【作者】 赵剑；

【导师】 蔡原；

【作者基本信息】 中国医科大学 ， 卫生毒理学， 2004， 硕士

【摘要】 前言 铅是人类广泛接触的有毒重金属之一，对机体的几乎所有组织器官都可造成损害。乙醇是常用的有机溶剂及酒类饮料的主要成份，乙醇对机体多个系统都有影响。铅与乙醇均是接触频率高，暴露人群广泛的化学物质。在铅作业工人、尤其是青年工人中多有嗜酒行为，有人在对铅接触者的调查中发现90％的男性工人有饮酒习惯。乙醇可促进骨骼中铅的释放，使中毒症状明显加重。饮酒者发生铅中毒的比例较高。因此，研究铅和乙醇的联合毒作用具有重要的理论和实际意义。 近年来，关于铅和乙醇对全身各系统的联合作用的研究逐渐深入，对于不同的组织器官，铅和乙醇的联合作用类型不尽相同，有的表现为拮抗作用，更多的表现为协同作用。虽然目前关于铅和乙醇联合毒作用的报道较多，但对免疫系统影响的研究还很少，尤其是对整体动物的研究更为少见。免疫系统是维护机体健康、正常执行生理生化功能的重要保证，也是化学毒物经常攻击的靶组织。为了阐明铅与乙醇联合作用对雄性小鼠免疫系统的影响，本课题拟用体内、体外实验的方法，观察铅和乙醇对小鼠免疫功能的联合作用并初步探讨其作用机理，为保护接触者的健康，进行有效防治提供理论依据。 实验材料与方法 1．实验动物分组与染毒 昆明种小鼠，雄性，体重22±2g，小鼠饲养1w后按体重随机分为4组，每组10只；动物室温度18-23℃，相对湿度45-55％，自由饮水摄食。以醋酸铅和乙醇为受试物，分别按以下剂量染毒，将小鼠按0.1ml/10gbw醋酸铅和乙醇灌胃，每天1次，连续4w。分别为： 对照组：蒸馏水灌胃； 醋酸铅组：醋酸铅水溶液灌胃，250mg／kgbw； 乙醇组：乙醇灌胃，3.16g／kgbw； 联合作用组:用25Om扩kg醋酸铅+3 .16扩kg乙醇组灌胃。 2.观察指标及检测方法 2.1一般指标:体重、脾重、胸腺重并计算脏器系数 2.ZT淋巴细胞增殖功能测定:C。认刺激淋巴细胞转化试验(M竹法) 2.3抗体生成细胞检测:定量溶血分光光度测定法(QHS法) 2.4试管凝集试验 2.5巨噬细胞功能检测 2.5.1大鼠腹腔灌洗液巨噬细胞(PM)数计数 2.5.2大鼠腹腔灌洗液巨噬细胞吞噬功能测定:中性红比色法 2 .6脾脏组织病理学检测 2.6.1光镜观察:脾脏经10%福尔马林固定，常规制片，HE染色，光镜下观察; 2.6.2电镜观察:脾脏经2.5%戊二醛固定，树脂包埋，超薄切片，醋酸铀及柠檬酸铅染色，透射电镜下观察超微结构的改变。 3.数据处理 采用SPsS统计软件进行统计学分析，实验数据用又土s表示，各指标比较，用单因素方差分析，随后采用LSD法进行两两比较。实验结果 1.各组小鼠体重、脾重、胸腺重及脏器系数 各染毒组体重均较对照组低，但铅组、乙醇组与联合作用组之间没有差别;各染毒组之间脾的绝对重量及相对重量都无差异。各染毒组胸腺重均较对照组低，且铅组和联合组的胸腺绝对重量与对照组相比有差异，但各染毒组脏器系数与对照组相比无差异。 2.铅和乙醇对小鼠细胞免疫的影响 各染毒组的细胞增殖功能都显著下降，而联合作用组的增殖功能与铅组和乙醇组相比下降更明显。 3.铅和乙醇对小鼠体液免疫的影响 染铅组和联合作用组的分泌抗体功能显著下降，乙醇组的分泌抗体功能下降不明显，而联合作用组较乙醇组下降明显;试管凝集试验中，各染毒组抗体水平都下降，但各染毒组之间无差别。 4.铅和乙醇对小鼠巨噬细胞吞噬功能的影响 各染毒组巨噬细胞数与对照组相比没有差别;但铅组与乙醇组的吞噬功能明显降低，而联合作用组较铅组与乙醇组又升高，且与乙醇组有显著性差异。 5.脾脏组织病理学改变 5.1光镜下改变 对照组细胞分布均匀，结构正常。染铅组小鼠脾脏淋巴组织减少，融合细胞较多;染乙醇组小鼠脾脏淋巴组织减少，可见细胞溶解;联合组小鼠脾脏淋巴组织减少，细胞溶解更明显。 5.2电镜下改变 对照组琳巴细胞边界清楚，核、线粒体及内质网均正常。染铅组小鼠淋巴细胞核间隙增宽，出现核固缩、核碎裂，线粒体严重空泡变性;染乙醇组小鼠脾脏淋巴细胞大量核固缩、核碎裂，偶见细胞坏死，线粒体严重空泡变性，内质网扩张，有空泡形成;联合组小鼠脾脏淋巴细胞出现核固缩、核碎裂，线粒体空泡变性等异常表现，较铅组和乙醇组更为明显。讨论 动物的体重变化，可以反映动物中毒后机体综合性改变，是一个客观简便的量化指标。在本实验中，各染毒组体重均较对照组明显降低，说明铅和乙醇对小鼠产生了毒作用。脾和胸腺是免疫系统比较重要的器官，本实验中各染毒组胸腺重均较对照组低，且铅组和联合组的绝对重量与对照组相比有差异，而脏器系数各组之间无差异，说明铅和乙醇对胸腺可能没有影响，而胸腺重量的降低可能是由于小鼠体重的降低所致。本实验中脾的绝对重量、脏器系数各染毒组之间无差异，这可能与免疫抑制有关。 免疫系统是维护机体健康、正常执行生理生化功能的重要保证，也是化学毒物经常攻更多还原

