B和C基因型重组乙型肝炎病毒的构建及其相关研究
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摘要
研究目的:
1.构建B和C基因型重组乙型肝炎病毒及检测其在Huh7细胞内的复制和表达。
2.探讨B和C基因型乙型肝炎病毒对Huh7细胞全基因组表达模式的影响是否相似。
3.评价针对重组B和C基因型HBV S区目的小发夹RNAs(shRNAs)在存在错配的情况下对HBsAg和HBeAg表达的抑制作用,对HBsAg mRNA水平的抑制作用,及对HBV DNA复制的抑制程度。
研究方法:
1.根据相关文献设计扩增HBV全基因组的引物,分别从感染B和C基因型的患者血清中提取HBV DNA作为模板,应用高保真热启动Taq DNA聚合酶扩增HBV全基因组;
2.SacⅠ酶切扩增的全长HBV和pUC19,应用T4 DNA连接酶使HBV插入pUC19上的SacⅠ酶切位点,命名为pUC19-BHBV和pUC19-CHBV;
3.应用SapⅠ酶切pUC19-HBV和pHY106真核表达载体,将获得的全长HBV在与线性的pHY106连接,使全长HBV插入pHY106的SapⅠ酶切位点,命名为pHY106-BHBV和pHY106-CHBV;
4.将pHY106-BHBV和pHY106-CHBV分别转染Huh7细胞,以pHY106空载体转染作对照;①分别于转染后24h、48h和72h取细胞培养上清,-20℃冻存,用于ELISA检测HBsAg和HBeAg表达;②转染后72h,裂解细胞,提取细胞内HBV核心颗粒内HBV复制中间体,用于Southern blot检测;③转染后72h,用PBS洗细胞5次,裂解细胞,应用Real-time PCR定量检测Huh7细胞内HBV DNA水平。
5.基因芯片技术:B或C基因型重组乙型肝炎病毒(即pHY106-BHBV,pHY106-CHBV)转染Huh7细胞,以pHY106空载体为对照,48h后提取细胞mRNA,逆转录为cDNA,与芯片杂交,用GenePix Pro3.0软件分析Cy3和Cy5两种荧光信号的强度,计算每个基因点在本次实验中的表达差异值Ratio,Ratio=Cy5/Cy3;筛选出Ratio大于2或小于0.5的基因点,这些数据所代表的基因在与两种探针杂交时表现出较大的差异;
6.实时定量PCR:pHY106-BHBV,pHY106-CHBV转染Huh7细胞48h,以pHY106空载体为对照,提取细胞总RNA,逆转录为cDNA,应用实时定量PCR对基因芯片差异表达的部分基因进行验证。
7.将shRNAs分别和B或C基因型重组HBV,即pHY106-BHBV和pHY106-CHBV共转染HepG2细胞;于转染后24h、48h、72h、96h和120h分别取细胞培养上清,-20℃冻存,留作ELISA检测HBsAg和HBeAg表达水平;
8.shRNAs分别和B或C基因型重组HBV转染HepG2细胞72h后,应用Trizol法提总RNA,逆转录为cDNA,进行RT-PCR,对HBsAg mRNA水平进行半定量检测;于转染后72h,裂解细胞,提取HBV核心颗粒内的HBV复制中间体进行Southernblot实验。
结果:
1.成功构建了pHY106-BHBV和pHY106-CHBV两个表达载体;
2.pHY106-BHBV和pHY106-CHBV转到Huh7细胞后,检测到了HBsAg和HBeAg的表达,HBsAg表达于48h达高峰,HBeAg的表达高峰晚于HBsAg约24h;
3.应用Southem blot检测到了细胞内HBV核心颗粒内的HBV复制中间体,包括rcDNA,dsDNA和ssDNA;
4.应用Real-time PCR定量检测发现转染到Huh7细胞内的pHY106-BHBV和pHY106-CHBV HBV DNA拷贝数高,于72h达高峰,可达8log_(10);
5.pHY106-BHBV转染Huh7细胞48h后,从4097个基因中筛选出差异表达基因共60条,其中1条基因表达明显增强,59条基因表达降低明显;
6.pHY106-CHBV转染Huh7细胞48h后,筛选出差异表达基因共122条,其中34条基因表达明显增强,88条基因表达降低明显;
7.实时定量PCR验证上述芯片结果,证实pHY106-BHBV转染Huh7细胞48h后,IFNGR2、GPR125、DDEF2和PI3K mRNA表达水平均不同程度下调,相对表达率分别为0.8、0.5、0.3和0.4,与基因芯片结果基本一致;pHY106-CHBV转染Huh7细胞48h后,EEF2、INF592表达下调,相对表达率分别为0.8和0.6,与基因芯片结果符合。
8.shRNA·458和shRNA·635对B和C基因型HBV HBsAg和HBeAg表达具有很强抑制作用,转染后48h显现明显的抑制作用,于72h抑制作用达高峰,对HBsAg和HBeAg表达抑制水平分别可达到80%和50%左右;
9.shRNA共转染72h,shRNA·458和shRNA·635对B和C基因型HBV HBsAgmRNA具有明显的抑制作用,抑制水平在60%～70%左右;
10.Southern blot结果发现shRNA·458和shRNA·635对B和C基因型HBV的复制具有明显的抑制作用。
结论:
1.本研究构建成的HBV真核重组表达载体pHY106-BHBV和pHY106-CHBV能够在Huh7细胞内复制和表达HBsAg和HBeAg,适合用于B和C基因型HBV的致病机制、病毒与机体的相互作用、新药开发等方面的研究工作。
2.B和C基因型乙型肝炎病毒均能对Huh7细胞表达谱产生明显的改变,B和C基因型乙型肝炎病毒对Huh7细胞表达谱的改变明显不同,仅发现DDEF2在两者均下调,这表明B和C基因型乙型肝炎病毒对机体的影响可能存在不同的方式。
3.shRNA·458和shRNA·635是良好的抑制B和C基因型HBV的有效工具;一定范围的错配仍然能够发挥shRNA的抑制HBV的作用。
Objective:
1. To construct recombinant full length B and C genotype hepatitis B virus, examining the ability of replication and expression of them in Huh7 cells.
2. Exploring whether the change of global gene expression pattern is similar or not in huh7 cells transfected with B or C genotype HBV.
3. To assess the function of shRNAs inhibiting HBsAg and HBeAg expression, HBsAg mRNA production, and HBV DNA replicative intermediate in B and C genotype HBV under the condition of mismatching between shRNAs and HBV.
Methods
1. Designing the primers for full length HBV genome, exstracting the HBV DNA from two patients infected with B and C genotype hepatits B virus respectively as template, amplifying the full length HBV with high fidelity hot-start Tag DNA polymerase;
2. full length HBV and pUC19 were cut with SacⅠ, then inserting HBV into the site of Sac I in pUC19 with T4 DNA ligase, named as pUC19-BHBV and pUC19-CHBV;
3. pUC19-HBV and pHY106 were cut by Sap I , then inserting HBV into the site of Sap I in pHY106 with T4 DNA ligase, named as pHY106-BHBV and pHY106-CHBV;
4. pHY106-BHBV and pHY106-CHBV were transfected into Huh7 cells, pHY106 as control;①Collected supernatant of culture at 24 h, 48 h, 72 h after transfection for examining HBsAg and HBeAg with an ELISA kit;②lysated the cells, exstracted the HBV replicative intermediate from HBV core for Southern blot at 72 h after transfection;③72 h after transfection, washed cells with PBS buffer for 5 times, then lysated the cells, examined the HBV DNA level by real-time PCR quantitatively;
5. cDNA microarray: Huh7 cells were transfected with recombinant B or C genotype HBV, namely pHY106-BHBV and pHY106-CHBV, pHY106 as control; 48 h after transfection, mRNA of cells were extracted, and reverse transcripted to cDNA, then hybrided with chip; The fluorescence signal intensity of Cy3 and Cy5 was analyzed with the GenePix Pro 3.0 software; the ratio of Cy5/Cy3 represent the differential value, the ratio above 2 or below 0.5 was regarded as significant data;
6. Real-time PCR: Huh7 cells were transfected with pHY106-BHBV or pHY106-CHBV, pHY106 as control; 48 h after transfection, the total RNA were extracted, and reverse transcripted to cDNA, validated the partial results of microarray with real-time PCR;
7. shRNA and pHY106-BHBV or pHY106-CHBV cotransfected HepG2 cells, the supernatant of the culture at 24 h, 48 h, 72 h, 96 h and 120 h after cotransfection was collected and frozen in -20℃for examining HBsAg and HBeAg level by an ELISA kit;
8. 72 h after shRNA and pHY106-BHBV or pHY106-CHBV cotransfed, the total RNA of cells was extracted and reverse-transcripted, then examining the level of HBsAg mRNA by RT-PCR (reverse transcript polymerase chain reaction); 72 h after cotransfection, cells were lysated, HBV DNA replicative intermediate from HBV core was extracted and detected by Southern blot.
Results:
1. Constructing the recombinant full length B and C genotype HBV successfully;
2. The expressiong of HBsAg and HBeAg were detectable after pHY106-BHBV and pHY106-CHBV transfected into Huh7 cells, the peak time was 48 h for HBsAg expression, the peak time for HBeAg expression is about 24 h late compared with that of HBsAg;
3. Southern blot detected the HBV replicative intermediate from HBV core, including rcDNA, dsDNA and ssDNA.
4. pHY106-BHBV and pHY106-CHBV could replicate after transfected into Huh7 cells, the HBV DNA leve get the peak at 72 h after transfection, the peak was about 8 log 10.
