【作者】 黄莹；

【导师】 张国成；

【作者基本信息】 第四军医大学 ， 儿科学， 2004， 硕士

【摘要】 【目的】 病毒性心肌炎（Viral myocarditis，VMC）是小儿常见疾病，发病机制尚未完全阐明，目前认为早期主要为病毒直接侵犯心肌，后期则以病毒持续感染和免疫损害为主。在VMC的发生发展中病毒感染是重要环节，柯萨奇B3病毒（Coxsakievirus B3，CVB3）为主要病原体之一，目前尚缺乏针对CVB3有效的抗病毒药物。近年大量研究证实，中药在抗CVB3上具有一定优势。芪芩Ⅰ号是经过反复研究筛选出的组方，由黄芪、黄芩等数味中药组成，具有清热解毒、益气养阴、活血化瘀功效。本实验主要研究芪芩Ⅰ号对小鼠CVB3-VMC的防治作用及其抗CVB3的作用机制。 【方法】 将正常小鼠随机分为两组：正常对照组、药物毒性实验组。正常对照组给予生理盐水，药物毒性实验组则给两倍治疗剂量芪芩Ⅰ号以观察其急性药物毒性反应。 将500TCID₅₀CVB3稀释液0.1ml注射至BALB／c小鼠腹腔，建立小鼠VMC动物模型。VMC动物随机分为三组：病毒对照组、第四军医大学硕士学位论文芙答I号组、黄茂组。病毒对照组给予生理盐水，治疗组分别给予不同的药物。 各组（除药物毒性实验组以外）按不同时间点检测小鼠的心肌收缩力指标左室内压（LVP）和左室内压最大变化率（LvdP/dtm以）、观察小鼠心肌组织病理改变并半定量分析其程度、观察第7天小鼠心肌细胞超微结构的变化、记录各组小鼠的死亡情况。 进一步用细胞病变抑制法研究茂答I号抗CvB3的作用机制:①直接灭活CVB3的作用:将不同浓度的茂答I号与10oTexnsoeVB3混合3h，攻击细胞，观察细胞病变（ePE）;②抑制细胞内CVB3复制作用:用1 00TCIDsoCVB3攻击细胞lh，弃去病毒液，加入不同浓度的茂答I号，观察CPE;③阻断CVB3吸附细胞的作用:将不同浓度的茂答I号加入细胞中3h，弃去药液，用100TCID5oCVB3攻击细胞lh，观察CPE。以上均设病毒对照组。 用免疫组化SABC法及TUNEL法原位检测茂答工号对小鼠心肌细胞TNF一a的表达及心肌细胞凋亡的影响。【结果】小鼠生存率的变化 小鼠死亡的高峰期为7一10天，第10天后未再发生死亡。至第10天各组小鼠的累积生存率分别是:病毒对照组39.08%，黄茂组48石1%，茂答I号组76.19%。统计分析显示茂答I号组的生存率明显高于病毒对照组（P=0.0081）及黄茂组（尸=0.035）。在21天的实验观察过程中，药物毒性试验组的生存率为100%，正常对照组的生存率为100%。第四军医大学硕士学位论文2小鼠心脏血流动力学指标的变化 分别于病毒感染后第3、5、7天，检测各组LVP、LVdP/dtmax的变化情况:①病毒对照组与正常对照组相比在第3天LVP变化不明显，但其LVdp/d tmax比正常组己明显下降（P<0.01），第5、7天两指标均明显下降（P<0.01）。②黄茂组在第5天时LvdP/dt脚ax值比病毒组明显增大（P<0.05），第7天LVP、LVdP/dtlnax均高于病毒组（P<0.05）。③茂答I号组小鼠的LVP、LVdp/dtmax与病毒组相比在各时间点上均有明显升高（P<0.05）;与黄茂组比较亦升高，至第5天显示有统计学差异（P<0 .05），第7天时LVP虽无差异，但Lv如/dtmax仍有统计学差异（P<0.01）。3小鼠心肌组织形态学的变化3.1光镜检查结果 分别于第3、5、7、10、14、21天处死小鼠，取心脏组织，切片，常规HE染色，光镜下观察显示:①正常对照组小鼠心肌组织结构层次明显，心肌细胞核清晰可见，核仁清楚，胞浆丰富，胞膜完整，肌纤维间无炎性细胞。②病毒对照组小鼠第5天心肌组织有炎细胞浸润，第7¹4天心肌组织有大量炎细胞浸润及点片状坏死灶。病变以第7¹0天最显著。③黄蔑组小鼠第7¹4天心肌组织病变积分比病毒对照组明显减轻（P<0.01）。④茂琴I号组小鼠第3²1天心肌组织病变积分比病毒对照组显著减轻（尸<0.001）;在第5一10天时比黄茂组减轻（P<0.05）。⑤药物毒性试验组小鼠心肌同正常对照组，无病变。3.2电镜检查结果 取第7天小鼠的心脏作电镜观察，病毒对照组小鼠心肌电镜检查显示心肌细胞肌丝溶解，线粒体肿胀、靖断裂，肌浆网扩张、其内发现病毒样颗粒;毛细血管血管内皮细胞肿胀、坏死。两治第四军医大学硕士学位论文疗组上述病变轻微，血管内皮细胞无肿胀、坏死且未发现病毒样颗粒，其中以茂答I号组更为明显。4蔑等I号抑制CVB3的作用 ①直接灭活CvB3作用:蔑芬I号药液浓度为2.875mg/ml和1.44m留ml时，病毒攻击细胞后30h药物组细胞病变（CPE）为阴性，与病毒对照组相比有显著差异（P<0.05）。 ②抑制细胞内CVB3复制作用:蔑答I号药液为上述两个浓度时，病毒攻击细胞后30h药物组CPE为阴性，与病毒对照组相比有显著差异（P<0.05）。 ③阻断CVB3吸附细胞的作用:茂答I号药液为上述两个浓度时，病毒攻击细胞后30h药物组CPE为（+）:与病毒对照组相比有显著差异（P<0 .05）。5芭菩I号对VMC小鼠心肌细胞TNF一住表达的影响 常规SABC法检测小鼠心肌细胞中TNF一a表达，结果显示VMC小鼠心肌细胞中有丁NF一a的表达，第5天最为明显。茂答I号组小鼠心肌细胞中阳性信号比病毒对照组明显减弱（P<0.05）。6芭等I号对VMC小鼠心肌细胞凋亡的影响 用TUNEL法原位检测VMC小鼠心更多还原

