IR@PKUHSC  > 北京大学解放军302医院教学医院
学科主题内科学
中药制剂肃毒星的体外和动物体内抗乙型肝炎病毒的作用及其相关机制研究
姚伟明
2014-05-14
导师徐东平
专业内科学
授予单位北京大学
授予地点北京大学解放军302医院教学医院
学位硕士
关键词肃毒星 乙型肝炎病毒 抗病毒 中草药
其他题名Antiviral effects and related mechanisms of Chinese herbal extracts-Suduxing on hepatitis B virus in vitro and in vivo
分类号R512.6
摘要目的 目前临床上批准用于抗乙型肝炎病毒(HBV)的药物主要分为两大类,一是核苷或核苷酸类似物,通过直接抑制病毒逆转录酶抑制病毒DNA的复制;二是干扰素类,通过免疫调节作用发挥其抗HBV活性。然而核苷或核苷酸类似物可耐药,停药后易复发;干扰素类副作用较多,难以彻底清除病毒;联合应用二者也未取得理想的疗效。近年来越来越多的研究则显示许多传统的中草药显示了强效的抗HBV活性,具有良好的临床应用前景。本研究目的在于观察一种新型中药制剂肃毒星的体内外抗HBV作用及其抗病毒机制。方法 以HepG2.2.15细胞(D基因型/ayw亚型野生HBV稳定复制细胞系)和HepG2.A64细胞(C基因型/adr亚型恩替卡韦耐药HBV稳定复制细胞系)作为评价肃毒星抗HBV作用的体外模型,将携带1.3个拷贝HBV基因组信息的重组8型腺相关病毒载体(rAAV-1.3HBV DNA)经小鼠的尾静脉注射建立乙肝小鼠体内评价模型。应用实时荧光定量PCR(Q-PCR)方法检测细胞内及血清中HBV DNA复制水平的变化;用酶联免疫吸附试验(ELISA)检测细胞培养上清和血清中HBsAg和HBeAg含量的变化。基因芯片技术分析肃毒星和恩替卡韦(ETV)药物处理HepG2.2.15细胞前后胞内基因的差异表达情况;采用免疫组织化学技术检测各组小鼠肝脏内HBcAg阳性的肝细胞数变化情况;流式细胞分析技术分析各组小鼠脾脏内T淋巴细胞的活化增殖情况。结果 肃毒星在浓度为10.0μg/mL时对HepG2.2.15细胞和HepG2.A64细胞HBV DNA,HBsAg和HBeAg的抑制率分别为:75.1%,51.0%,64.1%和65.2%,42.9%,63.9%。阳性对照药恩替卡韦(ETV)在浓度为10.0μmol/L时对HepG2.2.15细胞和HepG2.A64细胞HBV DNA,HBsAg和HBeAg的抑制率则分别为:94.7%, 36.6%, 19.8%和52.7%,9.4%, 14.7%。突变株相对于野生株对肃毒星和ETV药物敏感性变化倍数为别为1.2和712.5倍。实验组小鼠腹腔注射剂量为45 mg kg-1 d-1的肃毒星两周后,血清HBV DNA下降1.39个Log值,而血清HBsAg和HBeAg分别较给药前下降48.9% 和 51.7%;阳性对照组小鼠注射剂量为1.0 mg kg-1 d-1 的ETV两周后血清HBV DNA下降2.07个Log值,而血清HBsAg和HBeAg则无明显下降趋势。肃毒星处理HepG2.2.15细胞后,差异表达基因采用KEGG通路分析,发现有10种差异表达倍数在2倍以上的胞内小分子蛋白参与HBV感染相关分子调节网络,抑制HBV复制,而这些分子在ETV处理HepG2.2.15细胞后无显著性的表达差异。免疫组织化学分析显示实验组小鼠肃毒星治疗两周后肝脏内HBcAg阳性的肝细胞数较ETV阳性对照组和生理盐水阴性对照组显著性减少。流式细胞分析结果显示实验组小鼠脾脏内CD107a+CD3+CD8-,CD3+CD4+CD69+的T淋巴细胞比例较对照组显著性升高, 而CD3+CD4+CD62L+的T淋巴细胞比例较对照组显著性降低。结论 肃毒星具有强效的体内外抗HBV活性,抑制病毒DNA的复制,抗原的表达。其体外抗病毒作用机制为诱导胞内小分子差异表达,干扰HBV感染相关分子调节网络而发挥抗HBV作用;体内抗病毒机制可通过活化CD4+ T细胞,激活宿主免疫系统而控制HBV感染。
英文摘要Objective: The most commonly-used antiviral agents for HBV have been divided into two categories: nucleos(t)ide analogues(NAs) and interferons(IFNs).The former has the problem of drug resistance through long-term therapies and relapse after drug-withdrawal, while the later is characterized by the limitation of multiple side effects and unable to completely eradicate viruses. The combination of NAs and IFNs treatment do not achieve expectable results. Currently, an increasing number of researches demonstrated that many traditional Chinese herbs exhibited potent anti-HBV activity. The present study aimed to investigate anti-hepatitis B virus (HBV) effect and related mechanisms of Suduxing, a medicine extracted from Chinese herbs for intravenous use. Methods: HBV-replicating cell lines HepG2.2.15 (Wild-type) and HepG2.A64 (Entecavir-resistant) were used for in vitro test. C57BL/6 mice infected by adeno-associated virus carrying 1.3 mer wild-type HBV genome were used for in vivo test. HBV DNA was quantitated by real-time PCR. HBV antigens were examined by ELISA or immunohistochemistry. Differentially-expressed genes were detected by GeneChip. Immunocytes were analyzed by flow cytometry. Results: Inhibitory rates of Suduxing (10 µg/mL) on HBV DNA, HBsAg and HBeAg production were 75.1%, 51.0%, 64.1% in HepG2.2.15 cells and 65.2%,42.9%, 63.9% in HepG2.A64 cells. By contrast, inhibitory rates of entecavir (10 µmol/L) on HBV DNA, HBsAg and HBeAg were 94.7%, 36.6%, 19.8% in HepG2.2.15 cells and 52.7%, 9.4%, 14.7% in HepG2.A64 cells. The 50% inhibitory concentration of Suduxing and entecavir had 0.2-fold and 712.5-fold increase respectively for entecavir-resistant HBV compared to that for wild-type HBV. Suduxing-treated mice had 1.39 log10 IU/mL decrease of serum HBV DNA, and 48.9% and 51.7% decrease of serum HBsAg and HBeAg levels. Entecavir-treated mice had 2.07 log10 IU/mL decrease of HBV DNA, but without significant decrease of HBV antigens. The number of HBcAg-positive hepatocytes was significantly decreased in Suduxing-treated mice compared to entecavir or normal saline-treated mice. GeneChip analysis showed that 10 genes involved in HBV infection-related molecular interaction network were significantly up- or down-regulated in Suduxing-treated HepG2.2.15 cells rather than entecavir-treated cells. CD107a+CD3+CD8- and CD3+CD4+69+ cell frequencies were significantly increased, and CD3+CD4+CD62L+ cell frequency was significantly decreased in Suduxing-treated mice compared to control mice. Conclusion: Suduxing had potent inhibitory effects on replication and expression of both wild-type and entecavir-resistant HBV. Its effects were associated with the influence on HBV infection-related molecule interaction network in vitro and CD4+ T cell activation in vivo.
语种中文
出处http://xuewei.bjmu.edu.cn/simpsearch.action?keyword=中药制剂肃毒星的体外和动物体内抗乙型肝炎病毒的作用及其相关机制研究&dbid=72
相关网址查看原文
文献类型学位论文
条目标识符http://ir.bjmu.edu.cn/handle/400002259/105427
专题北京大学解放军302医院教学医院
作者单位北京大学解放军302医院教学医院
推荐引用方式
GB/T 7714
姚伟明. 中药制剂肃毒星的体外和动物体内抗乙型肝炎病毒的作用及其相关机制研究[D]. 北京大学解放军302医院教学医院. 北京大学,2014.
条目包含的文件
条目无相关文件。
个性服务
推荐该条目
保存到收藏夹
查看访问统计
导出为Endnote文件
谷歌学术
谷歌学术中相似的文章
[姚伟明]的文章
百度学术
百度学术中相似的文章
[姚伟明]的文章
必应学术
必应学术中相似的文章
[姚伟明]的文章
相关权益政策
暂无数据
收藏/分享
所有评论 (0)
暂无评论
 

除非特别说明,本系统中所有内容都受版权保护,并保留所有权利。