5. 48 h after pHY106-BHBV transfected Huh7 cells, 60 differentially expressed genes were screened from total 4097 genes, among of them, one was overexpressed, the other 59 genes were expressed at lower levels;
6. 122 differentially expressed genes were screened from total 4097 genes after pHY106-CHBV transfected Huh7 cells, among of them, 34 genes were expressed at higher levels, the other 88 genes were expressed at lower levels;
7. IFNGR2, GPR125, DDEF2 and PI3K which were down-regulatory genes after pHY106-BHBV transfected Huh7 cells with microarray analysis were validated by real-time PCR, the relative expression ratio of them were 0.8, 0.5, 0.3 and 0.4 respectively, which was consistent with the results of microarray; EEF2 and INF592 which were down-regulatory genes after pHY106-CHBV transfected Huh7 cells with microarray analysis were validated by real- time PCR, the relative expression ratio of them were 0.8 and 0.6, respectively, which was consistent with the results of microarray; only DDEF2 are found down-regulated in both microarrays;
8. HBsAg and HBeAg expression were inhibited obviously by shRNA-458 and shRNA·635, the inhibitory action was detectable at 48 h after cotransfection, the peak time was 72 h, the most inhibitory ratio was approximately 80% and 50% in HBsAg and HBeAg; there was no difference was observed about the the inhibitory action of shRNA-458 and shRNA-635 on B or C genotype HBV;
9. shRNA-458 and shRNA-635 had the similar inhibitory action on the production of HBsAg mRNA from B and C genotype HBV after 72 h of cotransfection, the inhibitory ratio was 60%-70%;
10. HBV DNA replicative intermediate from B and C genotype was inhibited obviously by the two shRNAs also.
Conclusions:
1. pHY106-BHBV and pHY106-CHBV can replicate and express HBsAg and HBeAg in Huh7 cells, which is suitable for studying the pathogenesis of B and C genotype HBV, the interaction between HBV and host, as well as exploiting new drugs againt HBV.
2. B and C genotype HBV all can change the expression pattern of Huh7 cells; but the change of expression pattern of Huh7 cells by B and C genotype HBV are obviously different, only DDEF2 are found down-regulated in both; which demonstrate B and C genotype HBV may differently influence on host. shRNA·458 and shRNA·635 have the powerfully inhibitory action on the expression
3. of HBsAg and HBeAg, the production HBsAg mRNA and HBV replicativeintermediate from B and C genotype HBV although 2-4 bases mismatch betweenshRNAs and HBV, so shRNA-458 and shRNA-635 are two useful tools for inhibitHBV.
1.构建B和C基因型重组乙型肝炎病毒及检测其在Huh7细胞内的复制和表达。
2.探讨B和C基因型乙型肝炎病毒对Huh7细胞全基因组表达模式的影响是否相似。
3.评价针对重组B和C基因型HBV S区目的小发夹RNAs(shRNAs)在存在错配的情况下对HBsAg和HBeAg表达的抑制作用,对HBsAg mRNA水平的抑制作用,及对HBV DNA复制的抑制程度。
研究方法:
1.根据相关文献设计扩增HBV全基因组的引物,分别从感染B和C基因型的患者血清中提取HBV DNA作为模板,应用高保真热启动Taq DNA聚合酶扩增HBV全基因组;
2.SacⅠ酶切扩增的全长HBV和pUC19,应用T4 DNA连接酶使HBV插入pUC19上的SacⅠ酶切位点,命名为pUC19-BHBV和pUC19-CHBV;
3.应用SapⅠ酶切pUC19-HBV和pHY106真核表达载体,将获得的全长HBV在与线性的pHY106连接,使全长HBV插入pHY106的SapⅠ酶切位点,命名为pHY106-BHBV和pHY106-CHBV;
4.将pHY106-BHBV和pHY106-CHBV分别转染Huh7细胞,以pHY106空载体转染作对照;①分别于转染后24h、48h和72h取细胞培养上清,-20℃冻存,用于ELISA检测HBsAg和HBeAg表达;②转染后72h,裂解细胞,提取细胞内HBV核心颗粒内HBV复制中间体,用于Southern blot检测;③转染后72h,用PBS洗细胞5次,裂解细胞,应用Real-time PCR定量检测Huh7细胞内HBV DNA水平。
5.基因芯片技术:B或C基因型重组乙型肝炎病毒(即pHY106-BHBV,pHY106-CHBV)转染Huh7细胞,以pHY106空载体为对照,48h后提取细胞mRNA,逆转录为cDNA,与芯片杂交,用GenePix Pro3.0软件分析Cy3和Cy5两种荧光信号的强度,计算每个基因点在本次实验中的表达差异值Ratio,Ratio=Cy5/Cy3;筛选出Ratio大于2或小于0.5的基因点,这些数据所代表的基因在与两种探针杂交时表现出较大的差异;
6.实时定量PCR:pHY106-BHBV,pHY106-CHBV转染Huh7细胞48h,以pHY106空载体为对照,提取细胞总RNA,逆转录为cDNA,应用实时定量PCR对基因芯片差异表达的部分基因进行验证。
7.将shRNAs分别和B或C基因型重组HBV,即pHY106-BHBV和pHY106-CHBV共转染HepG2细胞;于转染后24h、48h、72h、96h和120h分别取细胞培养上清,-20℃冻存,留作ELISA检测HBsAg和HBeAg表达水平;
8.shRNAs分别和B或C基因型重组HBV转染HepG2细胞72h后,应用Trizol法提总RNA,逆转录为cDNA,进行RT-PCR,对HBsAg mRNA水平进行半定量检测;于转染后72h,裂解细胞,提取HBV核心颗粒内的HBV复制中间体进行Southernblot实验。
结果:
1.成功构建了pHY106-BHBV和pHY106-CHBV两个表达载体;
2.pHY106-BHBV和pHY106-CHBV转到Huh7细胞后,检测到了HBsAg和HBeAg的表达,HBsAg表达于48h达高峰,HBeAg的表达高峰晚于HBsAg约24h;
3.应用Southem blot检测到了细胞内HBV核心颗粒内的HBV复制中间体,包括rcDNA,dsDNA和ssDNA;
4.应用Real-time PCR定量检测发现转染到Huh7细胞内的pHY106-BHBV和pHY106-CHBV HBV DNA拷贝数高,于72h达高峰,可达8log_(10);
5.pHY106-BHBV转染Huh7细胞48h后,从4097个基因中筛选出差异表达基因共60条,其中1条基因表达明显增强,59条基因表达降低明显;
6.pHY106-CHBV转染Huh7细胞48h后,筛选出差异表达基因共122条,其中34条基因表达明显增强,88条基因表达降低明显;
7.实时定量PCR验证上述芯片结果,证实pHY106-BHBV转染Huh7细胞48h后,IFNGR2、GPR125、DDEF2和PI3K mRNA表达水平均不同程度下调,相对表达率分别为0.8、0.5、0.3和0.4,与基因芯片结果基本一致;pHY106-CHBV转染Huh7细胞48h后,EEF2、INF592表达下调,相对表达率分别为0.8和0.6,与基因芯片结果符合。
8.shRNA·458和shRNA·635对B和C基因型HBV HBsAg和HBeAg表达具有很强抑制作用,转染后48h显现明显的抑制作用,于72h抑制作用达高峰,对HBsAg和HBeAg表达抑制水平分别可达到80%和50%左右;
9.shRNA共转染72h,shRNA·458和shRNA·635对B和C基因型HBV HBsAgmRNA具有明显的抑制作用,抑制水平在60%～70%左右;
10.Southern blot结果发现shRNA·458和shRNA·635对B和C基因型HBV的复制具有明显的抑制作用。
结论:
1.本研究构建成的HBV真核重组表达载体pHY106-BHBV和pHY106-CHBV能够在Huh7细胞内复制和表达HBsAg和HBeAg,适合用于B和C基因型HBV的致病机制、病毒与机体的相互作用、新药开发等方面的研究工作。
2.B和C基因型乙型肝炎病毒均能对Huh7细胞表达谱产生明显的改变,B和C基因型乙型肝炎病毒对Huh7细胞表达谱的改变明显不同,仅发现DDEF2在两者均下调,这表明B和C基因型乙型肝炎病毒对机体的影响可能存在不同的方式。
3.shRNA·458和shRNA·635是良好的抑制B和C基因型HBV的有效工具;一定范围的错配仍然能够发挥shRNA的抑制HBV的作用。
Objective:
1. To construct recombinant full length B and C genotype hepatitis B virus, examining the ability of replication and expression of them in Huh7 cells.
2. Exploring whether the change of global gene expression pattern is similar or not in huh7 cells transfected with B or C genotype HBV.
3. To assess the function of shRNAs inhibiting HBsAg and HBeAg expression, HBsAg mRNA production, and HBV DNA replicative intermediate in B and C genotype HBV under the condition of mismatching between shRNAs and HBV.
