DNA双链断裂与修复：一个潜在的乙肝病毒整合的分子机制
发布时间：2018-07-23 08:14
【摘要】： 目的与逆转录病毒感染不同,乙型肝炎病毒的整合不是病毒复制所必须,HBV本身也不编码整合酶,整合过程需要宿主细胞酶系的参与。尽管如此,乙肝相关性肝癌组织标本中HBV DNA整合的检出率高达80%。大量研究显示:HBV DNA的整合能引起插入突变、DNA缺失、染色体重排甚至基因组的不稳定性。此外,整合型HBV DNA导致原癌基因的激活及抑癌基因的失活也备受研究者关注。然而,乙肝病毒整合入宿主基因组的分子机制至今尚未阐明。在过去的三十年中,学者们主要集中在寻找HBV DNA在宿主染色体上的优势整合位点以及与肿瘤发生发展相关的整合靶基因,如癌基因、抑癌基因、信号转导因子、细胞周期调控因子等。迄今为止,已有60余种基因被成功鉴定为HBV DNA整合的靶基因,这其中包括维甲酸受体(Retinoic acid receptors; RAR)、细胞周期蛋白A(cyclin A)、端粒酶催化亚单位(human telomerase reverse transcriptase; hTERT)等一些与原发性肝癌密切相关的基因。hTERT更是被认为是HBV整合的优势靶位。研究结果还表明,HBV DNA的整合可发生在除13、X和Y外的所有染色体上。鉴于HBV整合过程中呈现出来的多样性和复杂性,目前的观点多数认为HBV在染色体上的整合是随机的。这一随机性体现在两个方面:一方面,整合到宿主细胞的HBV DNA往往不是完整的病毒序列,在已描述的HBV DNA整合片段中未检测到两个完全一致的序列;另一方面,HBV DNA整合入宿主基因组的部位是随机分布的。然而,随着研究的进一步深入,有结果发现DNA损伤的诱发尤其是DNA双链断裂以及DNA修复的干预均可大大增加乙肝病毒基因组的整合频率。于是有人设想,DNA双链断裂(Double-strand break; DSB)可能是HBV DNA整合的一个潜在的靶位点。这样,人们必然要问,HBV DNA的整合是如何发生的?整合之前宿主细胞内发生了哪些事件?HBV DNA的整合可以控制吗?如果我们弄清了这些过程,就可以采取有效措施干预乙肝病毒的整合,从而从源头上预防病毒整合所致的基因组不稳定性以及肿瘤的发生。我们知道,针对DNA损伤,人体内存在着一套保持基因组完整性(integrity)和忠实性(fidelity)的DNA修复机制。研究业已表明,机体可通过同源重组(homologous recombination; HR)和非同源末端连接(non-homologous end-joining; NHEJ)两种修复途径对DSB进行修复,避免因为复制停顿而引起的细胞死亡。两种修复途径各自拥有其门控蛋白(gatekeeper),HR的gatekeeper是Rad52;NHEJ的gatekeeper是Ku70/Ku80。DSB发生后,两类门控基因竞争结合DNA断端,从而引导两种不同的修复途径。本课题旨在验证DSB是HBV DNA整合的一个潜在的优势靶点,并试图从损伤修复这一全新的视角入手,探讨两种DSB修复途径与HBV整合的关系,通过调控启动DSB两种修复途径的门控蛋白Rad52和Ku70/Ku80,促使机体采用无错(error free)的HR途径,而不采用易错(error prone)的NHEJ途径,遏制乙肝病毒的整合,力图从源头上干预病毒整合所致的基因组不稳定性及肝癌的发生。 方法应用分子克隆技术构建I-SceⅠ系统中的真核表达载体pEGFP2,将其转染人胚胎肝细胞株L-02和肝癌细胞株HepG2,G418筛选出稳定转染株,从而人为地将18个碱基的I-SceⅠ归位内切酶识别序列(5’-TAG GGA TAA CAG GGT AAT-3’)引入到细胞的基因组中。随后将I-SceⅠ系统中表达I-SceⅠ内切酶的真核表达载体pCMV-3NSL-I-SceⅠ瞬时转染到L-02和HepG2细胞中,诱发产生位点特异性的DSB细胞模型。瞬时转染后24 h,γ-H2AX识别抗体法检测DSB发生情况,巢式PCR进一步确定DSB发生在基因组I-SceⅠ识别序列处。收集同济医院肝病门诊45例慢性乙型肝炎患者血清标本(HBsAg阳性,HBV DNA107拷贝),用于制备人HBV体外感染的肝细胞模型。为了释放细胞膜上更多的LDL受体(一种介导HBV吸附、穿入细胞的受体)接受病毒颗粒,参照文献,在接种HBV血清之前除去细胞表面的绑定脂蛋白。随后将HBV血清接种到L-02和HepG2细胞中,孵育,使细胞感染乙肝病毒。感染后的细胞继续按常规培养,加入适量胰岛素和地塞米松促进病毒对宿主细胞的整合。ELISA方法检测细胞培养上清中HBsAg和HBeAg的水平,细胞于被感染的第8天(病毒整合时间),用巢式PCR方法扩增出插入到I-SceⅠ酶切位点中的核苷酸序列,胶回收纯化后直接测序,并将测得的序列与HBV基因组进行BLAST比对分析。应用siRNA在线设计工具,针对DSB两条修复途径所涉及的门控基因(Rad52、Ku70和Ku80)各选择了2个靶位点,构建了相应的siRNA表达载体(针对Rad52的psiRNA1和psiRNA2;针对Ku70的psiRNA3和psiRNA4;针对Ku80的psiRNA5和psiRNA6)以及作为阴性对照的psiRNA7。酶切鉴定和测序法确定质粒构建成功后,将其转染人肝癌细胞株HepG2。RT-PCR和Western Blot分别用来检测psiRNAs在转录水平和翻译水平干扰靶基因的效果,筛选出有效干预门控基因的siRNA用于后续研究。为了探讨门控蛋白对整合的影响,将筛选出来的psiRNA在接种HBV血清前转染肝癌细胞株HepG2,余处理同前,运用荧光显微镜和流式细胞仪检测I-SceⅠ系统中绿色荧光蛋白表达情况,从而观察干扰后细胞中HR和NHEJ的比例变化情况;Real-time PCR法检测位点特异性乙肝病毒整合,比较各试验组乙肝病毒整合量的情况。 