没有明显抗菌活性,仅在早期有微弱抑制微生物生长的活性。具有抗氧化特性,对DPPH和ABTS清除效果显著,在0.6mg/ml其清除活性达到了90.83%和93.12%,热变性制备的多肽在0.05mg/ml对ABTS的清除活性达到了70.90%。(3)大鲵分泌物的毒性及抗肿瘤活性：大鲵皮肤分泌物可溶组分对小鼠有致死活性,LD50(腹腔)值为32.95mg/kg,其活性组分为热不稳定蛋白质。解剖死亡个体发现肺部组织发生明显变化,有充血现象,抽搐可能会导致小鼠死亡。大鲵分泌物对小鼠产生一些局部影响,可诱导小鼠显著的疼痛活动,促进血管通透性增加,导致持续性肿胀,没有引起明显的出血活性,可使肌肉组织肿胀坏死。体内抑制腹水瘤细胞的生长,减少腹水瘤细胞的数量,延长存活期,分泌物水溶物能明显改善小鼠血液指标,对肝脏结构没有带来明显损伤。体外对肿瘤细胞和正常细胞均有抑制生长活性,其抑制活性对不同的细胞存在差异,抑制活性机制不是促使肿瘤细胞的凋亡。(4)大鲵蜕皮的组成及治疗烧烫伤的活性：大鲵蜕皮含有丰富的氨基酸、矿物元素和8种脂肪酸。大鲵蜕皮大剂量软膏可明显提高创面愈合率,缩短创面愈合时间,加速创面愈合,抑制促炎因子活性可能是发挥作用的机制之一。更多还原

【Abstract】 The Chinese Salamander Andrias davidianus is a specific endangered animal as the second-class protected species. We had established16giant salamander protected areas and some nature reserves (giant salamander as a protected directory). The research on the suitable environment and artificial propagation of the giant salamander is conducted. It has gradually established the artificial breeding system, which can protect the species effectively.There are some deficiencies in current protection of giant salamander:(1) The confusion of germ plasm resources and inbreeding leads to the degradation of giant salamander, which is not conducive to long-term survival of the species. So it’s necessary to analyze genetic diversity and phylogenetic relationships among the parents of the artificial breeding of Giant Salamander to provide reasonable protection strategy.(2)There’s lack of sustainable protection approach. It is a natural physiological phenomenon that giant salamander produces the skin appendages such as shedding and secretion many times in life, belonging to the renewable materials for rational development and utilization. The rational use of these is more suitable for the long-term protection of giant salamander resources. This study establishes a non-injury sampling technique to extract genomic DNA using the skin appendages like sheding and secretion of giant salamander. And then we screen marker-site of SSR and analyze the genetic diversities and phylogenetic relationships of giant salamander parents, which provide the basis for the long-term protection strategy of the giant salamander. It provides us the support for the full and rational use of the giant salamander skin appendages with the analysis of their composition and physicochemical properties, combing the animal model to evaluate the specific effect:(1) The genetic diversity analysis:Skin secretion and shedding is a good source for non-destructive genetic sampling in the giant salamande. The amplification of the12S sequences confirmed that the DNA obtained can be used for the analysis of genetic structure.34microsatellite sequences was amplified from tissue samples of genomic DNA, and10microsatellite sequence with polymorphism which can be used in the analysis of genetic diversity of giant salamander resources. The artificial breeding of giant salamander parents in Zhangjiajie giant salamander core protected areas have a wealth of genetic diversity, whose cluster analysis shows it’s fall into four populations.(2) Physical and chemical properties of the secretion and activity analysis:highly hydrated gel material, the main component is protein, soluble in only5%(V/V) acetic acid and protease solution. It has a strong viscosity; average molecular weight is3.61×106. The secretions of the soluble fraction have the protease hydrolysis activity and phospholipase A2activity. It does not have significant antibacterial activity and only weak inhibition of microbial growth activity at an early stage.The oxidation resistance characteristics clearly significant effect on DPPH and ABTS scavenging activity,90.83%and93.12%at0.6mg/ml. The peptides by thermal denaturation at0.05mg/ml are the ABTS scavenging activity with70.90%.(3) The anti-tumor activity of the secretion:The giant salamander skin secretions of soluble fraction have lethal activity on mouse; LD50(intraperitoneal) value is32.95mg/kg. The active group is heat-labile protein. Anatomy of the death individual found that lung tissue changes significantly and have the phenomenon of congestion, convulsions which may cause the death of mouse. The secretion produce local effects on animals including the activity of significant pain, increase vascular permeability, persistent swelling, allow muscle tissue swelling and necrosis, but no significant bleeding activity. In vivo, it can Inhibit the growth of ascites tumor cells, reduce the number of ascites tumor cells and prolong survival, it could significantly improve blood parameters in mice, did not bring about significant changes in liver structure. In vitro, inhibiting the growth of both cancer cells and normal cells are different in different cells, and the mechanism is not to promote the apoptosis of tumor cells.(4) The giant salamander shedding composition and treatment on burns:giant salamander shedding contains rich amino acids, mineral elements, and eight kinds of fatty acids. Large dose of the giant salamander shedding ointment can be significantly improved wound healing rate, shortened wound healing time and accelerate wound healing. The inhibition of inflammation cytokines may be one of the mechanisms.更多还原