Methods
1. Designing the primers for full length HBV genome, exstracting the HBV DNA from two patients infected with B and C genotype hepatits B virus respectively as template, amplifying the full length HBV with high fidelity hot-start Tag DNA polymerase;
2. full length HBV and pUC19 were cut with SacⅠ, then inserting HBV into the site of Sac I in pUC19 with T4 DNA ligase, named as pUC19-BHBV and pUC19-CHBV;
3. pUC19-HBV and pHY106 were cut by Sap I , then inserting HBV into the site of Sap I in pHY106 with T4 DNA ligase, named as pHY106-BHBV and pHY106-CHBV;
4. pHY106-BHBV and pHY106-CHBV were transfected into Huh7 cells, pHY106 as control;①Collected supernatant of culture at 24 h, 48 h, 72 h after transfection for examining HBsAg and HBeAg with an ELISA kit;②lysated the cells, exstracted the HBV replicative intermediate from HBV core for Southern blot at 72 h after transfection;③72 h after transfection, washed cells with PBS buffer for 5 times, then lysated the cells, examined the HBV DNA level by real-time PCR quantitatively;
5. cDNA microarray: Huh7 cells were transfected with recombinant B or C genotype HBV, namely pHY106-BHBV and pHY106-CHBV, pHY106 as control; 48 h after transfection, mRNA of cells were extracted, and reverse transcripted to cDNA, then hybrided with chip; The fluorescence signal intensity of Cy3 and Cy5 was analyzed with the GenePix Pro 3.0 software; the ratio of Cy5/Cy3 represent the differential value, the ratio above 2 or below 0.5 was regarded as significant data;
6. Real-time PCR: Huh7 cells were transfected with pHY106-BHBV or pHY106-CHBV, pHY106 as control; 48 h after transfection, the total RNA were extracted, and reverse transcripted to cDNA, validated the partial results of microarray with real-time PCR;
7. shRNA and pHY106-BHBV or pHY106-CHBV cotransfected HepG2 cells, the supernatant of the culture at 24 h, 48 h, 72 h, 96 h and 120 h after cotransfection was collected and frozen in -20℃for examining HBsAg and HBeAg level by an ELISA kit;
8. 72 h after shRNA and pHY106-BHBV or pHY106-CHBV cotransfed, the total RNA of cells was extracted and reverse-transcripted, then examining the level of HBsAg mRNA by RT-PCR (reverse transcript polymerase chain reaction); 72 h after cotransfection, cells were lysated, HBV DNA replicative intermediate from HBV core was extracted and detected by Southern blot.
Results:
1. Constructing the recombinant full length B and C genotype HBV successfully;
2. The expressiong of HBsAg and HBeAg were detectable after pHY106-BHBV and pHY106-CHBV transfected into Huh7 cells, the peak time was 48 h for HBsAg expression, the peak time for HBeAg expression is about 24 h late compared with that of HBsAg;
3. Southern blot detected the HBV replicative intermediate from HBV core, including rcDNA, dsDNA and ssDNA.
4. pHY106-BHBV and pHY106-CHBV could replicate after transfected into Huh7 cells, the HBV DNA leve get the peak at 72 h after transfection, the peak was about 8 log 10.
5. 48 h after pHY106-BHBV transfected Huh7 cells, 60 differentially expressed genes were screened from total 4097 genes, among of them, one was overexpressed, the other 59 genes were expressed at lower levels;
6. 122 differentially expressed genes were screened from total 4097 genes after pHY106-CHBV transfected Huh7 cells, among of them, 34 genes were expressed at higher levels, the other 88 genes were expressed at lower levels;
7. IFNGR2, GPR125, DDEF2 and PI3K which were down-regulatory genes after pHY106-BHBV transfected Huh7 cells with microarray analysis were validated by real-time PCR, the relative expression ratio of them were 0.8, 0.5, 0.3 and 0.4 respectively, which was consistent with the results of microarray; EEF2 and INF592 which were down-regulatory genes after pHY106-CHBV transfected Huh7 cells with microarray analysis were validated by real- time PCR, the relative expression ratio of them were 0.8 and 0.6, respectively, which was consistent with the results of microarray; only DDEF2 are found down-regulated in both microarrays;
8. HBsAg and HBeAg expression were inhibited obviously by shRNA-458 and shRNA·635, the inhibitory action was detectable at 48 h after cotransfection, the peak time was 72 h, the most inhibitory ratio was approximately 80% and 50% in HBsAg and HBeAg; there was no difference was observed about the the inhibitory action of shRNA-458 and shRNA-635 on B or C genotype HBV;
9. shRNA-458 and shRNA-635 had the similar inhibitory action on the production of HBsAg mRNA from B and C genotype HBV after 72 h of cotransfection, the inhibitory ratio was 60%-70%;
10. HBV DNA replicative intermediate from B and C genotype was inhibited obviously by the two shRNAs also.
Conclusions:
1. pHY106-BHBV and pHY106-CHBV can replicate and express HBsAg and HBeAg in Huh7 cells, which is suitable for studying the pathogenesis of B and C genotype HBV, the interaction between HBV and host, as well as exploiting new drugs againt HBV.
2. B and C genotype HBV all can change the expression pattern of Huh7 cells; but the change of expression pattern of Huh7 cells by B and C genotype HBV are obviously different, only DDEF2 are found down-regulated in both; which demonstrate B and C genotype HBV may differently influence on host. shRNA·458 and shRNA·635 have the powerfully inhibitory action on the expression
3. of HBsAg and HBeAg, the production HBsAg mRNA and HBV replicativeintermediate from B and C genotype HBV although 2-4 bases mismatch betweenshRNAs and HBV, so shRNA-458 and shRNA-635 are two useful tools for inhibitHBV.
引文
2.Kramvisa A,Kew M,Francois G.Hepatitis B virus genotypes.Vaccine,2005,23:2409-2423.
3.Okamoto H,Tsuda F,Sakugawa H,Sastrosoewignjo R,Imai M,Miyakawa Y,Mayumi M.Typing hepatitis B vires by homology in nucleotide sequence:Comparaison of surface antigen subtypes.J Gen Virol,1988,69:2575-2583.
4.Fung S,Lok A.Hepatitis B virus genotypes:Do they pay a role in the outcome of HBV infection.Hepatology,2004,40:790-792.
5.Stuyver L,De Gendt S,Van Geyt C,Zoulim F,Fried M,Schinazi R,Rossau R.A new genotype of hepatitis B virus:Complete genome and phylogenetic relatedness.J Gen Virol,2000,81:67-74.
6.Fung S K,and Lok A S.Hepatitis B virus genotypes:do they play a role in the outcome of HBV infection? Hepatology,2004,40:790-792.
7. Kao J H. Hepatitis B viral genotypes: clinical relevance and molecular characteristics. J. Gastroenterol. Hepatol,2002,17:643-650.
8. Magnius L O and Norder H. Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene. Intervirology, 1995,38:24-34.
9. Zeng G, Wang Z, Wen S, et al. Geographic distribution, virologic and clinical characteristics of hepatitis B virus genotypes in China. J Viral Hepat,2005,12(6):609-617.
10. Yuen MF, Sablon E, Yuan HJ, Wong DKH, Hui CK, Wong BCY, et al. The significance of hepatitis B genotype in acute exacerbation, HBeAg seroconversion, cirrhosis-related complications and hepatocellular carcinoma. Hepatology, 2003,37:562-567.
11. Chu CJ, Hussain M, Lok ASF. Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C. Gastroenterology, 2002,122:1756-1762.
12. Sumi H, Yokosuka O, Seki N, Arai M, Imazeki F, Kurihara T, et al. Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease. Hepatology,2003,37:19-26.
13.Fujie H, Moriya K, Shintani Y, Yotsuyanagi H, Iino S, Kimura S, et al. Hepatitis B genotypes and hepatocellular carcinoma in Japan. Gastroenterology, 2001, 120: 1564-1565.
14. Sakugawa H, Nakasone H, Nakayoshi T, Orito E, Mizokami M, Yamashiro T, et al. Preponderance of hepatitis B virus genotype B contributes to a better prognosis of chronic HBV infection in Okinawa, Japan. J Med Virol,2002,67:484-489.
15. Orito E, Mizokami M, Sakugawa H, Michitaka K, Ishikawa K, Ichida T, et al. A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV genotype research group. Hepatology, 2001,33:218-223.
16. Wai CY, Chu CJ, Hussain M, Lok AS. HBV genotype B is associated with a higher response rate to interferon therapy than genotype C. Hepatology,2002,36:1425-1430.
17. Kao JH, Wu NH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes and the response to interferon therapy. J Hepatol,2000,33:998-1002.
18. Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology,2000,118:554-559.
19. Kao JH, Chen PJ, Lai MY, Chen DS. Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. Gastroenterology,2003,124:327-334.
20. Duong TN, Horiike N, Michitaka K, et al.Comparison of genotypes C and D of the hepatitis B virus in Japan: a clinical and molecular biological study. J Med Virol,2004,72(4):551-557.
21. Sanchez-Tapias JM, Costa J, Mas A, et al. Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in western patients. Gastroenterology, 2002,123(6):1848-1856.
22. Thakur V, Guptan RC, Kazim SN, et al. Profile, spectrum and significance of HBV genotypes in chronic liver disease patients in the Indian subcontinent. J Gastroenterol Hepatol,2002,17(2): 165-170.
23.Nther S G, LI BC, MISKA S, et al. A Novel Method for Efficient Amplification of Whole Hepatitis B Virus Genomes Permits Rapid Functional Analysis and Reveals Deletion Mutants in mmunosuppressed Patients. Journal of virology, 1995,69(9): 5437.5444.
24. Bosch V, Kuhn C, and Schaller H. Hepatitis B virus replication, In E. Domingo, J. J. Holland, and P. Ahlquist (ed.), Retroviruses, viroids, and RNA recombination. RNA genetics,1988,2: 43-58.
