STAT1基因敲除小鼠模型在EV71感染研究中的应用及土拨鼠肝炎病毒持续性感染模型的建立与应用

发布时间：2018-05-29 02:02

本文选题：EV71感染 + STAT1基因敲除小鼠模型　；参考：《北京协和医学院》2017年硕士论文

【摘要】：目的研究STAT1基因敲除小鼠对EV71的敏感性,对EV71动物模型的优化提供数据基础。方法利用LPS刺激STAT1基因敲除C57BL/6小鼠对模型免疫反应稳定性进行检测,并利用微珠免疫分析(CBA)方法对血清免疫相关因子定量分析;利用EV71感染10日龄STAT1基因敲除小鼠后,观察感染症状及病理表现,利用实时荧光定量检测对肌肉病毒RNA定量分析。利用相同剂量、相同毒株的EV71感染10日龄ICR小鼠,与STAT1基因敲除小鼠感染结果进行对比。结果LPS刺激后,STAT1基因敲除小鼠血清IL-1α、L-1β、IL-6、IL-10、IL-12、TNF-α、MIP-1α分泌水平下降,与野生型小鼠相比具有统计学意义(P0.05);EV71感染后,STAT1基因敲除小鼠病理损伤加重,骨骼肌中EV71抗原水平升高;STAT1基因敲除小鼠骨骼肌中病毒载量升高(P0.05);与野生型C57BL/6小鼠及ICR小鼠相比,STAT1基因敲除小鼠表现出更严重的症状与更高的死亡率。结论STAT1在抗EV71感染的免疫过程中扮演重要角色,STAT1基因敲除可以提高小鼠对EV71病毒的敏感性。目的建立土拨鼠肝炎病毒(Woodchuck Hepatitis B virus,WHV)感染3日龄土拨鼠的持续性感染动物模型,利用模型对灵芝孢子粉药物治疗肝炎及免疫功能调节作用进行评价,为慢性肝炎的机制性研究以及抗肝炎病毒药物评价建立动物模型基础。方法核酸检测:根据土拨鼠肝炎病毒表面抗原(WHsAg)设计引物,利用PCR方法,以WHVDNA全基因组质粒梯度稀释作为模板检测引物扩增灵敏度,以混合健康土拨鼠血清的WHV病毒为模板检测引物的特异性,并从中筛选灵敏度高、无非特异性扩增条带及引物二聚体的引物;利用Real-time PCR方法,WHVDNA全基因组质粒梯度稀释作为标准曲线,对土拨鼠血清WHV进行核酸定量检测。表面抗原与核心抗体检测:利用本实验室免疫兔和蛋鸡制备并纯化的多克隆抗体,进行土拨鼠血清中表面抗原WHsAg与核心抗体WHcAb检测。细胞因子mRNA检测:Trizol法提取土拨鼠肝脏mRNA,反转录为cDNA后使用Real-time PCR方法进行检测。结果核酸检测:引物WHV N3 F与WHV N3 R,PCR检测灵敏度可达1 ×104copies/mL,且电泳未见明显非特异性条带及引物二聚体;WHVDNA全基因组质粒的熔点曲线峰值一致,Tm均值为82.18℃,病毒拷贝数与Real-time PCR Ct值的标准曲线R2值为0.992。土拨鼠血清病毒载量水平在感染后3个月开始高于检测下限,感染后4-5个月达到血清病毒载量高峰后有所降低,维持在104copies/mL左右,并持续至感染8个月。雌性土拨鼠血清WHV病毒载量高值水平及高载量持续时间高于雄性土拨鼠。给予灵芝孢子粉药物后土拨鼠血清AST检测数值降低。与给药组相比,对照组IL-4、IL-8、PD-L1、NKp46的mRNA含量有所增高。给药组CD8的mRNA含量高于对照组。对给药组与对照组土拨鼠病理检测结果显示给药组土拨鼠肝脏炎症减轻。结论本实验利用3日龄土拨鼠感染土拨鼠肝炎病毒,建立了土拨鼠肝炎病毒持续性感染动物模型。对土拨鼠肝炎病毒持续性感染动物模型的病毒载量、血清学指标、肝功能酶指标及病理进行了分析,为土拨鼠模型建立及肝炎防治研究提供了数据基础。利用土拨鼠肝炎病毒持续性感染动物模型对灵芝孢子粉药物抗肝炎及免疫调节功能进行评价,发现其对病毒导致的土拨鼠肝炎有减轻作用。通过检测分析灵芝孢子粉药物对土拨鼠肝脏细胞因子mRNA含量的影响,发现灵芝孢子粉药物在土拨鼠肝炎病毒持续性感染动物模型中可以提高细胞毒性T细胞的数量。
[Abstract]:Objective to study the sensitivity of STAT1 gene knockout mice to EV71 and to provide a data basis for the optimization of EV71 animal models. Methods using LPS to stimulate STAT1 gene knockout C57BL/6 mice were used to detect the stability of the model immune response and the quantitative analysis of serum immunization related factors by microbead immunoassay (CBA), and EV71 infected 10 days old S. After TAT1 knockout mice, the infection symptoms and pathological manifestations were observed and the quantitative analysis of RNA was detected by real-time fluorescence quantitative detection. The same dose, the EV71 infection of 10 day old ICR mice with the same strain was compared with the results of STAT1 gene knockout mice. The results of LPS knocking, STAT1 gene knockout mice serum IL-1 a, L-1 beta, IL-6. The levels of IL-10, IL-12, TNF-, and MIP-1 alpha decreased, compared with the wild type mice (P0.05). After EV71 infection, the pathological damage of the STAT1 knockout mice increased, the level of EV71 antigen in the skeletal muscle increased, and the viral load in the skeletal muscle of the STAT1 knockout mice increased (P0.05), compared with the wild type C57BL/6 mice and ICR mice. 1 gene knockout mice showed more serious symptoms and higher mortality. Conclusion STAT1 plays an important role in the immune process of anti EV71 infection. STAT1 gene knockout can increase the sensitivity of mice to EV71 virus. Objective to establish the persistent infection of Groundhog hepatitis virus (Woodchuck Hepatitis B virus, WHV) to infect 3 day old Groundhog. Animal model was used to evaluate the effect of Ganoderma lucidum spore powder on hepatitis and immune function, and to establish an animal model basis for the mechanism