黄芩、柴胡及其配伍的提取物对CVB_3m增殖及心肌组织细胞凋亡干预的比较研究

【作者】 刘芳；

【导师】 王雪峰；

【作者基本信息】 辽宁中医学院 ， 中西医结合临床， 2005， 博士

【摘要】 目的: 研究黄芩、柴胡及其配伍提取物体外抗CVB_3m病毒及对细胞活性影响,探讨不同药物的作用机制和靶点;研究黄芩、柴胡及其配伍提取物对CVB_3m诱导Balb/c小鼠心肌炎的治疗作用,并初步探讨其作用机制。 方法: 1.采用微量细胞培养法,以细胞病变法和MTT法判定细胞活性,观察黄芩、柴胡及其配伍提取物以及黄芩两种不同提取制剂体外抗CVB_3m病毒和对细胞活性的影响,确定黄芩、柴胡及其配伍提取物以及黄芩两种不同提取制剂抑制病毒指数和药物对细胞的预防和治疗保护率。 2.采用腹腔注射CVB_3m病毒诱导Balb/c小鼠心肌炎,应用RT-PCR技术检测病原核酸,结合光镜和电镜病理观察方法,鉴定小鼠病毒性心肌炎模型。 3.将黄芩、柴胡及其配伍的提取物以灌胃给药途径治疗CVB_3m心肌炎小鼠14天,观察三组药物对小鼠一般状态、体重、14天存活率、心脏质量/体质量、心脏组织CVB_3m病毒滴度及光镜和电镜病理改变的影响。 4.采用RT-PCR技术,检测各组小鼠给药后不同时间（3天、5天、7、10天、14天）心肌组织中CVB_3m—RNA的复制和IFN-mRNA的表达情况,探讨黄芩、柴胡及其配伍提取物干预病毒复制和诱生干扰素的作用。 5.分别采用TUNEL、免疫组化和RT-PCR技术,检测小鼠给药后不同时间点心肌组织细胞凋亡情况、Bcl-2和Bax的蛋白及mRNA表达情况,探讨黄芩、柴胡及其配伍提取物对病毒性心肌炎心肌组织细胞凋亡的干预。 结果: 一、体外实验 1.黄芩提取物浓度为1.563mg/ml时,抑制病毒指数大于2,而柴胡组和配伍组的抑病毒指数均小于2;三组药物都具有不同程度的预防保护作用,而柴胡组和配伍组的预防保护作用尤为明显,其预防保护率明显高于黄芩组,有显著性差异（P<0.05）;在病毒感染后,三更多还原

【Abstract】 Objeetive1. To study the therapeutic effects of the skullcap extract、 the Bupleurum extract and their compatibility extract on Coxsackievixus B₃a. （CVB₃m） in vitro, and to explore the antiviral mechanism, and to observe the influence on cell infected by CVB₃m.2. To observe the therapeutic effects of the skullcap extract、 the Bupleurum extract and their compatibility extract on the 3alb/c mice myocarditis infected by CVB₃ and to exnlore the mechanism of the effect . Methods1. We used microcell culture method to observe the effect of the skullcap extract、 the Bupleurum extract and their compatibility extract on antivirus and protecting cell. We detected the restrained indexes of the three kinds of drugs on CVB-3m and the cell protection rate of the three kinds of drugs when the cell was infected by CVB₃M2. Balb/c mice were inoculated intraperitoneally with CVB₃m and batch sacrificed late. On the base of the models , following experiment were carried.（1）The CVB₃m-RNA were detected by reverse transcription - polymerase chain reaction （RT-PCR）, the heart histopathologic （macro- and ultramicro） were observed by the light microscope and electron microscope.（2）Mice were randomly divided into 5 groups, including the model group、 the control group、 the skullcap extract group、 the Bupleurum extract group and the compatibility extract group. We cured the myocarditis of mice by intragastric administration for 14 days, and observed the body of weigh of mice, appetite, thesymptom and the death of mice. We detected the tite of CVB₃m, in heart、 rate between the heart mass and weigh of the body and macro-histopathologic and ultrandcro changes of hearts.（3） The CVB₃m-RNA and IFN mRNA in heart were examed by RT-PCR method to explore the effect of antivirus and induction of IFN by the three kinds of drugs.（4）The apoptosis in heart were examed by TDT/Dutp Nick End labeling （TUNBL）, and the apoptosis related protein and gene expression of Bc1-2 /Bax were detected by RT-PCR method and immunohistochemistry method .Result Experiments in vitro.1. The skullcap extract can directly kill CVB₃m. in vitro when his density is 1. 563 mg/ml, but the Bupleurum extract and their compatibility extract hadn’ t this effect. The three kinds of drugs can protect cell infected by CVB3.. The effect of the Bupleurum group and the compatibility group is better than the skullcap groups when the drugs were dministered in advance. The effect of the skullcap group and the compatibility group were better than the Bupleurum group when the drugs were dministered after infection.2. The restrained indexes of the crude extracting skullcap was more than 2 when his density is TD₀; The restrained indexes of the fine extracting skullcap was less than 2 when his density is TD₀、 1/2TD_O and 1/4TD₀. The two kinds of drugs can protecte cell infected by CVB₃m , and the effect of the crude extracting skullcap was better than the fine extracting skullcap. Experiments on animal.1. The positive percent of CVB₃m-BNA in heart is 100% during 3th ¹4th day after infected. The heart histopathologic change were inflammatory cell infiltration、 dropsy and necrosis of heart cell.2. The three kinds of drugs can increcse the body weight of myocarditis mice, appetite and the rate of survival; In increasing the rate of survival and the body weight, the difference of the three groups is not significant . The three kinds of drugs can inhibit replication of CVB₃m. in heart. The viral titer in heart of the skullcap group were lower than the model group in the3th、 5th、 7th and 10th day: The viral titer in heart of the Bupleurnm. group were lower than the model group in the 3th、 5th day: The viral titer in heart of the compatibility group were lower than the model group in the 5th、 7th day: The macro-, histopathologic and ultramicro changes of mice hearts of the three drugs were better than the model group too, but the effect of the Bupleurum group and the compatibility group was better than the skullcap group.3. The content of CVB₃m-RNA in the hearts of the three drugs更多还原