摘要
目的构建SD大鼠鱼藤酮慢性中毒模型,同时对SD大鼠组织标本（脑组织和肝脏）进行线粒体DNA突变的初步探索,为后续实验提供线索。方法将75只雄性SD鼠随机分为三组,实验组：0.5 mg/（kg·d）鱼藤酮皮下注射4~8周,共45只;空白组：不予任何特殊处理4~8周,共15只;对照组：相同体积的二甲基亚砜（DMSO）皮下注射4~8周,共15只。其中实验组随机分为1、2、3小组,每小组15只;空白组和对照组也分别分为1、2、3小组,每小组5只。每组内相应的1、2、3小组分别在实验的第4、6、8周处死老鼠并留取相应的组织标本（中脑组织和肝脏）,存储于-80℃冰箱中待用。在实验组的每个小组内分别随机选取3对组织标本,共9对;在空白组和对照组的每个小组内分别随机选取1对组织标本,共6对。随后进行组织DNA提取,采用2对引物进行长PCR并将PCR产物进行凝胶电泳分析,同时设计并利用线粒体DNA引物,用一代测序的方法进行线粒体全基因组测序。实验中观察各组大鼠的一般情况并进行行为学测试。结果1）实验组大鼠在实验中表现出较为明显的中毒症状。在体重增长速度上与对照组和空白组存在明显差异（P〈0.05）。在行为学上,实验组大鼠亦表现出了较为明显的行为学异常（P〈0.05）。2）大鼠脑以及肝脏组织的长PCR凝胶电泳结果未发现明显的异常条带;所有测序突变（缺失、插入、点突变）均为大鼠固有突变。结论 0.5 mg/（kg·d）鱼藤酮皮下注射4~8周可导致SD大鼠鱼藤酮慢性中毒。SD幼鼠的选择、鱼藤酮剂量及作用时间、一代测序等多种因素可能是导致本次实验未检测到有意义的线粒体DNA突变的原因。因此,后续实验应做相应改进。
Objective To construct SD rat model with chronic rotenone intoxication, and randomly select 15 SD rats specimens （brain and liver） for pre-experiment. DNA of these specimens were extracted, and sent to detect the presence of the mutation of mitochondrial DNA in order to provide clues for the follow-up study.Methods A total of 75 male SD rats were randomly divided into 3 groups, experiment group： 0.5 mg/（kg, d） rotenone subcutaneous injection （4-8 weeks）, including 45 rats. Blank group： no any special treatment （4~8 weeks） , including 15 rats, and the control group： the same volume of subcutaneous injection of DMSO （4~8 weeks）, including 15 rats. And rats in the experiment group were randomly divided into 1,2,3 subgroups, 15 rats in each; the blank group and control group were divided into 1,2,3 subgroups respectively, 5 rats in each. The rats in each group of the corresponding 1,2, 3 subgroups would be killed respectively in the experiments of 4,6,8 weeks, and we were going to take corresponding tissue samples （including the brain tissue with the midbrain and the liver tissue） , storage -80 ~C in the refrigerator. Each subgroup in the experiment group was randomly selected 3 pairs of tissue samples, a total of 9 pairs; Each subgroup in the blank group and control group were randomly selected 1 pair tissue sample with a total of 6 pairs. DNA in these tissues were extracted, using 2 primers for long PCR and PCR products for gel electrophoresis analysis, at the same time, the mitoehondrial DNA primers were designed and used, using the method of sequencing mitochondrial genome sequencing. In progress of the experiment, the general condition of rats in each group were observed and the simple behavioral test were performed. Results The rats of the experimental group showed obvious symptoms of poisoning. There were significant differences between the control group and the blank group （P〈0.05）. In the behavioral tests, the experimental group also showed a more obvious abnormal behavior
出处
《中国热带医学》
CAS
2016年第1期10-16,共7页
China Tropical Medicine
基金
海南省重点科技计划项目(No.ZDXM20130065)
