【作者】 庞然；

【导师】 张淑玲；

【作者基本信息】 华中科技大学 ， 内科学， 2010， 博士

【摘要】 第一部分蛇葡萄根乙醇提取物抗乙肝病毒效应研究目的初步探讨蛇葡萄根Ampelopsis sinica W. T. Wang体外抗乙肝病毒效果。方法以HBV全基因组转染的人肝癌细胞系HepG2 2.2.15作为抗病毒细胞模型,首先采用细胞病变效应检测法以及MTT法检测蛇葡萄根对该细胞的毒性作用,然后加入不同无毒浓度蛇葡萄根对细胞进行不同时间的干预。ELISA法和实时荧光定量PCR法分别检测上清液中HBsAg、HBeAg的分泌量和HBV DNA含量。结果200μg/ml及200μg/ml以下浓度蛇葡萄根对HepG2 2.2.15的活性没有显著影响(P＞0.05)。在无毒浓度范围内,不同浓度蛇葡萄根干预细胞后细胞所分泌的HBsAg、HBeAg和HBV DNA水平较未干预组低(P＜0.05),且药物浓度越高,对HBsAg、HBeAg和HBV DNA的分泌抑制作用越明显,呈浓度依赖性。但与作用时间没有明显依赖关系。结论蛇葡萄根通过抑制HBsAg、HBeAg和HBV DNA的分泌而初步显示其抗病毒效果。为临床抗病毒药物的研究提供了一定的理论依据。第二部分蛇葡萄根乙醇提取物基于荧光素酶报告基因的抗乙肝病毒作用机制初步研究目的为了明确蛇葡萄根抗病毒的作用机制。方法首先对蛇葡萄根抗乙肝病毒过程中的信号通路进行筛选,以带有荧光素酶报告基因的PathDetect Cis-/Trans-Reporting Systems (pNFκB-Luc, pAP-1-Luc, pISRE-Luc, p53-Luc, pFA2-Elk1, pFA2-cJun, pFA2-CHOP pFA2-CREB, pFR-Luc)转染HepG2细胞,蛇葡萄根干预后,检测荧光素酶活性,并用流式细胞仪检测蛇葡萄根干预HepG2 2.2.15细胞48h后,细胞凋亡情况；然后从HBV转录水平来阐明问题,我们构建了五种带有荧光素酶报告基因的HBV启动子质粒(Cp, Xp, S1p, S2p和Fp)以及五个启动子截短报告质粒,并分别转入人肝癌细胞系HepG2,蛇葡萄根干预后检测荧光素酶活性。结果蛇葡萄根能够抑制p53的活性(P<0.05),对HepG2 2.2.15细胞具有促凋亡作用；蛇葡萄根能选择性抑制HepG2细胞中HBV启动子活性(Cp, S1p和Fp)(P<0.05)以及截短质粒t1,t4和t5的活性(P<0.05)。结论蛇葡萄根的抗病毒作用可能与其抑制HBV转录及p53通路有关,并且可能是通过抑制p53通路诱导细胞凋亡从而抑制病毒复制。第三部分蛇葡萄根的提取工艺目的为了促进蛇葡萄根的深入开发和为临床进程提供科学依据。对该药用植物的各种提取制备工艺做一简述及比较。方法采用水煮法、回流法、渗漉法等5项提取工艺分别提取蛇葡萄根,测定提取液所含的小分子水溶性指标性成分没食子酸和提取物的干浸膏量。结果以本实验推荐的5#工艺方法提取的提取物,其杂质减少了约70%,但提取液中小分子水溶性指标性成分没食子酸的含量没有明显差异。结论用该工艺最终制备的蛇葡萄根提取液或干浸膏中有效物质的纯度相对于水煮液和仅仅用醇提的粗提取物大大提高。用该工艺方法提取精制的蛇葡萄根制成0.8g/ml的生药提取液,澄明度增高,可以直接制备溶液剂(水或醇的粗提取物均为混悬剂)。尤其是该溶液去掉了有一定刺激性的粘液质成分,同时,通过最大限度去掉粘液质和鞣质等杂质,其细胞毒性大为降低。更多还原