【Abstract】 PREFACEThe lead is one of the poisonous heavy metals of the extensive contacts of mankind. Impaired of almost all the organs can result in exposing lead, and the ethanol is the main composition of the organic solvent and wine beverage, the ethanol can influence many systems. The lead and ethanol are extensive chemistry poisons which frequency of contact is high, exposing widely in crowd. In lead workers, particularly is in the youth worker, many are alcohol user. It was reported that 90% contact persons of lead get accustomed to drink wine. The ethanol can promote the lead inside the skeleton of release, make the condition of illness to severity. The incidence of lead poisoning is high of alcohol user. There is a importance theories and actual meaning in studying the joint effect of lead and ethanol.In recent years, the types of joint effect of lead and ethanol are different in different organs. Some showed antagonistic action , others showed synergistic action. There are many studies about joint effect of lead and ethanol, but it is seldom seen to the study of immune system. It is more seldom seen to a research of the whole animal particularly. Immune system is a important assurance for health, and perform normal function of physiology, biochemistry. Immune system is also a target constitution assaulted by chemical poison. In order to study the influence of immune system of male mice made by joint effect of lead and ethanol, we apply in vivo, in vitro experiments, and observe joint effect of cellular immunity, humoral immunity of mice, and search for the mechanism of its function. We also provide theory for protection of the health of contact person, and prevention and cure methods for it.MATERIALS AND METHODS1. Animals and treatmentAdult health Kunming strain mice, 22 +2g body weight, was used for this study. Animals were kept in room under controlled environmental conditions, fed a rodent chow and water ad libitum. Following a one - week acclimatization period, mice were divided at random into four test groups of ten. Negative control group, distilled water 0. 1ml/10g body weight, g. t. ; the simple lead treated group, 250mg/kg lead acetate administrated respectively; the simply ethanol treated groups, 3. 16g/kg ethanol administrated respectively; the joint effects group, 250mg/kg lead acetate plus 3. 16g/kg ethanol administrated respectively. Mice were administrated lead acetate and ethanol by route of oral gavage during four weeks, one time per day. Weight was recorded every three days throughout the experiment.2. Observation and determination2.1 Body weight and spleen and thymus index.2.2 Tlymphocytes function were determined by blastogenic response induced by the mitogen , concanavalin A (Con A) , of spleen lymphocytes. MTT spectrometry method was used.2.3 The cell examination of analysis were determined by hemolysis spec-trophotography method.2.4 Coagulation - tube experiment.2.5 The number of macrophages in peritoneal lavage was counted with a heacytometer.2. 6 Engulfment functions of peritoneal macrophage were determined by means of engulf neutral red spectrometry method according to Xue Bin.2.7 IstopathologyTissues selected for histopathological examination were fixed in 10% neutral buffered formalin , embedded in paraffin, sectioned at 3|xm, and routinely stained with hematoxylin and eosin.Tissues selected for ultrastructure check were fixed in 2. 5% glutardialde-hyd solution, 700 A ultra - thin section, electron stained, observed by transmission electron microscope.3. Statistical analysisThe variances were analyzed by One - way ANOVA procedure to assess the significance of treatment effects. The results were regarded as significant as P < 0.05. Pairwise comparisons were made by q -test . The relationship between indexes were determined by correlation analyses.RESULTS1. Body weight, spleen weight, thymus weight and spleen coefficient, thy-mus coefficient.Body weights of narcotics group were significantly lower than the contr更多还原