结果酶切鉴定结果显示I-SceⅠ系统中的真核表达载体pEGFP2成功构建,将该系统引入L-02和HepG2细胞后24 h,γ-H2AX识别抗体技术(免疫荧光和免疫印迹)检测到DSB:免疫荧光标记技术结果显示γ-H2AX定位于细胞核,对照组细胞中仅有微量γ-H2AX表达, I-SceⅠ系统处理组细胞γ-H2AX表达水平显著增高;Western Blot同样显示γ-H2AX在实验组中表达明显高于对照组细胞(P 0.05);巢式PCR结果进一步证实在特定位点I-SceⅠ识别序列处发生的DSB。HBV血清接种细胞后,ELISA检测细胞培养上清中HBsAg和HBeAg的水平:接种后最初两天,L-02和HepG2细胞上清中均有较高浓度的HBsAg和HBeAg表达;随后,HBsAg和HBeAg表达明显下降,至接种后第四天,L-02细胞上清中HBsAg和HBeAg检测结果呈阴性(P/N 2.1),HepG2上清中HBsAg仍呈阳性表达,并以较低浓度维持相当长的一段时间。感染后第八天,巢式PCR产物直接测序结果经BLAST分析后获得HBV整合入HepG2细胞位点特异性DSB的直接证据。psiRNA1～psiRNA7经酶切与测序鉴定后成功导入HepG2细胞,RT-PCR结果经UVP凝胶成像分析系统分析结果显示:psiRNA1和psiRNA2作用后,HepG2细胞中Rad52 mRNA分别下降83.75%和56.50%;psiRNA3和psiRNA4对Ku70 mRNA的抑制率分别为62.45%和71.92%;psiRNA5和psiRNA6对Ku80 mRNA的抑制率为77.59%和60.41%。psiRNAs在翻译水平干预靶基因效果显示,psiRNA1和psiRNA2作用HepG2细胞后, Rad52蛋白分别下调70.92%和51.65%; psiRNA3和psiRNA4对Ku70蛋白表达的抑制率分别为54.02%和65.24%;psiRNA5和psiRNA6对Ku80蛋白的抑制率分别为67.14%和66.83%。由此可见,psiRNA1～6均可不同程度地干扰靶基因的表达。相对而言,psiRNA1、psiRNA4和psiRNA5分别较psiRNA2、psiRNA3和psiRNA6对靶基因的干预效果更佳。荧光显微镜及流式细胞仪结果显示:psiRNA1作用细胞后,EGFP表达明显低于未处理组,预示HR途径修复位点特异性DSB比例下调,而靶向Kus基因的串联shRNAs表达系统psiRNAkus(能同时表达针对Ku70的siRNA4和针对Ku80的siRNA5)处理组EGFP表达显著上调,这就意味着更多的DSB是经过HR来修复的。Real-time PCR结果观察到经psiRNAkus作用的细胞中的病毒整合量明显减少或完全缺如;反之,经psiRNA1作用的细胞中的乙肝病毒整合较未处理组明显增多。 结论DSB是一个潜在的、优势的HBV DNA整合靶位点;针对门控基因的siRNA能有效调控HR和NHEJ的修复比例; HBV DNA整合入DSB与NHEJ密切相关,通过对门控蛋白Ku70/Ku80和Rad52的调控,可达到调控HBV位点特异性整合的目的。本研究一定程度上丰富了HBV整合的分子机制,并为干预乙肝病毒整合、预防HBV DNA整合引发的基因组不稳定性及肝癌的发生提供一种全新的策略。
[Abstract]:Objective: different from retroviral infection, hepatitis B virus integration is not necessary for viral replication, and HBV itself does not encode integrase. Integration process requires the involvement of the host cell enzyme system. However, the detection rate of HBV DNA integration in HBCC specimens is up to 80%. and a large number of studies show that the integration of HBV DNA can cause the integration of HBV. Mutations, DNA deletion, chromosome rearrangement and even genomic instability. In addition, integrated HBV DNA leads to the activation of the proto oncogene and the inactivation of the tumor suppressor gene. However, the molecular mechanism of the integration of HBV into the host genome has not yet been elucidated. In the past thirty years, scholars have mainly focused on the search. To find the dominant integration site of HBV DNA on the host chromosome and the integrated target genes related to the development of tumor, such as oncogene, tumor suppressor gene, signal transduction factor, cell cycle regulator, and so on. So far, more than 60 genes have been identified as the target genes of HBV DNA integration, including Retinoic acid Receptors; RAR), Cell Cyclin A (cyclin A), telomerase catalytic subunit (human telomerase reverse transcriptase; hTERT), and