25. Pfaff E, Kainkert MQ, Theihnann L, and Schaller H. Large surface proteins of hepatitis B virus containing the pre-S sequence. J. Virol, 1986,52:396-402.
26. Pfaff E, Klinkert MQ, Theilmann L, Schaller H. Characterization of large surface proteins of hepatitis B virus by antibodies to pre-S encoded amino acids. Virology,1986,148:15-22.
27. Stibbe W, and Gerlich W H. Structural relationships between minor and major proteins of hepatitis B virus surface antigen. J. Virol,1983,46:626-628.
28. Tiollais P, Pourcel C, and Dejean A. The hepatitis B virus. Nature (London), 1985,317:489-495.
1.Beasley R,Lin CC,Hwang LY,and Chien CS.Hepatocellular carcinoma and hepatitis B virus:a prospective study of 22707 men in Taiwan.Lancet,1981,ⅱ:1129-1133.
2.Yuen MF,Lai CL.Treatment for chronic hepatitis B.Lancet Infect Dis 2001;1:232-241.
3.Shepard CW,Simard EP,Finelli L,Fiore AE,Bell BP:Hepatitis B virus infection:epidemiology and vaccination.Epidemiol Rev 2006,28:112-125.
4.Lau JY and Wright TL.Molecular virology and pathogenesis of hepatitis B.Lancet,1993,342(8883):1335-1340.
5.Wands JR and Blurn HE.Primary hepatocellular carcinoma.N.Engl.J.Med,1991,25(10):729-731.
6. Nassal M. Hepatitis B virus replication: novel roles for virus-host interactions. Intervirology, 1999,42:100-116.
7. Cheong JY, Cho SW, Hwang IL, et al. Association between chronic hepatitis B virus infection and interleukin-10, tumor necrosis factor-a gene promoter polymorphisms. Journal of Gastroenterology and Hepatology,2006,21(7):1163-1169.
8. Park BL, Kim YJ, Cheong HS, et al. Association of common promoter polymorphisms of MCP1 with hepatitis B virus clearance. Experimental and Molecular medicine,2006,38(6): 694-702.
9. Yang CY, Kuo TH, Ting LP. Human Hepatitis B Viral e Antigen Interacts with Cellular Interleukin-1 Receptor Accessory Protein and Triggers Interleukin-1 Response. The Journal of Biological Chemistry, 2006,281(45): 34525-34536.
10. Kim SY, Kim JC, Kim JK, et al. Hepatitis B virus X protein enhances NF kappa B activity through cooperating with VBP1. BMB Rep,2008,41(2):158-163.
11. Wang JH, Yun C, Kim S, et al. Reactivation of p53 in cells expressing hepatitis B virus X-protein involves p53 phosphorylation and a reduction of Hdm2. Cancer Sci, 2008,Feb 24 [Epub ahead of print].
12. Chung TW, Lee YC, Ko JH, et al. Hepatitis B Virus X protein modulates the expression of PTEN by inhibiting the function of p53, a transcriptional activator in liver cells. Cancer Res,2003,63(13): 3453-3458.
13. Kim S, Park SY, Yong H, et al. HBV X protein targets hBubR1, which induces dysregulation of the mitotic checkpoint. Oncogene,2008, Jan 14 [Epub ahead of print].
14. Oishi N, Shilagardi K, Nakamoto Y, et al. Hepatitis B virus X protein overcomes oncogenic RAS-induced senescence in human immortalized cells. Cancer Sci,2007, 98(10):1540-1548.
15. Lei JH, He XE, Yang X, et al. Effects of HBV X gene and arsenic trioxide on the expression of p53 in cultured HepG2 cells. Chinese Medical Journal,2007,120(24): 2181-2184.
16. Jung JK, Arora P, Pagano JS, et al. Expression of DNA methyltransferase 1 is activated by hepatitis B virus X protein via a regulatory circuit involving the p16INK4a-cyclin D1-CDK 4/6-pRb-E2F1 pathway. Cancer Res,2007,67(12): 5771-5778.
17. Su JM, Lai XM, Lan KH, et al. X protein of hepatitis B virus functions as a transcriptional corepressor on the human telomerase promoter. Hepatology,2007, 46(2):402-13.
18. Kim KH, Shin HJ, Kim K, et al. Hepatitis B virus X protein induces hepatic steatosis via transcriptional activation of SREBP1 and PPARgamma. Gastroenterology,2007, 132(5):1955-67.
19. Jun Chen and Aleem Siddiqui. Hepatitis B Virus X Protein Stimulates the Mitochondrial Translocation of Raf-1 via Oxidative Stress. Journal of Virology, 2007,81(12): 6757-6760.
20. Schena M, Shalon D, Dais RW, Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995 , 270:467-470.
21. Zhang MQ. Large-scale gene expression data analysis: a new challenge to computational biologists. Genome Res, 1999,9:681-688.
22. Bigger CB, Brasky KM, Lanford RE. DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J Virol,2001,75:7059-7066.
23. Coutinho-Camillo CM, Salaorni S, Sarkis AS,and Nagai MA. Differentially expressed genes in the prostate cancer cell line LNCaP after exposure to androgen and anti-androgen. Cancer Genet Cytogenet,2006,166(2):130-138.
24. Christine A, Iacobuzio-Donahue, Anirban Maitra, et al. Exploration of Global Gene Expression Patterns in Pancreatic Adenocarcinoma Using cDNA Microarrays. American Journal ofPathology, 2003,162(4):1151-1162.
25. Bourquin JP, Subramanian A, Langebrake C, et al. Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling. PNAS, 2006,103(9): 3339-3344.
26. Snyder M, Huang XY, and Zhang JJ. Identification of Novel Direct Stat3 Target Genes for Control of Growth and Differentiation. J Biol Chem,2008,283(7): 3791-3798.
1.Yuen MF,Lai CL.Treatment for chronic hepatitis B.Lancet Infect Dis 2001,1:232-241.
2.Shepard CW,Simard EP,Finelli L,Fiore AE,Bell BP:Hepatitis B virus infection:epidemiology and vaccination.Epidemiol Rev,2006,28:112-125.
3.Lau JY and Wright TL.Molecular virology and pathogenesis of hepatitis B.Lancet,1993,342(8883):1335-1340.
4.Wands JR and Blum HE.Primary hepatocellular carcinoma.N.Engl.J.Med,1991,25(10):729-731.
5.Torresi J and Locarnini S.Antiviral chemotherapy for the treatment of hepatitis B virus infections.Gastroenterology,2000,118(Suppl.1):83-103.
6.Zuckerman J N and Zuckerman AJ.Current topics in hepatitis B.J Infect,2000,41:130-136.
7.Lin OS and Keeffe E B.Current treatment strategies for chronic hepatitis B and C.Annu Rev Med,2001,52:29-49.
8.Charles E.Samuel.Antiviral Actions of Interferons.Clincal Microbiology Review,2001,14(4):778-809.
9.Palurnbo E.New drugs for chronic hepatitis B:a review.Am J Ther,2008,15(2):167-72.
10. Mondelli M. New treatment options in chronic hepatitis B. Infez Med,2008,16(1):5-14.
11. hepatitis B treated with interferon alpha. Gastroenterology, 1997,(5): 1660-1667.
12. Hoofhagle JH and Bisceglie AM. The treatment of chronic viral hepatitis. N. Engl. J. Med,1997,336(5):347-356.
13. Lai CL, Chien RN, Leung NW, et al. A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N. Engl.J. Med,1998,339(2):61-68.
14. Yao G, Wang B, Cui Z. Long-term effect of lamivudine treatment in chronic hepatitis B virus infection. Zhonghua Gan Zang Bing Za Zhi,1999,7(2):80-83.
15. Marathe R, Anandalakshmi R, Smith,TH, Pruss GJ and Vance VB. RNA viruses as inducers, suppressors and targets of post-transcriptional gene silencing. Plant Mol Biol, 2000,43:295-306.
16. Shlomai A and Shaul Y. Inhibition of hepatitis B virus expression and replication by RNA interference. Hepatology,2003,37:764-770.
17. Gitlin L, Karelsky S and Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature,2002,418:430-434.
18. Ying C, De Clercq E and Neyts J. Selective inhibition of hepatitis B virus replication by RNA interference. Biochem Biophys Res Commun,2003,309:482-484.
19. McCaffrey A P, Nakai H, Pandey K, Huang Z, Salazar F H, Xu H, Wieland S F, Marion P L and Kay M A. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol, 2003,21:639-644.
20. Uprichard S L, Boyd B, Althage A and Chisari F V. Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs. Proc Natl Acad Sci USA,2005,102: 773-778.
21. Bernstein E, Denli AM, Hannon GJ. The rest is silence. RNA,2001,7: 1509-1521.
22. Hammond SM, Caudy AA, Hannon GJ. Post-transcriptional gene silencing by double- stranded RNA. Nat Rev Genet,2001,2:110-119.
23. Olsen PH, Ambros V. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking lin-14 protein synthesis after the initiation of translation. Dev Biol, 1999,216: 671-680.
24. Cheng TL, Chang WT. Construction of simple and efficient DNA vector-based short hairpin RNA expression systems for specific gene silencing in mammalian cells. Methods Mol Biol,2007,408:223-241.
25.Gou D,Weng T,Wang Y,et al.A novel approach for the construction of multiple shRNA expression vectors.J Gene Meal,2007,9(9):751-763.
