柯萨奇病毒A16与小鼠病毒性心肌炎的关联性及其药物干预研究

发布时间：2018-05-30 16:02

本文选题：病毒性心肌炎 + 柯萨奇病毒A组16型　；参考：《安徽医科大学》2017年硕士论文

【摘要】：目的经过细胞培养并经RT-PCR技术鉴定得到柯萨奇病毒A组16型病毒,使用该病毒建立小鼠病毒性心肌炎的模型,通过对病毒性心肌炎小鼠心脏组织及血清学研究探讨:1、病原学本身及免疫内环境的紊乱与病毒性心肌炎的关联,2、利巴韦林在治疗感染CVA16小鼠的病毒性心肌炎的适宜剂量。方法1、通过细胞培养技术,培养对该病毒敏感的Vero细胞,再用其感染CVA16病毒,观察病毒的致病变能力,同时通过RT-PCR对病毒进行鉴定,将培养至第9代的病毒进行放大培养,经离心获得病毒上清液,进行无菌分装,同时进行TCID50实验检测病毒的浓度,至此获得已知毒力的CVA16病毒;2、使用上述获得所需的病毒去感染小鼠,实验采用腹腔注射,建立与病毒性心肌炎有关的小鼠模型,将实验分五组,实验组(A组)、对照组(B组)、药物干预高(C组)、中(D组)、低剂量组(E组),实验过程记录小鼠的体重、观察小鼠四肢活动情况,分别在生后第3,7,11,15天处理小鼠,获取血清及心脏组织,将获得的心脏组织部分进行HE染色,另外一部分心脏组织用于提取RNA,进行RT-PCR实验,进行病毒的鉴定,获取的血清采用ELASA试剂盒检测血清中的IL-6数值。所得定量数据,采用随机区组设计资料的方差分析和完全随机设计资料的方差分析进行统计学分析。结果1、经RT-PCR鉴定所获病毒为CVA16,并且经TCID50计算出该病毒的浓度是106TCID50/ml;2、小鼠的体重随着日龄增加,对照组小鼠体重及适宜剂量利巴韦林药物干预后较实验组小鼠体重增加的更明显,而高剂量及低剂量干预后与实验组小鼠没有明显的改善;3、小鼠的血清IL-6值是先升高,在第7天时最高,后逐渐降低的趋势,并且较正常组有明显升高;4、感染CVA16小鼠的心肌组织病理切片中可见明显的心肌炎性病变,适宜剂量利巴韦林干预的心肌炎症有明显好转,高剂量及低剂量利巴韦林药物干预后,心肌炎症较实验组无明显改善;5、从实验组小鼠的心肌组织随机提取RNA经RT-PCR鉴定获得与感染病原相同大小的条带。结论1、柯萨奇病毒A组16型可通过病原本身致病毒性心肌炎的发生,同时免疫内环境也在心肌有损伤发挥作用;2、适宜剂量的利巴韦林在治疗CVA16有关的心肌炎中有一定的临床意义。
[Abstract]:Objective to obtain coxsackie virus group A 16 virus by cell culture and RT-PCR technique, and to establish a mouse model of viral myocarditis by using this virus. Through the study of the heart tissue and serology in mice with viral myocarditis, the relationship between the etiology itself and the disturbance of the immune internal environment and viral myocarditis was discussed. The appropriate dosage of ribavirin in the treatment of viral myocarditis in mice infected with CVA16 was discussed. Methods 1. Vero cells which were sensitive to the virus were cultured by cell culture technique, and then infected with CVA16 virus. The virulence of the virus was observed. At the same time, the virus was identified by RT-PCR, and the virus cultured to passage 9 was amplified. The virus supernatant was obtained by centrifugation, aseptic loading was carried out, and the concentration of the virus was detected by TCID50 experiment, so that the known virulence of CVA16 virus was obtained. The above obtained virus was used to deinfect the mice, and the mice were injected intraperitoneally. A mouse model related to viral myocarditis was established. The mice were divided into five groups: group A, group B, control group, group C, group D and group E, respectively. The body weight of the mice was recorded during the experiment. The activity of limbs of mice was observed. The mice were treated at the 3rd day after birth for 1115 days. The serum and heart tissue were obtained. The obtained heart tissue was stained with HE, and the other part of heart tissue was used to extract RNAs for RT-PCR test. The virus was identified and the ELASA kit was used to detect the value of IL-6 in the serum. The quantitative data were analyzed statistically by ANOVA of random block design data and complete random design data. Results 1. The virus was identified as CVA16 by RT-PCR, and the concentration of the virus was calculated by TCID50 to be 106TCID50 / ml ~ (-2). The body weight of mice increased with the age of day. The weight of control group mice and the appropriate dosage of ribavirin increased more obviously than that of experimental group mice after the intervention of ribavirin. However, after high and low dose intervention, there was no significant improvement in serum IL-6 of the experimental group. The serum IL-6 increased first, reached the highest level on the 7th day, and then decreased gradually. Compared with the normal group, the myocardial tissue of CVA16 infected mice showed obvious myocarditis pathological changes, the myocardial inflammation of the suitable dose of ribavirin was improved obviously, and the high dose and low dose of ribavirin were treated with ribavirin. Myocardial inflammation did not improve significantly compared with the experimental group. RNA was randomly extracted from the myocardial tissue of the experimental group and identified by RT-PCR to obtain the same size bands as the infection pathogen. Conclusion 1. Coxsackie virus group A 16 can cause viral myocarditis by the pathogen itself. At the same time, the immune environment also plays a role in myocardial injury. The appropriate dosage of ribavirin has a certain clinical significance in the treatment of CVA16 related myocarditis.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.21

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