other genes, which are closely related to primary liver cancer, are considered to be the dominant targets for HBV integration. In view of the diversity and complexity presented in the HBV integration process, most of the current views believe that the integration of HBV on chromosomes is random. This randomness is reflected in two aspects: on the one hand, the HBV DNA integrated into the host cell is often not a complete virus sequence, and two are not detected in the described HBV DNA integrated fragments. On the other hand, the location of HBV DNA integration into the host genome is random. However, with further research, it is found that the induction of DNA damage, especially the DNA double strand breaks and the intervention of DNA repair, can greatly increase the integration frequency of the hepatitis B virus gene group. Therefore, it is conceived that the DNA double strand breaks have been conceived. (Double-strand break; DSB) may be a potential target site for HBV DNA integration. In this way, people must ask, how does the integration of HBV DNA occur? What happens in the host cell before integration? Can the integration of HBV DNA be controlled? If we understand these processes, we can take effective measures to interfere with HBV We know that there is a set of DNA repair mechanisms for maintaining genomic integrity (integrity) and faithfulness (fidelity) in the human body for DNA damage. Research has shown that the body can be reorganized by homologous (homologous recombination;) HR) and the non homologous terminal connection (non-homologous end-joining; NHEJ) repair the DSB to avoid the cell death caused by the reproduction of the pause. The two repair pathways each own their gated protein (gatekeeper), HR gatekeeper is Rad52; NHEJ gatekeeper is the two class of gated gene competition after Ku70/Ku80.DSB. Two different repair approaches are guided by the DNA broken end. The purpose of this study is to verify that DSB is a potential advantage target for the integration of HBV DNA, and attempts to explore the relationship between the integration of the two DSB repair pathways and the integration of the DSB repair pathways from the new perspective of the damage restoration, and by regulating the gated protein Rad52 and Ku70/Ku80 to start the two repair pathways of DSB. It encourages the organism to adopt the HR pathway without error (error free), instead of using the NHEJ pathway of error prone to contain the integration of HBV, and try to interfere with the genomic instability and the occurrence of liver cancer from the source of viral integration.