26.Hollinger FB.Hepatitis B virus genetic diversity and its impact on diagnostic assays.J Viral Hepat,2007,14(Suppl 1):11-15.
27.Echevarria JM,Avellon A.Hepatitis B virus genetic diversity.J Med Virol,2006,78(Suppl 1):36-42.
28.Clarke B and Bloor S.Molecular genotyping of hepatitis B virus.J Clin Virol,2002,25:41-45.
29.Norder H,Courouce A M,and Magnius L O.Molecular basis of hepatitis B virus serotype variations within the four major subtypes.J.Gen.Virol,1992,73:3141-3145.
30.Norder H,Courouce A M,and Magnius L O.Complete genomes,phylogenic relatedness and structural proteins of six strains of the hepatitis B virus,four of which represent two new genotypes.Virology,1994,198:489-503.
31.Okamoto H,Tsuda F,Sakugawa H,et al.Typing hepatitis B virus by homology in nucleotide sequence:comparison of surface antigen subtypes.J.Gen.Virol,1988,69:2575-2583.
32.Arauz-Ruiz P,Norder H,Robertson B H,et al.Genotype H:a new Amerindian genotype of hepatitis B virus revealed in Central America.J.Gen.Virol,2002,83:2059-2073.
33.Stuyver L,Gendt D S,Geyt C V,et al.A new genotype of hepatitis B virus:complete genome and phylogenetic relatedness.J.Gen.Virol,2000,81:67-74.
34.刘顺爱,魏红山,董庆鸣,et al.乙型肝炎病毒P区RNA干扰抑制2.2.15细胞乙型肝炎病毒表面抗原和e抗原分泌的实验研究.中华医学杂志,2005,85(43):3079-3083.
1.Ganem D and Prince A M.Hepatitis B virus infection-natural history and clinical consequences.New England Journal of Medicine,2004,350:1118-29.
2.Sureau C,Romet-Lemonne J L,James I,et al.Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA.Cell,1986,10:47(1):37-47.
3.Sells MA,Chen ML,and Acs G.Production of hepatitis B virus particles in HepG2cells transfected with cloned hepatitis B virus DNA.Proc.Nati.Acad.Sci.USA,1987,84:1005-1009.
4.Galibert F,Mandart E,Fitoussi F,Tiollais P,Chamay P.Nucleotide sequence of the hepatitis B virus genome(subtype ayw)cloned in E.coli.Nature,1979,281(5733):646-650.
5.Jacquard AC,Brunelle MN,Pichoud C,et al.In Vitro Characterization of the Anti-Hepatitis B Virus Activity and Cross-Resistance Profile of 2',3'-Dideoxy-3'-Fluoroguanosine.Antimicrobial Agents and Chemotherapy,2006,50:955-961.
6.Tenney D J,Levine SM,Rose RE,et al.Clinical Emergence of Entecavir-Resistant Hepatitis B Virus Requires Additional Substitutions in Virus Already Resistant to Lamivudine. Antimicrobial Agents and Chemotherapy,2004,48:3498-3507.
7. William E, Delaney IV, Huiling Yang, et al. Combinations of Adefovir with Nucleoside Analogs Produce Additive Antiviral Effects against Hepatitis B Virus In Vitro. Antimicrobial Agents and Chemotherapy,2004,48:3702-3710.
8. Danni C, Gilda C, STEPHEN Stephen L, et al. In Vitro Antihepadnaviral Activities of Combinations of Penciclovir, Lamivudine, and Adefovir. Antimicrobial Agents and Chemotherapy,2000,44: 551-560.
9. Chen Robert YM, Edwards Ros, Shaw Tim, et al. Effect of the G1896A Precore Mutation on Drug Sensitivity and Replication Yield of Lamivudine-Resistant HBV In Vitro. Hepatology,2003,37:27-35.
10. Pan XB, Wei L, Chen HS, et al. Liver-derived cell lines QSG-7701 and HepG2 support different HBV replication patterns. Arch Virol, 2007,152(6): 1159-1173.
11. De Meyer S, Gong ZJ, Suwandhi W,et al. Organ and species specificity of hepatitis B virus (HBV) infection: a review of literature with a special reference to preferential attachment of HBV to human hepatocytes. J Viral Hepat, 1997,4(3): 145-53.
12. Shih C, Li LS, Roychoudhury S, and Hot MH. In vitro propagation of human hepatitis B virus in a rat hepatoma cell line. Proc. Natl. Acad. Sci. USA, 1989,86: 6323-6327.
13. Hirschman SZ, Garfinkel E. of human cells in culture with hepatitis B virus. Trans Assoc Am Physicians, 1980,93:60-71.
14. Hirschman S, Price P, Garfinkel E, et al. Expression of cloned hepatitis B virus DNA in human cell cultures. Proc. Natl. Acad. Sci,USA, 1980,77:5507-5511.
15. Christman JK, Gerber M, Price P M, et al. Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA. Proc. NatL Acad. Sci. USA, 1982,79:1815-1819,
16. Takeshima H, Namiki M, Inokoshi J, et al. Stable expression of hepatitis B virus genome in a primate kidney cell. Arch Virol,1989;109(1-2):35-49
17. Kumon I, Onji M, Nadano S, et al. Detection of hepatitis B surface antigen in lymphoid follicle in patients with chronic hepatitis type B. Gastroenterol Jpn,1989,24(6):738.
18. Seifer M, Heermann KH, Gerlich WH. Expression pattern of the hepatitis B virus genome in transfected mouse fibroblasts. Virology, 1990,179( 1 ):287-299.
19. Miiller C, Bergmann KF, Gerin JL, et al. Production of hepatitis B virus by stably transfected monocytic cell line U-937: a model for extrahepatic hepatitis B virus replication. J Infect Dis, 1992,165(5):929-933.
20. Wang RY, Shen CN, Lin MH, et al. Hepatocyte-Like Cells Transdifferentiated from a Pancreatic Origin Can Support Replication of Hepatitis B Virus. Journal of Virology, 2005,79:13116-13128.
21.Galle PR. Hagelstem J, kommerell B, et al. In vitro experimental Infection of primary humane hepatocytes with hepatitis B virus.Gastroenterology, 1994,106(3): 664-673.
22. Scaglioni PP, Melegari M, Wands JR. Posttranscriptional regulation of hepatitis B virus replication by the precore protein. J Virol,1997,71(1):345-353.
23. Muller C, Bergmann KF, Gerin JL, et al. Pro-duction of hepatitis B virus by stabty transfected monocytic cell line U-937: a model for extrahepatic hepatitis B replication. J Infect Dis,1992,165(5):929-933.
24. Marie-Noelle Brunelle, Anne-Carole Jacquard, Christian Pichoud, et al. Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir. Hepatology,2005,411391-1398.
25. Delaney WE 4th, Isom HC. Hepatitis B virus replication in human hepG2 Cells mediated by hepatitis B virus recombination baculovirus. Hepatology, 1998, 28(4): 1134-1146.
26. Delaney WE 4th, MillerTG, Isom HC. Use of the hepatitis B virus recombinant baculovirus -hepG2 system to study the effects of (-)-2' ,3' -dideoxy 3' -thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA. Antimicrob Agents Chemother,1999,43(8):2017-2026.
27. Spfinzl MF, Oberwinkler H, Schaller H, et al. Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice: Crossing the species barrier. J Vim1,2001,75(11):5108-5118.
28.Sun D,Nass M.Stable HepG2-and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus.Journal of Hepatology,2006,45:636-645.
29.Christen V,Duong F,Bernsmeier C,et al.Inhibition of Alpha Interferon Signaling by Hepatitis B Virus.Journal of Virology,2007,81(1):159-165.
30.王松,唐红,何芳,等.肝富集转录因子在HBV基因转录与复制中的作用.四川大学学报(医学版),2006,37:35-39.
31.Zajakina A,Kozlovska T,Bruvere R,et al.Translation of hepatitis B virus(HBV)surface proteins from the HBV pregenome and precore RNAs in Semliki Forest virus-driven expression.Journal of General Virology,2004,85:3343-3351.
32.Tacke F,Gehrke C,Luedde T,et al.Basal Core Promoter and Precore utations in the Hepatitis B Virus Genome Enhance Replication Efficacy of Lamivudine-Resistant Mutants.Journal of Virology,2004,78:8524-8535.
33.Locarnini,S.,J.McMillan,and A.Bartholomeusz.The hepatitis B virus and common mutants.Semin.Liver Dis,2003,23:5-20.
34.Buckwold VE,Xu Z,Chen M,et al.Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication.J.Virol,1996,70:5845-5851.
35.Hunt CM,McGill JM,Allen M I,et al.Clinical relevance of hepatitis B viral mutations.Hepatology,2000,31:1037-1044.
1.Shlomai A,Shanl Y.Inhibition of hepatitis B virus expression and replication by RNA Interference.Hepatology,2003,37:764-770.
2.Ying CX,De CE,Neyts J.Seleetiveinhibition ofhepatitis B vires replication by RNA interference.Biochem Biophys Res Commun,2003,309:482-484.
3.Konishi M,Wu CH,Wu GY.Inhibition of HBV replication by siRNA in a stable HBV-producing cell line.Hepatolgy,2003,38:842-850.
4.H.Kayhan,E.Karatayli,A.R.Turkyilmaz,et al.Inhibition of hepatitis B virus replication by shRNAs in stably HBV expressed HEPG2.2.15 cell lines.Archives of Virology,2007,152:871-879.
