【作者】 杨德刚；

【导师】 缪晓辉；

【作者基本信息】 第二军医大学 ， 内科学， 2008， 博士

【摘要】 [研究背景]共价闭合环状DNA（cccDNA）是乙型肝炎病毒（HBV）在细胞内复制过程的初始模板,也是病毒在宿主细胞核内形成的第一个复制中间体,它的形成标志着病毒在宿主细胞内感染状态的建立和复制循环的开始。HBV cccDNA在肝细胞核内形成含量相对稳定的池,目前绝大多数临床上应用的抗HBV药物均难以清除细胞内的cccDNA,被认为是抗病毒治疗后乙型肝炎复发的重要原因。HBV cccDNA的检测对抗HBV药物的研发和应用评价,以及慢性乙型肝炎（CHB）患者的病情监测和抗病毒疗效评估等具有十分重要的临床意义。就现有抗HBV药物的疗效看来,尚无能够迅速抑制或是清除被感染肝细胞内HBV cccDNA的治疗药物或方案,针对HBVcccDNA的药物研发和疗效监测工作尚未取得明显进展。本研究以我们实验室前期建立的一套特异、灵敏的HBV cccDNA检测技术为基础,在HepG2.2.15细胞平台上对胡黄连苷促进清除HBV cccDNA的作用效果和特点进行了对比评估,并试图对相关机制作出初步探讨。本研究共分为三个部分:（一）胡黄连苷促进清除HepG2.2.15细胞内HBVcccDNA的作用效果和特点研究。以阿德福韦酯为对照,分别观察胡黄连苷和阿德福韦酯作用后HepG2.2.15细胞培养上清中HBV DNA含量变化,定量检测细胞内HBVDNA、cccDNA和pgRNA水平并计算相应抑制率,比较胡黄连苷与阿德福韦酯作用的不同效果和特点。（二）胡黄连苷对HepG2.2.15细胞内HBV cccDNA超螺旋分布的影响。采用专门的二维凝胶电泳方法观察胡黄连苷作用前后HepG2.2.15细胞内HBV cccDNA超螺旋数量分布有无变化,并与阿德福韦酯作用结果相对比,利用非参数秩和检验方法进行统计分析,探寻胡黄连苷对HBV cccDNA超螺旋数量分布的影响。（三）胡黄连苷对HepG2.2.15细胞内OAS1、STAT2、ISGF3γ、MyD88、MxA等候选基因转录水平的影响。对于胡黄连苷作用后有明显差异转录的基因再以siRNA技术选择性沉默阻断,以反向验证相关基因参与胡黄连苷抗HBV作用的可能性。第一部分胡黄连苷对HepG2.2.15细胞内HBV cccDNA含量的影响目的观察胡黄连苷抑制HepG 2.2.15细胞内HBV cccDNA的作用效果及特点。方法分别用含胡黄连苷50μg/ml、胡黄连苷100μg/ml或阿德福韦酯5μg/ml的细胞培养液作用于HepG2.2.15细胞,加药后2d、5d收集细胞及培养上清液,用实时荧光定量PCR方法检测上清和细胞内HBV DNA、细胞内HBV cccDNA和pgRNA水平,分别计算抑制率。结果①胡黄连苷50μg/ml作用后2d和5d,上清HBV DNA抑制率分别为49.74%和79.48%;细胞内HBV cccDNA抑制率43.55%和56.43%,rcDNA抑制率43.39%和63.86%,pgRNA抑制率54.72%和56.08%。②胡黄连苷100μg/ml作用后2d和5d,上清HBV DNA抑制率分别为51.11%和82.07%;细胞内HBVcccDNA抑制率41.13%和57.59%,rcDNA抑制率45.09%和67.31%,pgRNA抑制率52.68%和52.47%。③阿德福韦酯作用后2d和5d,上清HBV DNA抑制率分别为25.56%和92.44%;细胞内HBV cccDNA抑制率18.54%和47.19%,rcDNA抑制率21.20%和71.47%,pgRNA抑制率11.14%和37.61%。结论胡黄连苷能够明显抑制HepG2.2.15细胞内HBV复制水平,对HBV cccDNA也具有抑制作用,而且在作用时相上早于阿德福韦酯,可能存在不同的作用机制。第二部分胡黄连苷对HepG2.2.15细胞内HBV cccDNA超螺旋分布的影响目的观察HepG2.2.15细胞内HBV cccDNA超螺旋数量分布规律及胡黄连苷、阿德福韦酯对其影响。方法实验共分3组,分别用含胡黄连苷50μg/ml、阿德福韦酯5μg/ml和空白细胞培养液作用于HepG2.2.15细胞,加药后2d、5d收集细胞,提取细胞内HBV cccDNA,经二维凝胶电泳分离含有不同超螺旋数量的cccDNA,转至硝酸纤维素膜与³²P-dCTP标记的HBV DNA探针杂交、放射显影。凝胶成像系统扫描A（0～5个超螺旋）、B（6～10个超螺旋）、C（11～15个超螺旋）、D（16～20个超螺旋）区域条带灰度值,计算各区域灰度积分值所占构成比,用非参数秩和检验方法分析组间差异。结果①用药2d时胡黄连苷组各区构成比中位值为A 14.94%、B 33.32%、C 35.96%、D 14.05%,阿德福韦酯组各区构成比中位值为A 13.00%、B20.04%、C 25.55%、D 39.04%,空白对照组各区构成比中位值为A 12.36%、B21.04%、C 23.22%、D 40.77%。其中胡黄连苷组分别与空白对照组和阿德福韦酯组cccDNA超螺旋分布有统计学差异(Nemenyi检验,x_1,3²=12.86,x_1,2²=8.28,P＜0.01),而阿德福韦酯组与空白对照组cccDNA超螺旋分布差异无统计学意义(x_2,3²=2.58,P＞0.25)。②用药5d时胡黄连苷组各区构成比中位值为A 15.40%、B 33.48%、C 34.47%、D 15.07%,阿德福韦酯组各区构成比中位值为A 13.30%、B 20.52%、C 23.22%、D 42.12%,空白对照组各区构成比中位值为A 13.02%、B 21.24%、C 22.91%、D40.61%。其中胡黄连苷组分别空白对照组和阿德福韦酯组cccDNA超螺旋分布有统计学差异(Nemenyi检验,x_1,3²=11.61,x_1,2²=9.58,P＜0.01),阿德福韦酯组与空白对照组cccDNA超螺旋分布差异无统计学意义(x_2,3²=0.86,P＞0.75)。③空白对照组HepG2.2.15细胞内HBV cccDNA分子所含超螺旋数量分布并不均匀,以D区所占比例最多（约40%）,B区和C区次之（各约20%）,A区最少,2d和5d间数据分布差异没有显著性（Mann-Whitney U检验,U=0.321,P=0.748）。结论HepG2.2.15细胞内HBV cccDNA超螺旋数量呈现相对固定的异质性分布状态,阿德福韦酯处理2d和5d均未改变分布规律;胡黄连苷能够减少HepG2.2.15细胞内HBV cccDNA内部超螺旋数量,可能导致cccDNA结构稳定性的减低,促进HBV cccDNA的清除。