【作者】 李金龙；

【导师】 徐世文；

【作者基本信息】 东北农业大学 ， 临床兽医学， 2004， 硕士

【摘要】 镉是环境中广泛存在的有毒金属污染物，随着其污染的逐渐加重，已经影响到环境生态系统，特别对野生禽类的影响越来越重。国外的一些学者对野生的禽类如海鸥、野鸭、企鹅、麻雀等进行了研究，主要集中于镉在这些鸟类体内的蓄积情况和镉对肝、肾等器官的损伤，而关于免疫毒理的研究较少。本课题以体外培养的鸡脾脏淋巴细胞为试验对象，在培养液中加入终浓度为10μmol/LCdCl2进行染镉处理。通过对鸡脾脏淋巴细胞活性、细胞凋亡、抗氧化功能、DNA损伤及细胞内bcl-2、p53mRNA表达水平的检测，进而探讨镉致鸡脾脏淋巴细胞凋亡的机制。研究结果表明：1．应用台盼蓝排斥实验和MTT比色法首次测定了镉对鸡脾脏淋巴细胞的半数抑制浓度（IC50），即：24h IC50为17.09±0.63μmol/L，48h IC50为13.03±0.62μmol/L，72h IC50为10.15±0.36μmol/L，表明禽类免疫细胞对镉高度敏感，并且随着时间的延长，敏感性升高。2．应用流式细胞术与AO/EB双荧光染色法分别检测了镉致鸡脾脏淋巴细胞凋亡情况，从生化改变和形态学角度均证实镉能够引发鸡脾脏淋巴细胞凋亡，表明镉能够通过诱导禽类免疫细胞凋亡发挥免疫毒性作用。3．通过对鸡脾脏淋巴细胞内NO、MDA含量及NOS、SOD与GSH-Px活性的检测，证实镉能够引发鸡脾脏淋巴细胞内氧化胁迫，导致细胞发生凋亡，进而发挥其对禽类免疫细胞的毒性作用。4．首次应用碱性SCGE、DNA片段化定量分析及DNA Ladder分析等方法检测了镉对鸡脾脏淋巴细胞DNA的损伤效应，结果表明镉能够引发淋巴细胞DNA损伤，并呈现时间效应，此结果揭示镉对细胞DNA的损伤效应是镉免疫毒性的重要机制之一，同时也是引发细胞凋亡的重要因素。5．成功的摸索了检测鸡脾脏淋巴细胞内bcl-2、p53mRNA表达水平的半定量RT-PCR的条件，并首次应用此法检测了镉对细胞内bcl-2、p53mRNA表达水平的影响，揭示了镉能够干扰禽类免疫细胞内bcl-2、p53mRNA表达，进而诱导细胞凋亡。本试验从细胞毒性、细胞凋亡、氧化损伤、DNA损伤及癌基因表达等角度入手，在分子水平上阐明了镉对禽类免疫毒性机制，为保护野生动物资源，维护生态平衡，防治家禽镉中毒和促进畜牧业生产提供了理论依据。更多还原

【Abstract】 Cadmium was one of toxic metal elements, which was ubiquitous in environment. As an important environmental pollution material, cadmium has endangered human and animals’ health, it caused universal research and emphasis. Foreign researchers have studied the effects of cadmium on seagull, widgeon, penguin and sparrow, which mainly concentrated on accumulation and damage of cadmium in the organ of liver and kidney in vivo of birds, while little research about immunotoxicty has been reported. In this paper chicken splenic lymphocytes cultured in vitro was used as experiment materials, treated with 10μmol/L CdCl2. in order to study the mechanism of cadmium-induced apoptosis in chicken splenic lymphocytes, some items about the activity of cell, apoptosis, antioxide function, DNA damage and the expression level of Bcl-2 and p53 mRNA in chicken splenic lymphocytes were detected. The results showed as follows:It was the first time to detect the IC50 of cadmium on chicken splenic lymphocytes by the trypan blue method and MTT assay respectively. The results of 17.09±0.63μmol/L of 24h, 13.03±0.62μmol/L of 48h and 10.15±0.36μmol/L of 72h demonstrated that immunocyte of avian is supersensitive to cadmium, and the sensitivity increases along with times going.Flow cytometry assay and AO/EB double fluorescence coloration was used respectively to detect apoptosis induced by cadmium in chicken splenic lymphocytes from the aspects of biochemical change and morphological change. It revealed that cadmium exhibited immunotoxicity by the way of induction apoptosis of avian immunocyte.From the detection of the contents of NO and MDA, and the activity of NOS, SOD and GSH-Px, this study demonstrated that cadmium caused oxidative stress in chicken splenic lymphocytes, apoptosis, so as to exhibit immunotoxicity to avian immunocell.The effects of cadmium on DNA damage in chicken splenic lymphocytes were detected through different aspects by the method of alkaline SCGE, quantitative analysis of DNA fragmentation and DNA ladder analysis. The results showed that cadmium caused DNA damage of lymphocytes, which exhibited time-dependent effect. It revealed that the effects of cadmium on DNA damage may be one of important mechanisms of avian immunotoxicity on cadmium and also may be an important factor of apoptosis. <WP=11>The optimal conditions of semi-quantitive RT-PCR for detecting the expression level of Bcl-2 and p53 mRNA in chicken splenic lymphocytes was investigated, and it was the first time to use this method to detect the effects of cadmium on the expression level of Bcl-2 and p53 mRNA in chicken splenic lymphocytes. It demonstrate that cadmium interfered with the expression of Bcl-2 and p53 mRNA in avian immunocyte, furthermore, cadmium induced apoptosis.This research elucidated the mechanism of cadmium on immunotoxicity in avian. On the molecular level from the aspects of cell toxicity, apoptosis, oxidative stress, DNA damage and the expression of oncogenes. The mechanism would play an important role in wild animal resource protection, ecology balance maintenance, prevention and therapy in domestic poultry toxicity and promotion of animal husbandry industry.Postgraduate: Li JinlongSpeciality: Clinical Veterinary ScienceSupervisor: Vice-Prof.Xu Shiwen更多还原