5.Chen Z,Xu ZF,Ye JJ,et al.Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression.J Zhejiang Univ SCI,2005,6B(4):236-241
6.Ren XR,Zhou LJ,Luo GB,et al.Inhibition of hepatitis B virus replication in 2.2.15cells by expressed shRNA.J Viral Hepat,2005,12(3):236-242.
7.Ren X,Luo G,Xie Z,et al.Inhibition of multiple gene expression and virus replication of HBV by stable RNA interference in 2.2.15 cells.J Hepatol,2006,44(4):663-670.
8.Jia F,Zhang YZ,and Liu CM.Stable inhibition of hepatitis B virus expression and replication in HepG2.2.15 cells by RNA interference based on retrovirus delivery.J Biotechnol,2007,128(1):32-40.
9.Giladi H.Ketzinel-Gilad M,Rivkin L,et al.Small interfering RNA inhibits hepatitis B virus replication in mice.Mol Iher,2003,8(5):769-776.
10.Zhang XN,Wei X,Wang JD,et al.RNAi mediated inhibition of HBVreplication and expression. World J Gastroenteml,2004,10(20):2967-2971.
11. M cCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol,2003,21(6):639-647.
12. Y Wu, AL Huang, N Tang, et al. Specific anti-viral effects of RNA interference on replication and expression of hepatitis B virus in mice. Chin Med J (Engl), August 20, 2005;118(16):1351-1356.
13. Susan L. Uprichard, Bryan Boyd, et al. Clearance of hepatitis B virus from the liverof transgenic mice by short hairpin RNAs. PNAS,2005,102: 773-778
14. Chen CC, Ko TM, Ma HI, et al. Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA. Gene Ther, January 1, 2007; 14(1):11-19.
15. Tan C, Xuan BQ, Hong J, et al. RNA interference against hepatitis B virus with endoribonuclease-prepared siRNA despite of the target sequence variations.Virus Research,2007,126:172-178.
3.Okamoto H,Tsuda F,Sakugawa H,Sastrosoewignjo R,Imai M,Miyakawa Y,Mayumi M.Typing hepatitis B vires by homology in nucleotide sequence:Comparaison of surface antigen subtypes.J Gen Virol,1988,69:2575-2583.
4.Fung S,Lok A.Hepatitis B virus genotypes:Do they pay a role in the outcome of HBV infection.Hepatology,2004,40:790-792.
5.Stuyver L,De Gendt S,Van Geyt C,Zoulim F,Fried M,Schinazi R,Rossau R.A new genotype of hepatitis B virus:Complete genome and phylogenetic relatedness.J Gen Virol,2000,81:67-74.
6.Fung S K,and Lok A S.Hepatitis B virus genotypes:do they play a role in the outcome of HBV infection? Hepatology,2004,40:790-792.
7. Kao J H. Hepatitis B viral genotypes: clinical relevance and molecular characteristics. J. Gastroenterol. Hepatol,2002,17:643-650.
8. Magnius L O and Norder H. Subtypes, genotypes and molecular epidemiology of the hepatitis B virus as reflected by sequence variability of the S-gene. Intervirology, 1995,38:24-34.
9. Zeng G, Wang Z, Wen S, et al. Geographic distribution, virologic and clinical characteristics of hepatitis B virus genotypes in China. J Viral Hepat,2005,12(6):609-617.
10. Yuen MF, Sablon E, Yuan HJ, Wong DKH, Hui CK, Wong BCY, et al. The significance of hepatitis B genotype in acute exacerbation, HBeAg seroconversion, cirrhosis-related complications and hepatocellular carcinoma. Hepatology, 2003,37:562-567.
11. Chu CJ, Hussain M, Lok ASF. Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C. Gastroenterology, 2002,122:1756-1762.
12. Sumi H, Yokosuka O, Seki N, Arai M, Imazeki F, Kurihara T, et al. Influence of hepatitis B virus genotypes on the progression of chronic type B liver disease. Hepatology,2003,37:19-26.
13.Fujie H, Moriya K, Shintani Y, Yotsuyanagi H, Iino S, Kimura S, et al. Hepatitis B genotypes and hepatocellular carcinoma in Japan. Gastroenterology, 2001, 120: 1564-1565.
14. Sakugawa H, Nakasone H, Nakayoshi T, Orito E, Mizokami M, Yamashiro T, et al. Preponderance of hepatitis B virus genotype B contributes to a better prognosis of chronic HBV infection in Okinawa, Japan. J Med Virol,2002,67:484-489.
15. Orito E, Mizokami M, Sakugawa H, Michitaka K, Ishikawa K, Ichida T, et al. A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Japan HBV genotype research group. Hepatology, 2001,33:218-223.
16. Wai CY, Chu CJ, Hussain M, Lok AS. HBV genotype B is associated with a higher response rate to interferon therapy than genotype C. Hepatology,2002,36:1425-1430.
17. Kao JH, Wu NH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes and the response to interferon therapy. J Hepatol,2000,33:998-1002.
18. Kao JH, Chen PJ, Lai MY, Chen DS. Hepatitis B genotypes correlate with clinical outcomes in patients with chronic hepatitis B. Gastroenterology,2000,118:554-559.
19. Kao JH, Chen PJ, Lai MY, Chen DS. Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. Gastroenterology,2003,124:327-334.
20. Duong TN, Horiike N, Michitaka K, et al.Comparison of genotypes C and D of the hepatitis B virus in Japan: a clinical and molecular biological study. J Med Virol,2004,72(4):551-557.
21. Sanchez-Tapias JM, Costa J, Mas A, et al. Influence of hepatitis B virus genotype on the long-term outcome of chronic hepatitis B in western patients. Gastroenterology, 2002,123(6):1848-1856.
22. Thakur V, Guptan RC, Kazim SN, et al. Profile, spectrum and significance of HBV genotypes in chronic liver disease patients in the Indian subcontinent. J Gastroenterol Hepatol,2002,17(2): 165-170.
23.Nther S G, LI BC, MISKA S, et al. A Novel Method for Efficient Amplification of Whole Hepatitis B Virus Genomes Permits Rapid Functional Analysis and Reveals Deletion Mutants in mmunosuppressed Patients. Journal of virology, 1995,69(9): 5437.5444.
24. Bosch V, Kuhn C, and Schaller H. Hepatitis B virus replication, In E. Domingo, J. J. Holland, and P. Ahlquist (ed.), Retroviruses, viroids, and RNA recombination. RNA genetics,1988,2: 43-58.
25. Pfaff E, Kainkert MQ, Theihnann L, and Schaller H. Large surface proteins of hepatitis B virus containing the pre-S sequence. J. Virol, 1986,52:396-402.
26. Pfaff E, Klinkert MQ, Theilmann L, Schaller H. Characterization of large surface proteins of hepatitis B virus by antibodies to pre-S encoded amino acids. Virology,1986,148:15-22.
27. Stibbe W, and Gerlich W H. Structural relationships between minor and major proteins of hepatitis B virus surface antigen. J. Virol,1983,46:626-628.
28. Tiollais P, Pourcel C, and Dejean A. The hepatitis B virus. Nature (London), 1985,317:489-495.
1.Beasley R,Lin CC,Hwang LY,and Chien CS.Hepatocellular carcinoma and hepatitis B virus:a prospective study of 22707 men in Taiwan.Lancet,1981,ⅱ:1129-1133.
2.Yuen MF,Lai CL.Treatment for chronic hepatitis B.Lancet Infect Dis 2001;1:232-241.
3.Shepard CW,Simard EP,Finelli L,Fiore AE,Bell BP:Hepatitis B virus infection:epidemiology and vaccination.Epidemiol Rev 2006,28:112-125.
4.Lau JY and Wright TL.Molecular virology and pathogenesis of hepatitis B.Lancet,1993,342(8883):1335-1340.
5.Wands JR and Blurn HE.Primary hepatocellular carcinoma.N.Engl.J.Med,1991,25(10):729-731.
6. Nassal M. Hepatitis B virus replication: novel roles for virus-host interactions. Intervirology, 1999,42:100-116.
7. Cheong JY, Cho SW, Hwang IL, et al. Association between chronic hepatitis B virus infection and interleukin-10, tumor necrosis factor-a gene promoter polymorphisms. Journal of Gastroenterology and Hepatology,2006,21(7):1163-1169.
8. Park BL, Kim YJ, Cheong HS, et al. Association of common promoter polymorphisms of MCP1 with hepatitis B virus clearance. Experimental and Molecular medicine,2006,38(6): 694-702.
9. Yang CY, Kuo TH, Ting LP. Human Hepatitis B Viral e Antigen Interacts with Cellular Interleukin-1 Receptor Accessory Protein and Triggers Interleukin-1 Response. The Journal of Biological Chemistry, 2006,281(45): 34525-34536.
10. Kim SY, Kim JC, Kim JK, et al. Hepatitis B virus X protein enhances NF kappa B activity through cooperating with VBP1. BMB Rep,2008,41(2):158-163.
11. Wang JH, Yun C, Kim S, et al. Reactivation of p53 in cells expressing hepatitis B virus X-protein involves p53 phosphorylation and a reduction of Hdm2. Cancer Sci, 2008,Feb 24 [Epub ahead of print].
12. Chung TW, Lee YC, Ko JH, et al. Hepatitis B Virus X protein modulates the expression of PTEN by inhibiting the function of p53, a transcriptional activator in liver cells. Cancer Res,2003,63(13): 3453-3458.
