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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 a chronic poisoning model of rotenone in SD rats and explore the mutation of mitochondrial DNA in the tissue of SD rats (brain and liver) to provide clues for subsequent experiments. Methods Seventy-five male SD rats were randomly divided into three groups: experimental group: rotenone 0.5 mg / (kg · d) subcutaneously for 4-8 weeks, 45 rats in total; blank group: without any special treatment for 4-8 weeks Control group: the same volume of dimethyl sulfoxide (DMSO) subcutaneously for 4 to 8 weeks, a total of 15. The experimental group were randomly divided into 1,2,3 groups, each group of 15; blank group and control group were also divided into 1,2,3 groups, each group of 5. The corresponding 1,2,3 groups in each group were sacrificed at the 4th, 6th and 8th week of the experiment and the corresponding tissue samples (mesencephalic tissue and liver) were collected and stored in the refrigerator at -80 ℃ for later use. Three pairs of tissue samples were randomly selected from each group in the experimental group, and a total of nine pairs. Six pairs of tissue samples were randomly selected from each of the blank group and the control group. Subsequently, tissue DNA extraction was carried out. Two pairs of primers were used for long PCR and the PCR products were subjected to gel electrophoresis analysis. At the same time, mitochondrial DNA primers were designed and used to perform whole-genome mitochondrial DNA sequencing. In the experiment, the general situation of each group was observed and behavioral tests were conducted. Results 1) Experimental rats showed obvious symptoms of poisoning during the experiment. There was a significant difference in the rate of weight gain between the control group and the blank group (P <0.05). In behavioral, experimental rats also showed more obvious behavioral abnormalities (P <0.05). 2) No significant abnormal bands were found in the long-term PCR gel electrophoresis of rat brain and liver tissues; all of the sequencing mutations (deletion, insertion, point mutation) were inherent mutations in rats. Conclusion Subcutaneous injection of 0.5 mg / (kg · d) rotenone for 4-8 weeks can cause chronic poisoning of rotenone in SD rats. SD baby rat selection, dose and duration of rotenone, generation sequencing and other factors may lead to this experiment did not detect meaningful mutations in mitochondrial DNA causes. Therefore, follow-up experiments should be improved accordingly.