Methods the eukaryotic expression vector pEGFP2 in I-Sce I system was constructed by molecular cloning technology, and transfected into the human embryo liver cell line L-02 and the liver cancer cell line HepG2, and the stable transfection strain was screened by G418, thus the 18 base I-Sce I homing endonuclease identification sequence (5 '-TAG GGA TAA CAG GGT AAT-3 ") was introduced into the cell gene artificially. In the group, the eukaryotic expression vector pCMV-3NSL-I-Sce I expressing the I-Sce I endonuclease pCMV-3NSL-I-Sce I in the I-Sce I system was transiently transfected into L-02 and HepG2 cells to induce the DSB cell model of the loci specificity. After transient transfection, 24 h, gamma -H2AX identification antibody method was used to detect the occurrence of DSB, and the nested PCR further confirmed that DSB occurred in genome I-Sce I recognition. 45 cases of chronic hepatitis B patients in Tongji Hospital liver disease clinic (HBsAg positive, HBV DNA107 copy) were used to prepare the hepatocyte model of human HBV infection in vitro. In order to release more LDL receptors on the membrane of the cell (a kind of receptor that mediates HBV adsorption, through the receptor in the cells) to accept the virus particles, and to inoculate HBV blood with reference to the literature. Before clearing away the binding lipoprotein on the surface of the cell, HBV serum was inoculated into L-02 and HepG2 cells, incubated to infect HBV. The infected cells continued to be cultured in accordance with the conventional culture, adding appropriate insulin and dexamethasone to promote the integration of the virus to the host cells by.ELISA method to detect HBsAg and HBeAg in the cell culture supernatant Level, the cells were amplified by nested PCR method in the eighth day of infection (virus integration time), and the nucleotide sequences inserted into the I-Sce I enzyme cut site were amplified by the nested PCR method. After the gel was recovered and purified, the sequence was compared with the BLAST of the HBV genome. The siRNA online design tool was applied to the doors involved in the two repair pathways of DSB. The control genes (Rad52, Ku70 and Ku80) each selected 2 target loci, and constructed a corresponding siRNA expression vector (psiRNA1 and psiRNA2 for Rad52, psiRNA3 and psiRNA4 for Ku70, Ku80 psiRNA5 and sequencing for Ku80), and the successful transfection of the human hepatoma cells after the identification and sequencing method as negative control. HepG2.RT-PCR and Western Blot were used to detect the effect of psiRNAs at the transcriptional level and translation level to interfere with the target gene, and to screen out the effective intervention gene siRNA for the follow-up study. In order to explore the effect of the gated protein on the integration, the screened psiRNA was transfected to the liver cancer cell line HepG2 before inoculated with HBV sera. Before, the expression of green fluorescent protein (GFP) in I-Sce I system was detected by fluorescence microscopy and flow cytometry, and the changes in the proportion of HR and NHEJ in the cells after interference were observed, and the Real-time PCR method was used to detect the integration of HBV, and the hepatitis B virus integration was compared in the experimental groups.