13. Kim S, Park SY, Yong H, et al. HBV X protein targets hBubR1, which induces dysregulation of the mitotic checkpoint. Oncogene,2008, Jan 14 [Epub ahead of print].
14. Oishi N, Shilagardi K, Nakamoto Y, et al. Hepatitis B virus X protein overcomes oncogenic RAS-induced senescence in human immortalized cells. Cancer Sci,2007, 98(10):1540-1548.
15. Lei JH, He XE, Yang X, et al. Effects of HBV X gene and arsenic trioxide on the expression of p53 in cultured HepG2 cells. Chinese Medical Journal,2007,120(24): 2181-2184.
16. Jung JK, Arora P, Pagano JS, et al. Expression of DNA methyltransferase 1 is activated by hepatitis B virus X protein via a regulatory circuit involving the p16INK4a-cyclin D1-CDK 4/6-pRb-E2F1 pathway. Cancer Res,2007,67(12): 5771-5778.
17. Su JM, Lai XM, Lan KH, et al. X protein of hepatitis B virus functions as a transcriptional corepressor on the human telomerase promoter. Hepatology,2007, 46(2):402-13.
18. Kim KH, Shin HJ, Kim K, et al. Hepatitis B virus X protein induces hepatic steatosis via transcriptional activation of SREBP1 and PPARgamma. Gastroenterology,2007, 132(5):1955-67.
19. Jun Chen and Aleem Siddiqui. Hepatitis B Virus X Protein Stimulates the Mitochondrial Translocation of Raf-1 via Oxidative Stress. Journal of Virology, 2007,81(12): 6757-6760.
20. Schena M, Shalon D, Dais RW, Brown PO. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 1995 , 270:467-470.
21. Zhang MQ. Large-scale gene expression data analysis: a new challenge to computational biologists. Genome Res, 1999,9:681-688.
22. Bigger CB, Brasky KM, Lanford RE. DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J Virol,2001,75:7059-7066.
23. Coutinho-Camillo CM, Salaorni S, Sarkis AS,and Nagai MA. Differentially expressed genes in the prostate cancer cell line LNCaP after exposure to androgen and anti-androgen. Cancer Genet Cytogenet,2006,166(2):130-138.
24. Christine A, Iacobuzio-Donahue, Anirban Maitra, et al. Exploration of Global Gene Expression Patterns in Pancreatic Adenocarcinoma Using cDNA Microarrays. American Journal ofPathology, 2003,162(4):1151-1162.
25. Bourquin JP, Subramanian A, Langebrake C, et al. Identification of distinct molecular phenotypes in acute megakaryoblastic leukemia by gene expression profiling. PNAS, 2006,103(9): 3339-3344.
26. Snyder M, Huang XY, and Zhang JJ. Identification of Novel Direct Stat3 Target Genes for Control of Growth and Differentiation. J Biol Chem,2008,283(7): 3791-3798.
1.Yuen MF,Lai CL.Treatment for chronic hepatitis B.Lancet Infect Dis 2001,1:232-241.
2.Shepard CW,Simard EP,Finelli L,Fiore AE,Bell BP:Hepatitis B virus infection:epidemiology and vaccination.Epidemiol Rev,2006,28:112-125.
3.Lau JY and Wright TL.Molecular virology and pathogenesis of hepatitis B.Lancet,1993,342(8883):1335-1340.
4.Wands JR and Blum HE.Primary hepatocellular carcinoma.N.Engl.J.Med,1991,25(10):729-731.
5.Torresi J and Locarnini S.Antiviral chemotherapy for the treatment of hepatitis B virus infections.Gastroenterology,2000,118(Suppl.1):83-103.
6.Zuckerman J N and Zuckerman AJ.Current topics in hepatitis B.J Infect,2000,41:130-136.
7.Lin OS and Keeffe E B.Current treatment strategies for chronic hepatitis B and C.Annu Rev Med,2001,52:29-49.
8.Charles E.Samuel.Antiviral Actions of Interferons.Clincal Microbiology Review,2001,14(4):778-809.
9.Palurnbo E.New drugs for chronic hepatitis B:a review.Am J Ther,2008,15(2):167-72.
10. Mondelli M. New treatment options in chronic hepatitis B. Infez Med,2008,16(1):5-14.
11. hepatitis B treated with interferon alpha. Gastroenterology, 1997,(5): 1660-1667.
12. Hoofhagle JH and Bisceglie AM. The treatment of chronic viral hepatitis. N. Engl. J. Med,1997,336(5):347-356.
13. Lai CL, Chien RN, Leung NW, et al. A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. N. Engl.J. Med,1998,339(2):61-68.
14. Yao G, Wang B, Cui Z. Long-term effect of lamivudine treatment in chronic hepatitis B virus infection. Zhonghua Gan Zang Bing Za Zhi,1999,7(2):80-83.
15. Marathe R, Anandalakshmi R, Smith,TH, Pruss GJ and Vance VB. RNA viruses as inducers, suppressors and targets of post-transcriptional gene silencing. Plant Mol Biol, 2000,43:295-306.
16. Shlomai A and Shaul Y. Inhibition of hepatitis B virus expression and replication by RNA interference. Hepatology,2003,37:764-770.
17. Gitlin L, Karelsky S and Andino R. Short interfering RNA confers intracellular antiviral immunity in human cells. Nature,2002,418:430-434.
18. Ying C, De Clercq E and Neyts J. Selective inhibition of hepatitis B virus replication by RNA interference. Biochem Biophys Res Commun,2003,309:482-484.
19. McCaffrey A P, Nakai H, Pandey K, Huang Z, Salazar F H, Xu H, Wieland S F, Marion P L and Kay M A. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol, 2003,21:639-644.
20. Uprichard S L, Boyd B, Althage A and Chisari F V. Clearance of hepatitis B virus from the liver of transgenic mice by short hairpin RNAs. Proc Natl Acad Sci USA,2005,102: 773-778.
21. Bernstein E, Denli AM, Hannon GJ. The rest is silence. RNA,2001,7: 1509-1521.
22. Hammond SM, Caudy AA, Hannon GJ. Post-transcriptional gene silencing by double- stranded RNA. Nat Rev Genet,2001,2:110-119.
23. Olsen PH, Ambros V. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking lin-14 protein synthesis after the initiation of translation. Dev Biol, 1999,216: 671-680.
24. Cheng TL, Chang WT. Construction of simple and efficient DNA vector-based short hairpin RNA expression systems for specific gene silencing in mammalian cells. Methods Mol Biol,2007,408:223-241.
25.Gou D,Weng T,Wang Y,et al.A novel approach for the construction of multiple shRNA expression vectors.J Gene Meal,2007,9(9):751-763.
26.Hollinger FB.Hepatitis B virus genetic diversity and its impact on diagnostic assays.J Viral Hepat,2007,14(Suppl 1):11-15.
27.Echevarria JM,Avellon A.Hepatitis B virus genetic diversity.J Med Virol,2006,78(Suppl 1):36-42.
28.Clarke B and Bloor S.Molecular genotyping of hepatitis B virus.J Clin Virol,2002,25:41-45.
29.Norder H,Courouce A M,and Magnius L O.Molecular basis of hepatitis B virus serotype variations within the four major subtypes.J.Gen.Virol,1992,73:3141-3145.
30.Norder H,Courouce A M,and Magnius L O.Complete genomes,phylogenic relatedness and structural proteins of six strains of the hepatitis B virus,four of which represent two new genotypes.Virology,1994,198:489-503.
31.Okamoto H,Tsuda F,Sakugawa H,et al.Typing hepatitis B virus by homology in nucleotide sequence:comparison of surface antigen subtypes.J.Gen.Virol,1988,69:2575-2583.
32.Arauz-Ruiz P,Norder H,Robertson B H,et al.Genotype H:a new Amerindian genotype of hepatitis B virus revealed in Central America.J.Gen.Virol,2002,83:2059-2073.
33.Stuyver L,Gendt D S,Geyt C V,et al.A new genotype of hepatitis B virus:complete genome and phylogenetic relatedness.J.Gen.Virol,2000,81:67-74.
34.刘顺爱,魏红山,董庆鸣,et al.乙型肝炎病毒P区RNA干扰抑制2.2.15细胞乙型肝炎病毒表面抗原和e抗原分泌的实验研究.中华医学杂志,2005,85(43):3079-3083.
1.Ganem D and Prince A M.Hepatitis B virus infection-natural history and clinical consequences.New England Journal of Medicine,2004,350:1118-29.
2.Sureau C,Romet-Lemonne J L,James I,et al.Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA.Cell,1986,10:47(1):37-47.
3.Sells MA,Chen ML,and Acs G.Production of hepatitis B virus particles in HepG2cells transfected with cloned hepatitis B virus DNA.Proc.Nati.Acad.Sci.USA,1987,84:1005-1009.
4.Galibert F,Mandart E,Fitoussi F,Tiollais P,Chamay P.Nucleotide sequence of the hepatitis B virus genome(subtype ayw)cloned in E.coli.Nature,1979,281(5733):646-650.
5.Jacquard AC,Brunelle MN,Pichoud C,et al.In Vitro Characterization of the Anti-Hepatitis B Virus Activity and Cross-Resistance Profile of 2',3'-Dideoxy-3'-Fluoroguanosine.Antimicrobial Agents and Chemotherapy,2006,50:955-961.
