【作者】 何燕；

【导师】 万新祥；

【作者基本信息】 南方医科大学 ， 药理学， 2010， 硕士

【摘要】 海鞘(Ascidian)是属于脊索动物门(Chordates),尾索动物亚门(Uronordata),海鞘纲(Ascidiancea),在自然界中已知有2000余种,主要分布在热带及亚热带海域。近年来对海鞘的化学成分及其生理活性的研究是海洋天然产物研究的热门领域,研究已发现海鞘类生物中含有生物碱类、肽类、吲哚类、重金属螯合剂、多硫化物、大环内酯、萜类等数十种化合物,且大部分都具有较强的生理活性,如抗肿瘤、抗病毒、抗菌、诱导肌浆网释钙、抑制钙调蛋白活性等,其中有关抗肿瘤及抗病毒的研究报道较多。国内相关的研究与报道较少,本实验室是国内唯一开展海鞘抗乙肝病毒作用相关研究的实验机构。我国海域广阔,尤其是南海有着丰富的海鞘资源,皱瘤海鞘[Styela plicata(lesueur)]便是其中之一。本课题组利用地理优势之便在海洋药物的研究方面针对海鞘的抗病毒作用进行了初步研究,发现皱瘤海鞘的醇提物在体内外均提示有抗乙肝病毒药理作用。现在已经成功分离出两个活性单体,为多肽成分,并且分离出其生物总碱和其正丁醇部位,建立了其有效部位群的指纹图谱。为了进一步评估皱瘤海鞘抗乙型肝炎病毒(Hepatitis B virus,HBV)的作用机理,本课题对皱瘤海鞘如何抑制乙肝病毒DNA的复制,以及其究竟作用于病毒复制的哪一个环节进行深入的研究。第一节皱瘤海鞘对乙型肝炎病毒DNA拷贝的影响目的：本课题以体外细胞学研究为依据,通过皱瘤海鞘醇提取物对体外HepG22.215细胞中HBsAg和HBeAg,及细胞毒性的影响来研究皱瘤海鞘的体外药效学,且进一步定量检测细胞培养基中的HBV DNA拷贝量来研究皱瘤海鞘对乙肝病毒DNA的抑制作用；从而揭示皱瘤海鞘醇提取物体外抑制乙肝病毒复制的作用机制方法：1取HepG2 2.2.15细胞培养后,加入不同浓度的皱瘤海鞘醇提取物和拉米夫定药物,在37℃,5%CO2培养5天,收集上清,用ELISA法及MTT法检测乙肝病毒表面抗原(HBsAg)和e抗原(HBeAg)的OD值及细胞增殖能力。2取HepG2 2.2.15细胞培养后,加入不同浓度的皱瘤海鞘醇提取物和拉米夫定药物,在37℃,5%CO2培养,收集48、96h上清,用荧光定量聚合酶链反应(PCR)检测HBV DNA拷贝量。结果：1皱瘤海鞘各浓度组与细胞组之间比较结果有显著的统计学差异(P=0.000,P<0.05),皱瘤海鞘醇提取物的浓度>0.5mg·ml-1时,对乙型肝炎表面抗原(HBsAg)和乙肝e抗原(HBeAg)都有良好的抑制作用,而且随着药物浓度的增大其抑制效果越明显。2药物作用细胞后上清中HBV DNA拷贝量的定量检测,结果显示在48h、96h两个时间点时不同浓度皱瘤海鞘提取物对细胞中的HBV DNA表达有抑制作用,且随着浓度的增加而增强(P<0.001)。对HBV DNA分泌的抑制亦随着浓度的增加而增强以2 mg·ml-1时最强。在48、96h时分别达到44.13%、46.40%。不同浓度拉米夫定作用后细胞中HBV DNA的表达有抑制作用,且随着浓度的增加而增强。而其对HBV DNA的分泌的抑制也随着浓度的增加而增强,拉米夫定浓度在1 mg·ml-1时,其48、96h分别达到了17.41%、18.37%。二者在抑制HBV DNA分泌的作用上有相似的效果。第二节皱瘤海鞘对乙型肝炎病毒DNA聚合酶活性的影响目的：本部分通过对乙肝病毒DNA聚合酶活性的研究进一步探索皱瘤海鞘提取物抑制乙肝病毒复制的作用机制,进而明确皱瘤海鞘醇提取物体外抑制乙肝病毒复制的作用机制。方法：通过对HepG2.215细胞中的HBV DNA颗粒提纯后,在最佳反应条件下进行聚合酶链反应(PCR),在底物中加入具有放射性的32P-dCTP,并在不同浓度皱瘤海鞘醇提取物和拉米夫定药物的作用下反应,最后检测HBV DNA中掺入的32P-dCTP放射量,由于HBV DNA双链是通过DNA聚合酶进行复制或延伸,如果聚合酶活性受到影响则掺入双链中的32P-dCTP的放射量就会减少,从而间接得到药物对HBV DNA聚合酶活性的影响。结果：当皱瘤海鞘醇提取物浓度达到1 mg·ml-1以上时,与空白组比较具有显著性差异(P<0.05)。拉米夫定在浓度达到1 mg·ml-1时,与空白组比较也有统计学意义(P<0.05),由此可知皱瘤海鞘醇提取物和拉米夫定对HBV DNA聚合酶活性都有影响。且二者有相似的影响作用。本次实验证实了皱瘤海鞘醇提取物是通过影响乙肝病毒DNA聚合酶的活性从而抑制了HBV DNA的分泌,其在体外细胞试验中显示了明显的抑制乙肝病毒作用。本次实验在前人研究的基础上进一步明确了皱瘤海鞘抗乙肝病毒的作用机制,而实验的结果提示我们皱瘤海鞘将可能会是一种新型有效的治疗慢性乙肝药物。本课题实验为开发这种新型有效的抗病毒海洋药物提供了实验基础和理论依据。更多还原