study of chronic hepatitis and the evaluation of anti hepatitis virus drug. Method nucleic acid detection was designed according to the primer of WHsAg of Groundhog hepatitis virus (HBV), and the PCR method was used to make WHVDNA whole. The gradient dilution of the genomic plasmid was used as a template to detect primer amplification sensitivity, and the specificity of the primers was detected with the WHV virus in the mixed healthy earth mouse sera as a template, and the sensitivity was high, not not the specific amplification strip and primer two primers, and the Real-time PCR method was used as the standard for the gradient dilution of the whole genome plasmid of WHVDNA as the standard. The quasi curve, the nucleic acid quantitative detection of WHV in the groundhog serum. Surface antigen and core antibody detection: using the polyclonal antibody prepared and purified from the rabbits and laying hens in our laboratory, the surface antigen WHsAg and the core antibody WHcAb in the groundhog serum were detected. The cytokine mRNA was detected by the Trizol method to extract the mRNA of the dialing rat liver, and the reverse transcription was C After DNA, Real-time PCR method was used to test the results. Results the nucleic acid detection: primers WHV N3 F and WHV N3 R, the sensitivity of PCR detection can reach 1 x 104copies/mL, and there is no obvious non specific band and primer two polymer. The standard curve R2 value of the 0.992. groundhog serum virus load level is higher than the lower detection limit after 3 months of infection. After 4-5 months of infection, it reaches the peak of the peak of serum viral load and maintains at about 104copies/mL, and continues to the infection for 8 months. The high level of the WHV virus load and the high load duration of the female groundhog sera are high. In male chidren. The serum AST detection value of Groundhog mice decreased after giving ganoderma spore powder. Compared with the drug group, the mRNA content of IL-4, IL-8, PD-L1 and NKp46 in the control group was higher. The mRNA content of CD8 in the drug group was higher than that of the control group. The pathological examination of the dialing group and the control group showed that the liver inflammation of the dialing group was reduced. In this experiment, the animal model of the persistent infection of the groundhog hepatitis virus was established by using the 3 day old earth chuck infected chuck hepatitis virus. The viral load, the serological index, the liver function enzyme index and the pathology of the animal model of the persistent infective animal model of the groundhog hepatitis virus were analyzed to provide the groundhog model and the study on the prevention and control of hepatitis. Based on the data basis, the anti hepatitis and immunomodulatory function of Ganoderma lucidum spore powder drugs were evaluated using the animal model of the persistent infection of the groundhog hepatitis virus, and it was found that it had a mitigate effect on the virus induced groundhog hepatitis. The effect of ganoderma spore powder on the content of cytokine mRNA in the liver of Groundhog was detected and analyzed, and Ganoderma lucidum was found. Sub powder drugs can increase the number of cytotoxic T cells in animal models of persistent infection of Groundhog virus.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R-332;R725.1

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