慢性病毒性肝炎小鼠动物模型的建立及其特征分析

发布时间：2018-03-24 22:14

本文选题：MHV-3　切入点：C3H/HeJ　出处：《华中科技大学》2006年硕士论文

【摘要】： 目的: 慢性病毒性肝炎是一种严重危害人类健康的常见病,多发病,目前尚缺乏理想的实验动物模型来阐明其具体的发病机制,从而使得对其防治的研究受到很大的局限。本课题拟克服现有模型所存在的不足,建立一种更为理想的慢性病毒性肝炎实验动物模型,为慢性病毒性肝炎发病机制的研究以及防治提供新的理论依据。在此模型基础上我们对其外周血及脾脏T细胞亚群的动态变化做初步观察,以探讨病毒性肝炎慢性迁延免疫机制,并为其免疫治疗提供一些新的思路。方法: 取6~-8周龄雌性C3H/HeJ小鼠60只,将纯化MHV-3病毒进行稀释,以10pfu/200ul/只行C3H/HeJ小鼠腹腔注射,然后对其一般情况、生存曲线进行观察,并于病毒感染后4、6、8、9、10、12、15、20、25、30及40天各取C3H小鼠三只行肝脏组织病理学、血液生化指标以及肝脏组织病毒滴度的变化进行检测。同时采用流式细胞仪检测感染后相应时间点外周血、脾细胞悬液中T淋巴细胞亚群包括CD3~+CD4~+CD8~-、CD3~+CD4~-CD8~+、CD3~+CD4~+CD25~+及CD3~+CD4~-CD8~-T(DN T)细胞的细胞数及各自在T淋巴细胞中所占比率。以生理盐水注射组小鼠作为对照。结果: C3H/HeJ小鼠感染MHV-3后,6天左右开始出现少部分小鼠死亡,约63%存活,存活下来的小鼠一般情况较正常对照组差,肝脏组织呈现持续炎性改变,血清ALT、AST升高而TP、ALB有所下降,且肝脏组织病毒滴度增高,呈现持续病毒复制,与人类慢性病毒性肝炎的发生发展极为类似。其外周血中及脾脏中CD3~+CD4~+CD25~+及CD3~+CD4~-CD8~-T(DN T)细胞在T细胞中的比率有升高的趋势。结论: 本研究所建立的实验动物模型与人类慢性病毒性肝炎的发生发展极为类似,在目前尚无更好的模型的前提下,不失为一种较为理想的慢性病毒性肝炎实验动物模型。其外周血及脾脏中CD3~+CD4~+CD25~+及CD3~+CD4~-CD8~-T(DN T)细胞比率的升高趋势提示这两类细胞可能在慢性病毒性肝炎发生、发展中起着一定的作用。本研究的意义: 本实验所选鼠肝炎3型病毒(MHV-3)易于体内、体外培养, C3H/HeJ小鼠遗传背景清晰,所建模型成本相对低廉,重复性好,同时也克服了已有慢性病毒性肝炎动物模型体内缺乏病原体复制而与临床情况显著不同的局限性,使系统性、动态性研究慢性病毒性肝炎的遗传控制因素及分子生物学、分子免疫学发病机理等成为可能,为其防治提供新的理论依据。在目前尚无完善的慢性病毒性肝炎动物模型的情况下,不失为一种较好的慢性病毒性肝炎动物模型。目前免疫调节T细胞包括CD3~+CD4~+CD25~+及CD3~+CD4~-CD8~-T(DN T)等的研究是一个热点,我们在此模型基础上对外周血及脾脏中T细胞亚群的动态变化进行了初步观察,发现在此模型中这两群调节T细胞的比率都呈现上升趋势,提示这两类细胞可能在慢性病毒性肝炎发生、发展中起着一定的作用,为慢性病毒性肝炎中免疫调节T细胞的具体作用及机制的深入研究打下基础。
[Abstract]:Objective:
Chronic viral hepatitis is a common disease seriously harming human health, disease, lack of ideal animal models to elucidate its pathogenesis, which makes the study on its prevention and treatment are very limited. This paper intends to overcome the shortcomings of the existing model, establish a more chronic virus hepatitis A animal model, provide a new theoretical basis for the study of the pathogenesis of chronic viral hepatitis prevention and control. Based on this model we dynamic changes of peripheral blood and spleen T cell subsets do preliminary observation, to explore the immune mechanism of chronic viral hepatitis, and provide some new ideas for the immune therapy.
Method:
60 female C3H/HeJ mice aged 6~-8 weeks, the purified MHV-3 virus was diluted to 10pfu/200ul/ C3H/HeJ mice by intraperitoneal injection, and then the general condition, survival curves were observed, and in 40 days after infection of 4,6,8,9,10,12,15,20,25,30 and the three C3H mice liver histopathology, blood biochemical indices and the change of liver the organization of virus titer was detected. At the same time were detected by flow cytometry after infection at corresponding time of peripheral blood, spleen cell suspension in T lymphocyte subsets including CD3~+CD4~+CD8~-, CD3~+CD4~-CD8~+, CD3~+CD4~+, CD25~+ and CD3~+CD4~-CD8~-T (DN T) the number of cells in T lymphocytes and their proportion. In the saline group mice were used as controls.
Result:
The C3H/HeJ mice infected with MHV-3 after 6 days began to appear a few mice died, about 63% survive, survive the general condition of mice compared with normal control group, the liver tissue showed persistent inflammatory changes, serum ALT, AST and TP increased, ALB decreased, and the liver tissue virus titer increased, showing sustained viral replication with human chronic viral hepatitis is very similar. The occurrence and development of CD3~+CD4~+CD25~+ and CD3~+CD4~-CD8~-T in peripheral blood and spleen (DN T) cell ratio in T cells increased.
Conclusion:
The experimental animal model with human chronic viral hepatitis established in the study of the occurrence and development of extremely similar, there is no better model under the premise, it is an ideal experimental animal model of chronic viral hepatitis. CD3~+CD4~+CD25~+ and CD3~+CD4~-CD8~-T in peripheral blood and spleen (DN T) increased the percentage of cells that these two kinds of cells may occur in patients with chronic viral hepatitis, plays a certain role in the development of the significance of this study:
The selected mouse hepatitis virus type 3 (MHV-3) to the in vivo, in vitro, C3H/HeJ mice model clear genetic background, relatively low cost, good repeatability, and overcomes the shortcomings of existing chronic viral hepatitis animal model lacking pathogen replication but significantly different from the clinical condition of the limitations of the system. The genetic factors controlling the dynamics of chronic viral hepatitis and molecular biology, molecular immunology and other possible pathogenesis, provide a new theoretical basis for its prevention and treatment. At present there is no perfect chronic viral hepatitis animal model, it is a good animal model of chronic viral hepatitis. The immune regulation of T cells including CD3~+CD4~+CD25~+ and CD3~+CD4~-CD8~-T (DN T) and so on is a hot spot, the dynamic changes of peripheral blood and spleen T cell subsets based on us A preliminary observation, found that the two group of regulatory T cell ratio showed an upward trend in this model, suggesting that these two cell types may occur in patients with chronic viral hepatitis, which plays a certain role in the development, lay the foundation for further study the specific role and mechanism of regulation of T cell immunity in chronic viral hepatitis..

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R-332

【参考文献】