第三部分胡黄连苷对HepG2.2.15细胞内部分基因转录水平的影响目的观察胡黄连苷能否影响HepG2.2.15细胞内部分候选基因的转录,探讨其与抗HBV效应的关系。方法①选取既往报道的能够促进感染细胞内HBV pgRNA降解的部分基因如2’,5’-寡腺苷酸合成酶1（OAS1）、信号转导与转录活化因子2（STAT2）、干扰素刺激基因因子3γ（ISGF3γ）、髓样细胞分化蛋白（MyD88）和MxA蛋白作为候选基因,实时荧光定量RT-PCR方法检测胡黄连苷作用后48h HepG2.2.15细胞内OAS1、STAT2、ISGF3γ、MyD88和MxA基因转录产物mRNA水平变化,以β-actin基因mRNA作为检测内参照。②用MyD88 siRNA诱导沉默HepG2.2.15细胞内MyD88基因转录,实时荧光定量PCR方法检测胡黄连苷作用下HepG2.2.15细胞内MyD88 mRNA、HBV pgRNA及培养上清中HBV DNA水平变化。③用MyD88siRNA诱导沉默HepG2.2.15细胞内MyD88基因转录,实时荧光定量PCR方法检测胡黄连苷作用下HepG2.2.15细胞内MyD88 mRNA、HBV cccDNA水平变化,二维电泳方法观察cccDNA超螺旋分布情况并作组间比较。结果①胡黄连苷作用后48hHepG2.2.15细胞内各候选基因转录水平与对照组的比值分别为1.17（OAS1）、0.87（STAT2）、0.93（ISGF3γ）、5.14（MyD88）和0.83（MxA）,其中MyD88基因转录水平显著升高（P＜0.01）,其余各候选基因转录水平变化无显著性差异（P＞0.05）。②MyD88 siRNA能够有效沉默胡黄连苷诱导的MyD88转录（沉默效率86.35%）,同步检测胡黄连苷对培养上清中HBV DNA和细胞内pgRNA抑制率下降为5.02%、6.67%,而空转染组胡黄连苷对培养上清中HBV DNA和细胞内pgRNA抑制率为49.82%、51.36%,提示MyD88转录沉默后胡黄连苷抗HBV活性的部分阻断。③MyD88siRNA有效沉默MyD88基因转录后（沉默效率85.79%）,胡黄连苷作用48h对HepG2.2.15细胞内HBV cccDNA抑制率为46.24%,空转染组抑制率为52.73%;MyD88 siRNA组cccDNA超螺旋数量分布为A 17.08%、B 33.82%、C 34.20%、D14.89%,空白对照组为A 13.82%、B 20.44%、C 23.14%、D 42.60%,MyD88 siRNA组与与空白对照组cccDNA超螺旋分布趋势仍有显著差别(Nemenyi检验,x_1,3²=24.74,P＜0.01)。结论胡黄连苷作用可以诱导HepG2.2.15细胞内MyD88基因活跃转录,并通过MyD88途径促进HBV pgRNA的降解。但是MyD88并未参与胡黄连苷早期抑制HBV cccDNA的过程,胡黄连苷体外抑制HBV cccDNA、减少HepG2.2.15细胞内cccDNA超螺旋数量的作用可能依赖其它宿主基因的参与。更多还原

【Abstract】 Covalently closed circular DNA（cccDNA）,serving as the original template of hepatitis B virus（HBV） replication,is the first replicative intermediate emerging during the life cycle of hepadnavirus,which indicates establishment of viral infection and origination of the replication cycle.A stable pool of cccDNA,has been found in each nucleus of persistently-infected hepatocyte,and is thought to be one of the main factors responsible for reoccurrence of chronic hepatitis B after withdrawal of antiviral agents. Therefore,determination of HBV cccDNA does not only play an vital role in the development and evaluation of aritiviral agents,but also may provide a useful guide to the efficacy of antiviral treatment and the likelihood of long-term response in patients with hepatitis B,and may aid in the decision of when to cease therapy.However,poor efficacy has been achieved aiming at clearance or inhibition of HBV cccDNA with current anti-HBV therapies,and few visible progresses were made in drug development and efficacy monitoring work against HBV cccDNA.In this research,we aimed to confirm the effectiveness and characteristics of picrosides inhibiting cccDNA in HepG2.2.15 cells,based on the specific and sensitive strategy developed earlier by our laboratory to determine HBV cccDNA.This research was composed of three parts:Ⅰ.Comparative study on the effectiveness and characteristics in inhibiting HBV cccDNA in HepG2.2.15 cells: picrosides versus adefovir dipivoxil.