Results the results of enzyme digestion showed that the eukaryotic expression vector pEGFP2 in I-Sce I system was successfully constructed, and the system was introduced into L-02 and HepG2 cells after 24 h, and gamma -H2AX identification antibody technique (immunofluorescence and immunoblotting) detected by DSB: immunofluorescence technique, the results showed that gamma -H2AX was located in the nucleus, and only a small amount of gamma -H2AX in the control group. The expression of gamma -H2AX in the I-Sce I system treated group was significantly higher, and Western Blot also showed that the expression of gamma -H2AX in the experimental group was significantly higher than that of the control group (P 0.05). The nested PCR results further confirmed that the ELISA detection of the cell culture supernatant was HBs after the positive specific location point I-Sce I identification sequence occurred. Level of Ag and HBeAg: high concentrations of HBsAg and HBeAg were expressed in L-02 and HepG2 cell supernatants at the first two days after inoculation. Subsequently, the expression of HBsAg and HBeAg decreased significantly, and the results of HBsAg and HBeAg in the L-02 cell supernatant were negative (P/N 2.1) at the fourth day after inoculation (P/N 2.1), and maintained at a lower concentration and maintained at a lower concentration. After eighth days of infection, the direct sequencing of the nested PCR products after BLAST analysis obtained the direct evidence of HBV integration into the specific DSB of the HepG2 cell site,.PsiRNA1 ~ psiRNA7 was successfully introduced into HepG2 cells after enzyme digestion and sequencing, and the results of RT-PCR results by UVP gel imaging analysis showed: psiRNA1 and psi. After the action of RNA2, the Rad52 mRNA in HepG2 cells decreased by 83.75% and 56.50%, respectively, and the inhibition rates of psiRNA3 and psiRNA4 on Ku70 mRNA were 62.45% and 71.92%, respectively, the inhibition rate of psiRNA5 and psiRNA6 to Ku80 mRNA was 77.59%. The inhibition rates of psiRNA3 and psiRNA4 on the expression of Ku70 protein were 54.02% and 65.24%, and the inhibition rates of psiRNA5 and psiRNA6 to Ku80 protein were 67.14% and 66.83%., respectively. PsiRNA1 ~ 6 could interfere with the expression of target genes in varying degrees. The effect of siRNA6 on the target gene was better. The results of fluorescence microscopy and flow cytometry showed that the expression of EGFP was significantly lower than that of the untreated group after psiRNA1 action cells, indicating that the specific DSB ratio of the HR pathway repair site was down, and the shRNAs expression system psiRNAkus of the target Kus gene (can also express siRNA4 for Ku70 and aimed at Ku80. " SiRNA5) the expression of EGFP in the treatment group was significantly up-regulated, which meant that more DSB was the.Real-time PCR repaired by HR to observe that the viral integration in the cells treated by psiRNAkus was significantly reduced or completely absent, whereas the integration of HBV in psiRNA1 affected cells was significantly higher than that in the untreated group.
Conclusion DSB is a potential, dominant HBV DNA integration target site, and siRNA for gated gene can effectively regulate the ratio of HR and NHEJ. The integration of HBV DNA into DSB and NHEJ is closely related to the regulation of Ku70/Ku80 and Rad52. The purpose of this study is to regulate the specific integration of the locus. The molecular mechanism of integration will provide a new strategy for intervention of hepatitis B virus integration, prevention of genomic instability caused by HBV DNA integration and occurrence of liver cancer.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R373
本文编号：2138765
[Abstract]:Objective: different from retroviral infection, hepatitis B virus integration is not necessary for viral replication, and HBV itself does not encode integrase. Integration process requires the involvement of the host cell enzyme system. However, the detection rate of HBV DNA integration in HBCC specimens is up to 80%. and a large number of studies show that the integration of HBV DNA can cause the integration of HBV. Mutations, DNA deletion, chromosome rearrangement and even genomic instability. In addition, integrated HBV DNA leads to the activation of the proto oncogene and the inactivation of the tumor suppressor gene. However, the molecular mechanism of the integration of HBV into the host genome has not yet been elucidated. In the past thirty years, scholars have mainly focused on the search. To find the dominant integration site of HBV DNA on the host chromosome and the integrated target genes related to the development of tumor, such as oncogene, tumor suppressor gene, signal transduction factor, cell cycle regulator, and so on. So far, more than 60 genes have been identified as the target genes of HBV DNA integration, including Retinoic acid Receptors; RAR), Cell Cyclin A (cyclin A), telomerase catalytic subunit (human telomerase reverse transcriptase; hTERT), and other genes, which are closely related to primary liver cancer, are considered to be the dominant targets for HBV integration. In view of the diversity and complexity presented in the HBV integration process, most of the current views believe that the integration of HBV on chromosomes is random. This randomness is reflected in two aspects: on the one hand, the HBV DNA integrated into the host cell is often not a complete virus