【作者】 李晓光；

【导师】 王慧芬； 成军；

【作者基本信息】 中国人民解放军军医进修学院 ， 内科学， 2008， 博士

【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8log₁₀;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。更多还原

【Abstract】 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.Methods1. 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 expression3. 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.更多还原