高表达NQO1转基因鼠的筛选与抗PD作用研究

发布时间：2018-05-09 17:28

本文选题：NQO1 + MPTP　；参考：《华东师范大学》2014年硕士论文

【摘要】：目的：在C57BL/6J小鼠上构建稳定的NQO1(NAD(P)H:quinone oxidoreductase1: EC1.6.99.2)转基因小鼠种系,研究NQO1在小鼠各组织器官中的表达；以NQO1转基因小鼠为平台,建立MPTP(1-甲基-4苯基-1,2,3,6-四氢吡啶)的帕金森病(Parkinson's Disease, PD)模型,研究高表达的NQO1是否对神经元具有保护作用,从而进一步探究帕金森病的发病机理与治疗方法。方法：分别用已有的NQO1表达量不同的转基因小鼠Founder(Tg27、Tg39、Tg68)与WT小鼠进行配种、繁育,对所有子代小鼠进行标记,获得其DNA样本,利用PCR和琼脂糖凝胶电泳的方法进行NQO1基因型的鉴定；跟踪监测三个种系的转基因小鼠与WT小鼠的体重情况,比较之间的差别；将NQO1转基因小鼠杂合子进行配种繁育,利用Real-Time PCR的方法筛选出NQO1纯合子小鼠；利用蛋白印迹(Western Blot)的方法对不同种系小鼠和WT小鼠各组织器官中的NQO1含量进行比较。分别以NOQ1+/-转基因小鼠和WT小鼠为平台,使用7次急性腹腔注射的给药方式,建立MPTP的PD模型；利用NBT/Glycine法检测不同组别的MPTP给药组和对照组小鼠中各脑区中醌化蛋白含量的区别；免疫组化法观察比较NOQ1+/-转基因小鼠和WT小鼠MPTP给药组和对照组间黑质致密部(SNpc) TH神经元的损伤情况;检测比较了NOQ1+/-转基因小鼠和WT小鼠组织中的GSSG和GSH、 NADPH和NADP的含量。结果：保证繁育得到的每一只小鼠的信息(系别、性别、出生日期、基因型等)清晰,以便用于各类实验。体重检测没有发现NQO1转基因小鼠与WT小鼠的生长曲线间存在显著性差异(P0.05); NQOl在小鼠各组织器官中均有表达,三个系的转基因小鼠中NQO1的表达量间有所差异,但均明显高于WT小鼠。成功地以NQO1转基因小鼠以及WT小鼠为平台,建立了MPTP的PD模型；检测得到,MPTP给药组小鼠各脑区的醌化蛋白含量要显著高于对照组小鼠(**PP0.01)；在MPTP给药组中,NQO1转基因小鼠SNpc的TH神经元细胞的受损程度要显著高于WT小鼠(**P0.01)；NQ01小鼠脑内的GSSG和GSH含量与WT小鼠没有显著区别(P0.05),但NADP的含量,NQO1与WT间存在显著性差异(*P0.05)。结论：对运用MPTP建立的PD模型,NQO1高表达的转基因小鼠对PD的病理性症状未出现明显缓解,反而加重了神经元的受损程度。推测这种情况可能是由于NQO1小鼠体内的氧化还原酶类平衡失调而导致的。
[Abstract]:Objective: to construct a stable strain of NQO1(NAD(P)H:quinone oxidoreductase1: EC1.6.99.2) transgenic mice in C57BL/6J mice, to study the expression of NQO1 in various tissues and organs of mice, and to establish a PD model of Parkinson's disease (PD) with MPTP1-methyl-4-phenyl-1-trihydropyridine (MPTP1-methyl-4-phenyl-1-trihydropyridine). To investigate the protective effect of high expression NQO1 on neurons and to further explore the pathogenesis and treatment of Parkinson's disease. Methods: transgenic mice with different NQO1 expression levels were used to breed and breed WT mice. All the offspring mice were labeled and their DNA samples were obtained. The NQO1 genotypes were identified by PCR and agarose gel electrophoresis. The body weight of transgenic mice and WT mice of three strains were monitored and the differences were compared. The heterozygotes of NQO1 transgenic mice were bred and the homozygote mice of NQO1 were screened by Real-Time PCR method. The contents of NQO1 in tissues and organs of different strain mice and WT mice were compared by Western blotting method. The PD model of MPTP was established on the platform of NOQ1 r-transgenic mice and WT mice by seven times of acute intraperitoneal injection. NBT/Glycine method was used to detect the difference of quinonated protein content in the brain regions of mice treated with different groups of MPTP and the control group. Immunohistochemical method was used to observe and compare the injury of th neurons in the substantia nigra densification region between the MPTP group and the control group of NOQ1 / 尾 -transgenic mice and WT mice, and the contents of GSSG, GSH, NADPH and NADP in the tissues of NOQ1 / 尾 -transgenic mice and WT mice were compared. Results: the information (gender, date of birth, genotype, etc.) of each mouse was clear for use in all kinds of experiments. There was no significant difference in growth curve between NQO1 transgenic mice and WT mice by body weight test. NQOl was expressed in all tissues and organs of mice, and the expression of NQO1 in three lines of transgenic mice was different. But both of them were significantly higher than those of WT mice. The PD model of MPTP was successfully established on the platform of NQO1 transgenic mice and WT mice. The content of quinone protein in the brain regions of mice treated with MPTP was significantly higher than that in the control group, and the damage degree of th neurons in SNpc of MPTP group was significantly higher than that in WT mice. The contents of GSSG and GSH were not significantly different from those of WT mice (P 0.05), but there was significant difference between NADP and WT. Conclusion: the pathological symptoms of PD in transgenic mice with high expression of NQO1 in PD model established by MPTP were not significantly alleviated, but the damage degree of neurons was aggravated. This may be due to the imbalance of redox enzymes in NQO1 mice.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：Q78;R749

【相似文献】