sequence, and two are not detected in the described HBV DNA integrated fragments. On the other hand, the location of HBV DNA integration into the host genome is random. However, with further research, it is found that the induction of DNA damage, especially the DNA double strand breaks and the intervention of DNA repair, can greatly increase the integration frequency of the hepatitis B virus gene group. Therefore, it is conceived that the DNA double strand breaks have been conceived. (Double-strand break; DSB) may be a potential target site for HBV DNA integration. In this way, people must ask, how does the integration of HBV DNA occur? What happens in the host cell before integration? Can the integration of HBV DNA be controlled? If we understand these processes, we can take effective measures to interfere with HBV We know that there is a set of DNA repair mechanisms for maintaining genomic integrity (integrity) and faithfulness (fidelity) in the human body for DNA damage. Research has shown that the body can be reorganized by homologous (homologous recombination;) HR) and the non homologous terminal connection (non-homologous end-joining; NHEJ) repair the DSB to avoid the cell death caused by the reproduction of the pause. The two repair pathways each own their gated protein (gatekeeper), HR gatekeeper is Rad52; NHEJ gatekeeper is the two class of gated gene competition after Ku70/Ku80.DSB. Two different repair approaches are guided by the DNA broken end. The purpose of this study is to verify that DSB is a potential advantage target for the integration of HBV DNA, and attempts to explore the relationship between the integration of the two DSB repair pathways and the integration of the DSB repair pathways from the new perspective of the damage restoration, and by regulating the gated protein Rad52 and Ku70/Ku80 to start the two repair pathways of DSB. It encourages the organism to adopt the HR pathway without error (error free), instead of using the NHEJ pathway of error prone to contain the integration of HBV, and try to interfere with the genomic instability and the occurrence of liver cancer from the source of viral integration.
Methods the eukaryotic expression vector pEGFP2 in I-Sce I system was constructed by molecular cloning technology, and transfected into the human embryo liver cell line L-02 and the liver cancer cell line HepG2, and the stable transfection strain was screened by G418, thus the 18 base I-Sce I homing endonuclease identification sequence (5 '-TAG GGA TAA CAG GGT AAT-3 ") was introduced into the cell gene artificially. In the group, the eukaryotic expression vector pCMV-3NSL-I-Sce I expressing the I-Sce I endonuclease pCMV-3NSL-I-Sce I in the I-Sce I system was transiently transfected into L-02 and HepG2 cells to induce the DSB cell model of the loci specificity. After transient transfection, 24 h, gamma -H2AX identification antibody method was used to detect the occurrence of DSB, and the nested PCR further confirmed that DSB occurred in genome I-Sce I recognition. 45 cases of chronic hepatitis B patients in Tongji Hospital liver disease clinic (HBsAg positive, HBV DNA107 copy) were used to prepare the hepatocyte model of human HBV infection in vitro. In order to release more LDL receptors on the membrane of the cell (a kind of receptor that mediates HBV adsorption, through the receptor in the cells) to accept the virus particles, and to inoculate HBV blood with reference to the literature. Before clearing away the binding lipoprotein on the surface of the cell, HBV serum was inoculated into L-02 and HepG2 cells, incubated to infect HBV. The infected cells continued to be cultured in accordance with the conventional culture, adding appropriate insulin and dexamethasone to promote the integration of the virus to the host cells by.ELISA method to detect HBsAg and HBeAg in the cell culture supernatant Level, the cells were amplified by nested PCR method in the eighth day of infection (virus integration time), and the nucleotide sequences inserted into the I-Sce I enzyme cut site were amplified by the nested PCR method. After the gel was recovered and purified, the sequence was compared with the BLAST of the HBV genome. The siRNA online design tool was applied to the doors involved in the two repair pathways of DSB. The control genes (Rad52, Ku70 and Ku80) each selected 2 target loci, and constructed a corresponding siRNA expression vector (psiRNA1 and psiRNA2 for Rad52, psiRNA3 and psiRNA4 for Ku70, Ku80 psiRNA5 and sequencing for Ku80), and the successful transfection of the human hepatoma cells after the identification and sequencing method as negative control. HepG2.RT-PCR and Western Blot were used to detect the effect of psiRNAs at the transcriptional level and translation level to interfere with the target gene, and to screen out the effective intervention gene siRNA for the follow-up study. In order to explore the effect of the gated protein on the integration, the screened psiRNA was transfected to the liver cancer cell line HepG2 before inoculated with HBV sera. Before, the expression of green fluorescent protein (GFP) in I-Sce I system was detected by fluorescence microscopy and flow cytometry, and the changes in the proportion of HR and NHEJ in the cells after interference were observed, and the Real-time PCR method was used to detect the integration of HBV, and the hepatitis B virus integration was compared in the experimental groups.