【Abstract】 Part 1 Preliminary research on anti-hepatitis B virus effects of Ampelopsis Sinica Root in vitroObjective:The present study was performed in order to investigate the anti-HBV activity of the ethanol extract from A. sinica root in vitro.Methods:The stably HBV-transfected HepG2 2.2.15 cells were cellular model. First, the cytotoxic concentration of A. sinica root was detected by CPE and MTT assay. Then, the HepG2 2.2.15 cells were interfered by A. sinica root. The antiviral activity of A. sinica root was examined by detecting the levels of HBsAg, HBeAg and extracellular HBV DNAs in stable HepG2 2.2.15 cells by ELISA and real-time PCR assay respectively.Results:A. sinica root with concentration 200μg/ml and the following had no effect on cell viability (P< 0.05). It effectively suppressed the secretion of HBsAg and HBeAg from HepG2 2.2.15 cells in a dose-dependent manner, and it also suppressed the amount of extracellular HBV DNA (P< 0.05). But this inhibiting effect had no significant relationship with the action time.Conclusions:A. sinica root had anti-virus activity by inhibiting the secretion of HBsAg, HBeAg and HBV DNA from HepG2 2.2.15 cells. Part 2 Anti-hepatitis B virus mechanisms of extract from Ampelopsis Sinica Root in vitroObjective:To clarify the anti-virus mechanisms of extract from A. Sinica Root in vitro.Methods:First, screening the signaling pathways, the PathDetect Cis-/Trans-Reporting Systems (pNFκB-Luc, pAP-1-Luc, pISRE-Luc, p53-Luc, pFA2-Elkl, pFA2-cJun, pFA2-CHOP, pFA2-CREB, pFR-Luc) were transiently transfected into HepG2 cells, respectively. After 8 h of transfection, cells were treated with 40μg/ml A. Sinica Root for 48 h. The activity of each signaling pathway was determined by measuring the luciferase activities of the reporter in treated and untreated transfectants. After A. Sinica Root treatment, the percentage of apoptotic HepG2 2.2.15 cells were detected by flow cytometric analysis; then, constructing 5 promoter plasmids and 5 truncate plasmid. These plasmids were transiently transfected into HepG2 cells, respectively. After 8 h of transfection, cells were treated with 40μg/ml A. Sinica Root for 48 h. The activity was determined by measuring the luciferase activities of the reporter in treated and untreated transfectants.Results:A. Sinica Root selectively inhibited the activity of p53-associated signaling pathway and the activities of HBV promoters (Cp, S1p and Fp) as well as truncates (t1, t4, t5) significantly(P< 0.05), and could induce apoptosis of HepG2 2.2.15 cells.Conclusions:A. Sinica Root exerts anti-HBV effects via inhibition of HBV transcription and the p53-associated signaling pathway, possibly by inhibiting the p53 pathway to induced apoptosis. Part 3 A optimum technology of extracting bioactive components from Ampelopsis Sinica RootObjective:In order to promote the advanced development of Ampelopsis Sinica Root and provide a scientific evidence for its clinical progress, giving a summary and comparison about the medicine plant extract on a variety of technology of extracting.Methods:Based on water boiling method, reflux method, percolation method, etc, the Ampelopsis Sinica Root was extracted to detect the dry extract and the content of gallic acid, a small-molecule and water-soluble component from extract.Results:In this experiment, the recommended 5# process were used to extract the medicine plant, as a result, the impurities from extract reduced 70%, but the content of gallic acid had no significant difference.Conclusions:The purity of active substance in extract solution or dry extract from Ampelopsis Sinica Root extracted by 5# process was greatly increased. And the extract was made of crud drug with concentration 0.8g/ml, as well as an increased clarity. It could be directly prepared for solution agent. In particular, the extract solution didn’t have an element of stimulating mucus, so its cytotoxicity was significantly reduced.更多还原