DNA雙鏈斷裂與修復(fù)：一個潛在的乙肝病毒整合的分子機制
發(fā)布時間：2018-07-23 08:14
【摘要】： 目的與逆轉(zhuǎn)錄病毒感染不同,乙型肝炎病毒的整合不是病毒復(fù)制所必須,HBV本身也不編碼整合酶,整合過程需要宿主細胞酶系的參與。盡管如此,乙肝相關(guān)性肝癌組織標本中HBV DNA整合的檢出率高達80%。大量研究顯示:HBV DNA的整合能引起插入突變、DNA缺失、染色體重排甚至基因組的不穩(wěn)定性。此外,整合型HBV DNA導致原癌基因的激活及抑癌基因的失活也備受研究者關(guān)注。然而,乙肝病毒整合入宿主基因組的分子機制至今尚未闡明。在過去的三十年中,學者們主要集中在尋找HBV DNA在宿主染色體上的優(yōu)勢整合位點以及與腫瘤發(fā)生發(fā)展相關(guān)的整合靶基因,如癌基因、抑癌基因、信號轉(zhuǎn)導因子、細胞周期調(diào)控因子等。迄今為止,已有60余種基因被成功鑒定為HBV DNA整合的靶基因,這其中包括維甲酸受體(Retinoic acid receptors; RAR)、細胞周期蛋白A(cyclin A)、端粒酶催化亞單位(human telomerase reverse transcriptase; hTERT)等一些與原發(fā)性肝癌密切相關(guān)的基因。hTERT更是被認為是HBV整合的優(yōu)勢靶位。研究結(jié)果還表明,HBV DNA的整合可發(fā)生在除13、X和Y外的所有染色體上。鑒于HBV整合過程中呈現(xiàn)出來的多樣性和復(fù)雜性,目前的觀點多數(shù)認為HBV在染色體上的整合是隨機的。這一隨機性體現(xiàn)在兩個方面:一方面,整合到宿主細胞的HBV DNA往往不是完整的病毒序列,在已描述的HBV DNA整合片段中未檢測到兩個完全一致的序列;另一方面,HBV DNA整合入宿主基因組的部位是隨機分布的。然而,隨著研究的進一步深入,有結(jié)果發(fā)現(xiàn)DNA損傷的誘發(fā)尤其是DNA雙鏈斷裂以及DNA修復(fù)的干預(yù)均可大大增加乙肝病毒基因組的整合頻率。于是有人設(shè)想,DNA雙鏈斷裂(Double-strand break; DSB)可能是HBV DNA整合的一個潛在的靶位點。這樣,人們必然要問,HBV DNA的整合是如何發(fā)生的?整合之前宿主細胞內(nèi)發(fā)生了哪些事件?HBV DNA的整合可以控制嗎?如果我們弄清了這些過程,就可以采取有效措施干預(yù)乙肝病毒的整合,從而從源頭上預(yù)防病毒整合所致的基因組不穩(wěn)定性以及腫瘤的發(fā)生。我們知道,針對DNA損傷,人體內(nèi)存在著一套保持基因組完整性(integrity)和忠實性(fidelity)的DNA修復(fù)機制。研究業(yè)已表明,機體可通過同源重組(homologous recombination; HR)和非同源末端連接(non-homologous end-joining; NHEJ)兩種修復(fù)途徑對DSB進行修復(fù),避免因為復(fù)制停頓而引起的細胞死亡。兩種修復(fù)途徑各自擁有其門控蛋白(gatekeeper),HR的gatekeeper是Rad52;NHEJ的gatekeeper是Ku70/Ku80。DSB發(fā)生后,兩類門控基因競爭結(jié)合DNA斷端,從而引導兩種不同的修復(fù)途徑。本課題旨在驗證DSB是HBV DNA整合的一個潛在的優(yōu)勢靶點,并試圖從損傷修復(fù)這一全新的視角入手,探討兩種DSB修復(fù)途徑與HBV整合的關(guān)系,通過調(diào)控啟動DSB兩種修復(fù)途徑的門控蛋白Rad52和Ku70/Ku80,促使機體采用無錯(error free)的HR途徑,而不采用易錯(error prone)的NHEJ途徑,遏制乙肝病毒的整合,力圖從源頭上干預(yù)病毒整合所致的基因組不穩(wěn)定性及肝癌的發(fā)生。 