【Abstract】 Styela plicata is an ascidian which belongs to class Ascidiacea, subphylum urochordata, phylum Chordates, there are about 2000 kinds of ascidians have been found in the nature and they distribute in tropical and subtropical seas. Recent years, studying on the chemical composition and biological activity of ascidian has been one of the most popular domains in marine drug researches. Many chemical compositions such as peptid, alkaloide, macrolide, terpenoid, and so on, have been found in ascidian. Most of them have strong biological activity, such as anti-tumor, anti-virus, antibiosisi, immumity modulation and biocatalysisi, etc. And mechanisms on anti-tumor and anti-virus were better studed. But studies and reports were less seen in civil,our experiment institution was the only department which developed the research of Ascidiacea anti Hepatitis B virus in civil.In the vast ocean, specially in South China sea, there are abundant Styela plicata ascidian resources. On study of marine drugs, our research team made the basic research about the anti-virus effect of ascidian, the results showed the alcohol extract of Styela plicata has a definite inhibitory effect on hepatitis B virus in vivo and vitro. Now we had separated two activated monomers which are polypeptide, and we also separated its biochronometer and its n-butanol extraction position, we set up a finger print of Styela plicata’s validation section. This study aimed to explore the anti-HBV mechanisms of Styela plicata further.Section 1 The inhibition effects of Styela plicata on Hepatitis B virus DNAObjectiveThis study was carried out according to cytology in vitro, we identified the Styela plicata’s pharmacodynamic through inhiting HBsAg and HBeAg in HepG 2.215 cells,then tested HBV DNA copies in supernatant of cell culture by PCR to research the impacts and inhibition of HBV DNA.We analyzed the results and revealed the mechanisms of Styela plicata(lesueur) on anti -Hepatitis B virus in vitro.Methods1 HepG2 2.2.15 cells were incubated at 37℃5% CO2 5 days with different concentrations of Styela plicata and Lamivudine. By using ELISA to test HBsAg and HBeAg in culture supernatants, and MTT colorimetry to assay cell reproductive activity.2 HepG2 2.2.15 cells were incubated at 37℃5% CO2 with different concentrations of Styela plicata and Lamivudine, collecting culture supernanant of 48h and 96h.Using PCR to test the copies of HBV DNA.Results1 Differents concentrations of Styela plicata compared with cell group,the results had significant statistical difference(P=0.000,P<0.05).When Styela plicata’s concentrations was higher than 0.5 mg·ml-1,it had good inhibition effects on both HBsAg and HBeAg.And its inhibition effects were influenced by the concentrations of Styela plicata.The concentrations were higher,the inhibition effects were stronger.2 The quantitative test results of HBV DNA copies from the drug-treated supernatant shows that different concentrations of Styela plicata extract inhibit HBV DNA expression in cells. And the inhibition effect increased with concentration (P <0.001).The inhibition effect of the HBV DNA secretion increased with concentration. As the highest concentration(2 mg·ml-1), and it inhibit 44.13%,46.40% of HBV DNA secretion at 48、96 hours respectively. The different concentrations of lamivudine also inhibit HBV DNA replication in cells. And The inhibition increased with concentration also. The inhibition effects increase with concentrations. When the concentration get to 1 mg·ml-1, it inhibit 17.41%,18.37% of HBV DNA secretion at 48、96 hours respectively. Both Styela plicata and lamivudine had similar inhibition effects on HBV DNA secretion. Section 2 The influence of Styela plicata on Hepatitis B virus DNA pclymeraseObjectiveThis part researched the activity of HBV DNA pclymerase and explored the mechanisms of Hepatitis B virus replication inhibition by Styela plicata. Then identified how the Styela plicata inhibited HBV DNA replication.MethodsPuring HBV DNA drops in HepG2.215 cell,PCR was beginning at the best action conditions and adding 32P-dCTP,which had radioactivity, in substrates, acting with different concentrations of Styela plicata and Lamivudine. At last,testing the radiation quantity of 32P-dCTPctp in HBV DNA,the double strand of HBV DNA were copied and extended by DNA pclymerase,if the activity of pclymerase were impacted,the radioactivity of 32P-dCTP in double strand would reduce,so we could know the impacts of DNA pclymerase activity which caused by Styela plicata and LamivudingResultsWhen the concentration of Styela plicata and Lamivudine were 1 mg·ml-1,their radiation quantity of 32P-dCTP compared with blank group, respectively(P< 0.05, >0.001).And when Lamivudine’s concentration was 1 mg·ml-1,its radiation quantity of 32P-dCTP compared with blank group had significant difference. They both had statistical significance. From the results ,we knew Styela plicata extract product and Lamivudine both had impacts to activity of HBV DNA pclymerase, and their impacts were similar.The results of this experiment identied that Styela plicata extract product inhibited the secretion of HBV DNA from impacting activity of HBV DNA pclymerase. And its inhibition effects of HBV DNA replication was as well as Lamivudine. Our experiments based on predecessor’ researchs but we had improvements that we identified the mechamisms of Styela plicata anti HBV,and the results of this task remind that Styela plicata might be an effective anticiral medicine in treating chronic hepatitis B virus .All of our experiments provided tational theories to develop this new and efficacious anti-HBV marine drug.更多还原