Ⅱ.Effect of picrosides on the supercoil configuration of HBV cccDNA molecules in HepG2.2.15 cells.Ⅲ.Effect of picrosides on the transcription of OAS1,STAT2,ISGF3γ,MyD88 and MxA genes in HepG2.2.15 cellsPartⅠInhibitory Effect of Pierosides on Hepatitis B Virus Covalently Closed Circular DNA in HepG2.2.15 CellsObjectives To observe the effect of picrosides on HBV cccDNA in HepG2.2.15 cells.Materials and Methods HepG2.2.15 cells were separately incubated with culture medium containing 50μg/ml picrosides,100μg/ml picrosides or 5μg/ml adefovir dipivoxil for 2 and 5 days.HBV DNA in supernatant,intracellular cccDNA, intracellular relaxed circular DNA（rcDNA） and pregenomic RNA（pgRNA） were quantified by specific real-time PCR or RT-PCR.Results①Treatment with 50μg/ml picrosides for 2 and 5 days could reduce the production of HBV in the cell line,as shown by the decline of HBV DNA in the supernatant（49.74%and 79.48%）. Intracellular synthesis of cccDNA had also been markedly lowered by 43.55%and 56.43%,and intracellular rcDNA decreased by 43.39%and 63.86%,as well as intracellular pgRNA decreased by 54.72%and 56.08%.②Treatment with 100μg/ml picrosides for 2 and 5 days resulted in decline of HBV DNA（51.11%and 82.07%） in the supernatant,as well as intracellular cccDNA（41.13%and 57.59%）,rcDNA（45.09% and 67.31%） and pgRNA（52.68%and 52.47%）.③Comparative treatment with adeforvir dipivoxil for 2 and 5 days resulted in decline of HBV DNA（25.56%and 92.44%） in the supernatant,cccDNA（18.54%and 47.19%）,rcDNA（21.20%and 71.47%） and pgRNA（11.14%and 37.61%） in HepG2.2.15 cells.Conclusions Our research indicated that picrosides can interfere with the replication cycle of HBV, including the formation of cccDNA in HepG2.2.15 cells.The mechanism of picrosides on cccDNA may differ from adefovir dipivoxil’s in its earlier inhibition phase.PartⅡImpact of Picrosides on the Distribution of Supercoils inside Hepatitis B Virus cccDNA in HepG2.2.15 CellsObjectives To observe the distribution of superhelixes inside HBV cccDNA molecules in HepG2.2.15 cells and the effect of picrosides or adefovir dipivoxil on such distribution.Materials and Methods HepG2.2.15 cells were divided into 3 groups: Cells of normal control group were incubated with fresh complete culture medium, while the other two groups were incubated with culture medium containing 50μg/ml picrosides or 5μg/ml adefovir dipivoxil respectively for 2 and 5 days.HBV cccDNA molecules were extracted from HepG2.2.15 cells by methods based on Hirt-fractionation and then divided into different groups according to the distinct number of innate superhelixes inside each cccDNA molecule through special two-dimensional gel electrophoresis