6.Tenney D J,Levine SM,Rose RE,et al.Clinical Emergence of Entecavir-Resistant Hepatitis B Virus Requires Additional Substitutions in Virus Already Resistant to Lamivudine. Antimicrobial Agents and Chemotherapy,2004,48:3498-3507.
7. William E, Delaney IV, Huiling Yang, et al. Combinations of Adefovir with Nucleoside Analogs Produce Additive Antiviral Effects against Hepatitis B Virus In Vitro. Antimicrobial Agents and Chemotherapy,2004,48:3702-3710.
8. Danni C, Gilda C, STEPHEN Stephen L, et al. In Vitro Antihepadnaviral Activities of Combinations of Penciclovir, Lamivudine, and Adefovir. Antimicrobial Agents and Chemotherapy,2000,44: 551-560.
9. Chen Robert YM, Edwards Ros, Shaw Tim, et al. Effect of the G1896A Precore Mutation on Drug Sensitivity and Replication Yield of Lamivudine-Resistant HBV In Vitro. Hepatology,2003,37:27-35.
10. Pan XB, Wei L, Chen HS, et al. Liver-derived cell lines QSG-7701 and HepG2 support different HBV replication patterns. Arch Virol, 2007,152(6): 1159-1173.
11. De Meyer S, Gong ZJ, Suwandhi W,et al. Organ and species specificity of hepatitis B virus (HBV) infection: a review of literature with a special reference to preferential attachment of HBV to human hepatocytes. J Viral Hepat, 1997,4(3): 145-53.
12. Shih C, Li LS, Roychoudhury S, and Hot MH. In vitro propagation of human hepatitis B virus in a rat hepatoma cell line. Proc. Natl. Acad. Sci. USA, 1989,86: 6323-6327.
13. Hirschman SZ, Garfinkel E. of human cells in culture with hepatitis B virus. Trans Assoc Am Physicians, 1980,93:60-71.
14. Hirschman S, Price P, Garfinkel E, et al. Expression of cloned hepatitis B virus DNA in human cell cultures. Proc. Natl. Acad. Sci,USA, 1980,77:5507-5511.
15. Christman JK, Gerber M, Price P M, et al. Amplification of expression of hepatitis B surface antigen in 3T3 cells cotransfected with a dominant-acting gene and cloned viral DNA. Proc. NatL Acad. Sci. USA, 1982,79:1815-1819,
16. Takeshima H, Namiki M, Inokoshi J, et al. Stable expression of hepatitis B virus genome in a primate kidney cell. Arch Virol,1989;109(1-2):35-49
17. Kumon I, Onji M, Nadano S, et al. Detection of hepatitis B surface antigen in lymphoid follicle in patients with chronic hepatitis type B. Gastroenterol Jpn,1989,24(6):738.
18. Seifer M, Heermann KH, Gerlich WH. Expression pattern of the hepatitis B virus genome in transfected mouse fibroblasts. Virology, 1990,179( 1 ):287-299.
19. Miiller C, Bergmann KF, Gerin JL, et al. Production of hepatitis B virus by stably transfected monocytic cell line U-937: a model for extrahepatic hepatitis B virus replication. J Infect Dis, 1992,165(5):929-933.
20. Wang RY, Shen CN, Lin MH, et al. Hepatocyte-Like Cells Transdifferentiated from a Pancreatic Origin Can Support Replication of Hepatitis B Virus. Journal of Virology, 2005,79:13116-13128.
21.Galle PR. Hagelstem J, kommerell B, et al. In vitro experimental Infection of primary humane hepatocytes with hepatitis B virus.Gastroenterology, 1994,106(3): 664-673.
22. Scaglioni PP, Melegari M, Wands JR. Posttranscriptional regulation of hepatitis B virus replication by the precore protein. J Virol,1997,71(1):345-353.
23. Muller C, Bergmann KF, Gerin JL, et al. Pro-duction of hepatitis B virus by stabty transfected monocytic cell line U-937: a model for extrahepatic hepatitis B replication. J Infect Dis,1992,165(5):929-933.
24. Marie-Noelle Brunelle, Anne-Carole Jacquard, Christian Pichoud, et al. Susceptibility to antivirals of a human HBV strain with mutations conferring resistance to both lamivudine and adefovir. Hepatology,2005,411391-1398.
25. Delaney WE 4th, Isom HC. Hepatitis B virus replication in human hepG2 Cells mediated by hepatitis B virus recombination baculovirus. Hepatology, 1998, 28(4): 1134-1146.
26. Delaney WE 4th, MillerTG, Isom HC. Use of the hepatitis B virus recombinant baculovirus -hepG2 system to study the effects of (-)-2' ,3' -dideoxy 3' -thiacytidine on replication of hepatitis B virus and accumulation of covalently closed circular DNA. Antimicrob Agents Chemother,1999,43(8):2017-2026.
27. Spfinzl MF, Oberwinkler H, Schaller H, et al. Transfer of hepatitis B virus genome by adenovirus vectors into cultured cells and mice: Crossing the species barrier. J Vim1,2001,75(11):5108-5118.
28.Sun D,Nass M.Stable HepG2-and Huh7-based human hepatoma cell lines for efficient regulated expression of infectious hepatitis B virus.Journal of Hepatology,2006,45:636-645.
29.Christen V,Duong F,Bernsmeier C,et al.Inhibition of Alpha Interferon Signaling by Hepatitis B Virus.Journal of Virology,2007,81(1):159-165.
30.王松,唐红,何芳,等.肝富集转录因子在HBV基因转录与复制中的作用.四川大学学报(医学版),2006,37:35-39.
31.Zajakina A,Kozlovska T,Bruvere R,et al.Translation of hepatitis B virus(HBV)surface proteins from the HBV pregenome and precore RNAs in Semliki Forest virus-driven expression.Journal of General Virology,2004,85:3343-3351.
32.Tacke F,Gehrke C,Luedde T,et al.Basal Core Promoter and Precore utations in the Hepatitis B Virus Genome Enhance Replication Efficacy of Lamivudine-Resistant Mutants.Journal of Virology,2004,78:8524-8535.
33.Locarnini,S.,J.McMillan,and A.Bartholomeusz.The hepatitis B virus and common mutants.Semin.Liver Dis,2003,23:5-20.
34.Buckwold VE,Xu Z,Chen M,et al.Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication.J.Virol,1996,70:5845-5851.
35.Hunt CM,McGill JM,Allen M I,et al.Clinical relevance of hepatitis B viral mutations.Hepatology,2000,31:1037-1044.
1.Shlomai A,Shanl Y.Inhibition of hepatitis B virus expression and replication by RNA Interference.Hepatology,2003,37:764-770.
2.Ying CX,De CE,Neyts J.Seleetiveinhibition ofhepatitis B vires replication by RNA interference.Biochem Biophys Res Commun,2003,309:482-484.
3.Konishi M,Wu CH,Wu GY.Inhibition of HBV replication by siRNA in a stable HBV-producing cell line.Hepatolgy,2003,38:842-850.
4.H.Kayhan,E.Karatayli,A.R.Turkyilmaz,et al.Inhibition of hepatitis B virus replication by shRNAs in stably HBV expressed HEPG2.2.15 cell lines.Archives of Virology,2007,152:871-879.
5.Chen Z,Xu ZF,Ye JJ,et al.Combination of small interfering RNAs mediates greater inhibition of human hepatitis B virus replication and antigen expression.J Zhejiang Univ SCI,2005,6B(4):236-241
6.Ren XR,Zhou LJ,Luo GB,et al.Inhibition of hepatitis B virus replication in 2.2.15cells by expressed shRNA.J Viral Hepat,2005,12(3):236-242.
7.Ren X,Luo G,Xie Z,et al.Inhibition of multiple gene expression and virus replication of HBV by stable RNA interference in 2.2.15 cells.J Hepatol,2006,44(4):663-670.
8.Jia F,Zhang YZ,and Liu CM.Stable inhibition of hepatitis B virus expression and replication in HepG2.2.15 cells by RNA interference based on retrovirus delivery.J Biotechnol,2007,128(1):32-40.
9.Giladi H.Ketzinel-Gilad M,Rivkin L,et al.Small interfering RNA inhibits hepatitis B virus replication in mice.Mol Iher,2003,8(5):769-776.
10.Zhang XN,Wei X,Wang JD,et al.RNAi mediated inhibition of HBVreplication and expression. World J Gastroenteml,2004,10(20):2967-2971.
11. M cCaffrey AP, Nakai H, Pandey K, et al. Inhibition of hepatitis B virus in mice by RNA interference. Nat Biotechnol,2003,21(6):639-647.
12. Y Wu, AL Huang, N Tang, et al. Specific anti-viral effects of RNA interference on replication and expression of hepatitis B virus in mice. Chin Med J (Engl), August 20, 2005;118(16):1351-1356.
13. Susan L. Uprichard, Bryan Boyd, et al. Clearance of hepatitis B virus from the liverof transgenic mice by short hairpin RNAs. PNAS,2005,102: 773-778
14. Chen CC, Ko TM, Ma HI, et al. Long-term inhibition of hepatitis B virus in transgenic mice by double-stranded adeno-associated virus 8-delivered short hairpin RNA. Gene Ther, January 1, 2007; 14(1):11-19.
15. Tan C, Xuan BQ, Hong J, et al. RNA interference against hepatitis B virus with endoribonuclease-prepared siRNA despite of the target sequence variations.Virus Research,2007,126:172-178.