苦参碱抗牛乳头状瘤病毒感染C127细胞研究

发布时间：2018-02-15 07:47

本文关键词： 牛乳头状瘤病毒 C127细胞苦参碱细胞凋亡　出处：《东北农业大学》2015年硕士论文　论文类型：学位论文

【摘要】：牛乳头状瘤病毒(Bovine Papillomavirus,BPV)是牛乳头状瘤病的病原体,BPV能够引起牛的皮肤、黏膜上皮增生性病变,目前BPV已呈全球内流行趋势,带给了养牛业巨大的经济损失。当前防治牛乳头状瘤病的方法有外科手术治疗法、中药治疗法及疫苗免疫法等。本研究利用BPV感染C127细胞,建立细胞感染模型,进行牛乳头状瘤病防治药物的体外筛选,为牛乳头状瘤病的临床治疗提供理论基础及试验依据。试验选取临床典型的牛乳头状瘤组织,对其进行光镜及电镜观察,通过间接ELISA法检测肿瘤组织中BPV,并进行PCR扩增、测序确定其基因型;BPV野毒株感染C127细胞,利用光镜对比观察细胞形态学变化,透射电镜观察细胞内病毒粒子分布;以利巴韦林作为阳性对照药物,通过实时荧光定量PCR法检测苦参碱及氧化苦参碱对BPV直接灭活作用、抑制BPV在细胞内增殖作用及预防BPV对细胞的感染作用,评价苦参碱及氧化苦参碱对BPV的作用效果;利用细胞流式技术检测药物作用后,对染毒细胞的凋亡影响,探索药物作用机制。结果表明:光镜观察可见肿瘤组织呈多个指状突起,每个突起的中心为纤维组织和血管,突起外为多层鳞状上皮细胞,异型性低,未见明显角化,且无异常核分裂象。透射电镜观察可见肿瘤组织基底细胞、棘细胞及颗粒细胞均出现细胞形状不规则、线粒体空泡状等病理变化。间接ELISA法检测肿瘤组织BPV呈阳性反应;经PCR扩增、测序结果与分离自中国的BPV-2病毒SW株(Gen Bank:KC256805.1)L1基因的相似性高达99%。BPV野毒株感染C127细胞,细胞形状变小、细长、生长密集、排列紊乱,PCR检测在450bp左右出现特异条带。细胞透射电镜观察发现病毒粒子,并伴随高尔基体、粗面内质网、线粒体变形等病理变化。通过药物作用后染毒细胞CPE及实时荧光定量PCR检测结果表明具直接抗BPV作用的为:利巴韦林浓度为0.625μg/ml时抑制率最高,可达90%,苦参碱浓度为80μg/ml时抑制率最高,可达98%,与对照组相比差异极显著(P㩳0.01);具预防BPV感染作用的为:苦参碱浓度为80μg/ml时抑制率最高,可达95%,与对照组相比差异极显著(P㩳0.01);具抑制BPV在细胞内增殖作用的为:利巴韦林浓度为0.625μg/ml时抑制率最高,可达93%,苦参碱浓度为80μg/ml时抑制率最高,可达95%以上,与对照组相比差异极显著(P㩳0.01);而氧化苦参碱药物最大无毒浓度的三种作用途径对BPV的抑制率均低于30%,视为无效。BPV感染细胞24h时,己经开始抑制细胞凋亡,且细胞主要处于晚期凋亡,同时苦参碱的三种给药途径处理BPV,均对细胞晚期凋亡具有一定促进作用。结论:苦参碱直接作用于BPV可使其活性丧失,苦参碱又具有抑制BPV在细胞内增殖及预防BPV感染的作用。
[Abstract]:Bovine papillomavirus (BPV) is the pathogen of bovine papillomatosis. The current methods of prevention and treatment of bovine papillomatosis include surgical treatment, traditional Chinese medicine therapy and vaccine immunization. In this study, C127 cells were infected with BPV, and cell infection models were established. In order to provide theoretical basis and experimental basis for clinical treatment of bovine papillomatosis, the clinical typical bovine papillomatous tissue was selected and observed by light and electron microscope, and in vitro screening of drugs for the prevention and treatment of bovine papillomatosis was carried out in order to provide theoretical basis and experimental basis for clinical treatment of bovine papillomatosis. BPVs in tumor tissues were detected by indirect ELISA method, and PCR amplification was carried out. The genotypes of BPV-wild strains were determined by sequencing. The morphological changes of C127 cells were observed by light microscopy and the distribution of virus particles in the cells was observed by transmission electron microscope (TEM). Ribavirin was used as a positive control drug. The direct inactivation of BPV by matrine and oxymatrine was detected by real-time fluorescence quantitative PCR method, which inhibited the proliferation of BPV in cells and prevented the infection of BPV. To evaluate the effect of matrine and oxymatrine on BPV and to detect the apoptosis of exposed cells by flow cytometry. The results showed that the tumor tissue showed multiple digital processes, the center of each process was fibrous tissue and blood vessel, and the outer process was multilayer squamous epithelial cells, with low heterogeneity and no obvious keratosis. There were no abnormal mitotic signs. The basal cells of tumor tissues were observed by transmission electron microscope, the cells of spinous cells and granulosa cells were irregular, and the mitochondria were vacuolated. Indirect ELISA method was used to detect the positive reaction of BPV in tumor tissues. The results of PCR amplification showed that the similarity of the gene of Genin Bank:KC256805.1)L1 of BPV-2 virus SW strain isolated from China was as high as 99k.BPV wild strain infected C127 cells. The cells became smaller, longer and more dense in growth. The specific bands were detected by PCR at about 450 BP. The virus particles were observed by transmission electron microscope and accompanied by Golgi body and rough endoplasmic reticulum. The results of CPE and real-time fluorescence quantitative PCR analysis showed that the inhibition rate of ribavirin was the highest when the concentration of ribavirin was 0.625 渭 g / ml, and the results of real-time fluorescence quantitative PCR analysis showed that the inhibition rate of ribavirin was the highest when the concentration of ribavirin was 0.625 渭 g / ml. Up to 90%, matrine concentration is 80 渭 g / ml, the inhibition rate is the highest, up to 98, compared with the control group, the difference is very significant. The inhibitory rate of matrine was the highest when the concentration of matrine was 80 渭 g / ml, up to 95%, and the difference was significant compared with the control group. The inhibition rate of ribavirin was the highest when the concentration of ribavirin was 0.625 渭 g / ml, and that of matrine was 80 渭 g / ml, which was more than 95%. However, the inhibitory rate of the three pathways of maximal nontoxic concentration of oxymatrine on BPV was lower than that of 30%, which was regarded as ineffective. After 24 hours of infection, the apoptosis of the cells was inhibited, and the cells were mainly in the late stage of apoptosis. At the same time, the three ways of administration of matrine can promote the late apoptosis of BPV.Conclusion: matrine can inhibit the proliferation of BPV in cells and prevent BPV infection by directly acting on BPV.Conclusion: matrine has the function of inhibiting the proliferation of BPV in cells and preventing the infection of BPV.
【学位授予单位】：东北农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S853.7

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