process.Heterogeneous groups of cccDNA with different superhelix number were detected with ³²P-dCTP labeled cccDNA probe after transfer from gels to nitrocellulose blotting membranes by the Southern procedure. Integral gray values of different groups,named as A（containing 0-5 supercoils）,B （containing 6-10 supercoils）,C（containing 11-15 supercoils） and D（containing 16-20 supercoils） were converted into percentage value and then analyzed with nonparametric test.Results①The median percentage values of A to D distribution group in picrosides group on day 2 were 14.94%,33.32%,35.96%and 14.05%,respectively; while in adefovir dipivoxil group they were 13.00%,20.04%,25.55%and 39.04%and 12.36%,21.04%,23.22%and 40.77%in normal control group.The distribution of HBV cccDNA superhelixes in picrosides group were significantly different from that in adefovir dipivoxil group(Nemenyi Test,x_1,2²=8.28,P＜0.01) and normal control group(x_1,3²=12.86,P＜0.01),while no significant difference was found between adefovir dipivoxil group and normal control group(x_2,3²=2.58,P＞0.25).②The median percentage values of A to D in picrosides group on day 5 were 15.40%,33.48%, 34.47%and 15.07%,respectivel;while in adefovir dipivoxil group they were 13.30%, 20.52%,23.22%and 42.12%and 13.02%,21.24%,22.91%and 40.61%in normal control group.The distribution of HBV cccDNA superhelixes in picrosides group were significantly different from those of adefovir dipivoxil group(Nemenyi Test,x_1,2²=9.58, P＜0.01) and normal control group(x_1,3²=11.61,P＜0.01),but no significant difference was found between adefovir dipivoxil group and normal control group(x_2,3²=0.86, P＞0.75).③The distribution of superhelixes inside cccDNA molecules in HepG2.2.15 cells was found as a heterogeneous population,with group D showing the maximum percentage（about 40%）,group B and C a less percentage（but both above 20%） and group A the least（less than 15%）.No significant difference was found between data on day 2 and day 5（Mann-Whitney U test,U=0.321,P=0.748）.Conclusions The distribution of superhelixes numbers inside cccDNA molecules in HepG2.2.15 cells exists as a heterogeneous population,treatment with adefovir dipivoxil for 2 days and 5 days imposed no significant effect on the distribution pattern.Treatment with picrosides reduced the number of innate supercoils inside HBV cccDNA in HepG2.2.15 cells. Such alteration may cause subsequent reduction of cccDNA molecular stability which facilitates the clearance of cccDNA in nucleus.PartⅢEffect of picrosides on the transcription of OAS1,STAT2,ISGF3γ, MyD88 and MxA genes in HepG2.2.15 cellsObjectives To observe the effect of picrosides on the transcription of some host genes and its relationship with picrosides’ anti-HBV activity in HepG2.2.15 