Results the results of enzyme digestion showed that the eukaryotic expression vector pEGFP2 in I-Sce I system was successfully constructed, and the system was introduced into L-02 and HepG2 cells after 24 h, and gamma -H2AX identification antibody technique (immunofluorescence and immunoblotting) detected by DSB: immunofluorescence technique, the results showed that gamma -H2AX was located in the nucleus, and only a small amount of gamma -H2AX in the control group. The expression of gamma -H2AX in the I-Sce I system treated group was significantly higher, and Western Blot also showed that the expression of gamma -H2AX in the experimental group was significantly higher than that of the control group (P 0.05). The nested PCR results further confirmed that the ELISA detection of the cell culture supernatant was HBs after the positive specific location point I-Sce I identification sequence occurred. Level of Ag and HBeAg: high concentrations of HBsAg and HBeAg were expressed in L-02 and HepG2 cell supernatants at the first two days after inoculation. Subsequently, the expression of HBsAg and HBeAg decreased significantly, and the results of HBsAg and HBeAg in the L-02 cell supernatant were negative (P/N 2.1) at the fourth day after inoculation (P/N 2.1), and maintained at a lower concentration and maintained at a lower concentration. After eighth days of infection, the direct sequencing of the nested PCR products after BLAST analysis obtained the direct evidence of HBV integration into the specific DSB of the HepG2 cell site,.PsiRNA1 ~ psiRNA7 was successfully introduced into HepG2 cells after enzyme digestion and sequencing, and the results of RT-PCR results by UVP gel imaging analysis showed: psiRNA1 and psi. After the action of RNA2, the Rad52 mRNA in HepG2 cells decreased by 83.75% and 56.50%, respectively, and the inhibition rates of psiRNA3 and psiRNA4 on Ku70 mRNA were 62.45% and 71.92%, respectively, the inhibition rate of psiRNA5 and psiRNA6 to Ku80 mRNA was 77.59%. The inhibition rates of psiRNA3 and psiRNA4 on the expression of Ku70 protein were 54.02% and 65.24%, and the inhibition rates of psiRNA5 and psiRNA6 to Ku80 protein were 67.14% and 66.83%., respectively. PsiRNA1 ~ 6 could interfere with the expression of target genes in varying degrees. The effect of siRNA6 on the target gene was better. The results of fluorescence microscopy and flow cytometry showed that the expression of EGFP was significantly lower than that of the untreated group after psiRNA1 action cells, indicating that the specific DSB ratio of the HR pathway repair site was down, and the shRNAs expression system psiRNAkus of the target Kus gene (can also express siRNA4 for Ku70 and aimed at Ku80. " SiRNA5) the expression of EGFP in the treatment group was significantly up-regulated, which meant that more DSB was the.Real-time PCR repaired by HR to observe that the viral integration in the cells treated by psiRNAkus was significantly reduced or completely absent, whereas the integration of HBV in psiRNA1 affected cells was significantly higher than that in the untreated group.
Conclusion DSB is a potential, dominant HBV DNA integration target site, and siRNA for gated gene can effectively regulate the ratio of HR and NHEJ. The integration of HBV DNA into DSB and NHEJ is closely related to the regulation of Ku70/Ku80 and Rad52. The purpose of this study is to regulate the specific integration of the locus. The molecular mechanism of integration will provide a new strategy for intervention of hepatitis B virus integration, prevention of genomic instability caused by HBV DNA integration and occurrence of liver cancer.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R373
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