【Abstract】 [Objective]Viral myocarditis (VMC) is a common disease in children. Coxsackievirus B3 (CVB3) is an important pathogen that causes VMC, but the pathogenesis of VMC has been unknown until now. It is considered that virus could directly result in myocardial lesions at the early stage of VMC, and that a continuous virus infection and immunity destruction follow at the later stage of VMC. Few methods are effective on treating VMC up to now, while a lot of studies have proved that the treatment of traditional Chinese herbs is a new solution. Qiqin I is made from membranous milkvetch root, baikal skullcap root, etc. The efficacy of it is to clear away heat and toxic materials, to invigorate Qi and nourish Yin to activate blood circulation, and to dissipate blood stasis. The objective of this research was to investigate the therapeutic effect and mechanism of Qiqin I on CVB3-VMC in mice. [Methods]Firstly, the mice were divided into two groups at random: control group and drug toxicity group. Saline were perfused into micestomach in control group. The double therapeutic dosage of Qiqin I were perfused into mice stomach in drug toxicity group in order to observe acute toxicity.Secondly, BALB/c mice were infected (0.1ml) with 500TCID50CVB3, which were divided into three groups: viral control group, Qiqin I group, and the membranous milkvetch root group. The different drugs were given to Qiqin I group, and to membranous milkvetch root group. We detected LVP and LVdp/dtmax were detected at different time points, to observe myocardial lesions and to estimate myocardial histopathological scores. Ultrostructure of myocardial cell was observed when mice were killed at the 7th day after infection with 500TCID50CVB3, and mortality was recorded..Thirdly, we studied the role and mechanism of Qiqin I on prevention and cure CVB3 by cellular protected method.(1)CVB3 was inactivated directly. After Qiqin I of different concentrations and 100TCID50CVB3 were mixed for 3h, Hep-2 cell were infected by it for 1h, and CPE was observed;(2)Duplication of CVB3 was inhibited directly. After Hep-2 cell were attacked by 100TCID50CVB3 for 3h. Qiqin I of different concentration were added into it for lh, and CPE was observed;(3)Adsorption of CVB3 was inhibited. After Qiqin I of different concentrations was added for lh, Hep-2 cell was attacked by 100TCID50CVB3 for 3h, CPE was observed.We observed the expression levels of Qiqin I on TNF-a and the effect of Qiqin I on apoptosis. TdT -mediated dUTP nick and labeling (TUNEL) technique and immunohistochemistry staining were also used in the experiment.[Results]1 Survival rate of the experimental miceMortality rate reached the summit from the 7th day to the 10th day after the drugs were perfused into mice stomach. But, mice were alive after the 10th day. The survival rate of virus group, membranous milkvetch root group and Qiqin I group were 39.08%, 48.61% and 76.19 % respectively. The survival rate of Qiqin I group was increased significantly (P<0.05). After the 21st day, the survival rate of drug toxicity group reached 100%.2 Alteration of cardiac hemodynamics in MiceWe detected LVP and LVdp/dtwax at the 3rd, 5th and 7th day after BALB/c mice were infected with 500TCID50CVB3.(1)There was no significant difference in LVP between control group and virus group, but LVdp/dtmax of virus group decreased significantly (P<0.01). Both LVP and LVdp/dtmax of virus group decreased significantly at the 5th day and the 7th day (P<0.01)(2)Compared with virus group, LVdp/dtwax of membranous milkvetch root group increased significantly at the 5th day (P<0.05). And LVP and LVdp/dtmax of membranous milkvetch root group were higher than those of virus group at the 7th day (P<0.05)(3)Compared with virus group, LVP and LVdp/dtwax of Qiqin I group increased significantly in different time point (P<0.05). Even compared with membranous milkvetch root group, LVP of Qiqin I group increased significantly at the 5th day (P<0.05), and LVdp/dtmax of Qiqin I group increased significantly at the 5更多还原