Cells. Materials and Methods①A group of host genes including 2’,5’-oligoadenylate synthetase 1（OAS1）,interferon-stimulated gene factor 3-γ（ISGF3γ）,signal transducer and activator of transcription-2（STAT-2）,myeloid differentiation primary response gene 88（MYD88） and myxovirus resistance protein A（MxA） were selected as candidate genes according to their reported activity of decreasing pgRNA.Their mRNA levels with or without picrosides treatment were quantitated by realtime reverse transcriptase PCR（RT-PCR） withβ-actin mRNA as internal reference.②HepG2.2.15 cells were treated with picrosides,and effect of MyD88 gene being silenced by siRNA or not were assessed by quantifying intracellular HBV pgRNA and MyD88 mRNA level as well as total HBV DNA in supernatant by realtime PCR.③MyD88 gene being silenced by siRNA or not,intracellular HBV cccDNA and MyD88 mRNA level were quantitated by realtime PCR,while the distribution of superhelixes inside HBV cccDNA was evaluated by two-dimensional gel electrophoresis.Results①Ratio of those candidate gene mRNA levels to normal control were 1.17（OAS1）,0.87（STAT2）,0.93（ISGF3γ）, 5.14（MyD88） and 0.83（MxA）.The transcription of MyD88 gene was significantly activated by picrosides’ treatment（P＜0.01）,while no significant difference were observed in the transcription of other candidate genes between groups with or without picrosides treatement（P＞0.05）.②MyD88 siRNA can effectively silence MyD88 gene transcription with a ratio up to 86.35%,while the inhibition rate of HBV DNA in supernatant and intracellular pgRNA by picrosides declined to 5.02%and 6.67% respectively on day 2,indicating the partial block of picrosides’ anti-HBV activity by MyD88 siRNA.③The inhibition rate of intracellular cccDNA by 2 days’ picrosides treatment in HepG2.2.15 cells remained as 46.24%in despite of silenced MyD88 gene transcription（with a ratio up to 85.79%）,and the inhibition rate of intracellular cccDNA in mock-transfection HepG2.2.15 cells was 52.73%.The A,B,C and D distribution pattern of cccDNA superhelixes in MyD88 siRNA group were 17.08%,33.82%, 34.20%and 14.89%while in normal control group they were 13.82%,20.44%,23.14% and 42.60%,respectively.The distribution of HBV cccDNA superhelixes in MyD88 siRNA group were also significantly different from that of normal control group (Nemenyi Test,x_1,3²=24.74,P＜0.01).Conclusions Treatment with picrosides can activate transcription of MyD88 gene and may subsequently induce the degradation of pgRNA through MyD88 pathway,but MyD88 gene might not participate in the rapid, early inhibition of HBV cccDNA by picrosides.The inhibition effect of picrosides on HBV cccDNA in vitro and the reduction of superhelix number of cccDNA might rely on further activation of other host genes.更多还原