方法應(yīng)用分子克隆技術(shù)構(gòu)建I-SceⅠ系統(tǒng)中的真核表達載體pEGFP2,將其轉(zhuǎn)染人胚胎肝細胞株L-02和肝癌細胞株HepG2,G418篩選出穩(wěn)定轉(zhuǎn)染株,從而人為地將18個堿基的I-SceⅠ歸位內(nèi)切酶識別序列(5’-TAG GGA TAA CAG GGT AAT-3’)引入到細胞的基因組中。隨后將I-SceⅠ系統(tǒng)中表達I-SceⅠ內(nèi)切酶的真核表達載體pCMV-3NSL-I-SceⅠ瞬時轉(zhuǎn)染到L-02和HepG2細胞中,誘發(fā)產(chǎn)生位點特異性的DSB細胞模型。瞬時轉(zhuǎn)染后24 h,γ-H2AX識別抗體法檢測DSB發(fā)生情況,巢式PCR進一步確定DSB發(fā)生在基因組I-SceⅠ識別序列處。收集同濟醫(yī)院肝病門診45例慢性乙型肝炎患者血清標本(HBsAg陽性,HBV DNA107拷貝),用于制備人HBV體外感染的肝細胞模型。為了釋放細胞膜上更多的LDL受體(一種介導HBV吸附、穿入細胞的受體)接受病毒顆粒,參照文獻,在接種HBV血清之前除去細胞表面的綁定脂蛋白。隨后將HBV血清接種到L-02和HepG2細胞中,孵育,使細胞感染乙肝病毒。感染后的細胞繼續(xù)按常規(guī)培養(yǎng),加入適量胰島素和地塞米松促進病毒對宿主細胞的整合。ELISA方法檢測細胞培養(yǎng)上清中HBsAg和HBeAg的水平,細胞于被感染的第8天(病毒整合時間),用巢式PCR方法擴增出插入到I-SceⅠ酶切位點中的核苷酸序列,膠回收純化后直接測序,并將測得的序列與HBV基因組進行BLAST比對分析。應(yīng)用siRNA在線設(shè)計工具,針對DSB兩條修復(fù)途徑所涉及的門控基因(Rad52、Ku70和Ku80)各選擇了2個靶位點,構(gòu)建了相應(yīng)的siRNA表達載體(針對Rad52的psiRNA1和psiRNA2;針對Ku70的psiRNA3和psiRNA4;針對Ku80的psiRNA5和psiRNA6)以及作為陰性對照的psiRNA7。酶切鑒定和測序法確定質(zhì)粒構(gòu)建成功后,將其轉(zhuǎn)染人肝癌細胞株HepG2。RT-PCR和Western Blot分別用來檢測psiRNAs在轉(zhuǎn)錄水平和翻譯水平干擾靶基因的效果,篩選出有效干預(yù)門控基因的siRNA用于后續(xù)研究。為了探討門控蛋白對整合的影響,將篩選出來的psiRNA在接種HBV血清前轉(zhuǎn)染肝癌細胞株HepG2,余處理同前,運用熒光顯微鏡和流式細胞儀檢測I-SceⅠ系統(tǒng)中綠色熒光蛋白表達情況,從而觀察干擾后細胞中HR和NHEJ的比例變化情況;Real-time PCR法檢測位點特異性乙肝病毒整合,比較各試驗組乙肝病毒整合量的情況。 結(jié)果酶切鑒定結(jié)果顯示I-SceⅠ系統(tǒng)中的真核表達載體pEGFP2成功構(gòu)建,將該系統(tǒng)引入L-02和HepG2細胞后24 h,γ-H2AX識別抗體技術(shù)(免疫熒光和免疫印跡)檢測到DSB:免疫熒光標記技術(shù)結(jié)果顯示γ-H2AX定位于細胞核,對照組細胞中僅有微量γ-H2AX表達, I-SceⅠ系統(tǒng)處理組細胞γ-H2AX表達水平顯著增高;Western Blot同樣顯示γ-H2AX在實驗組中表達明顯高于對照組細胞(P 0.05);巢式PCR結(jié)果進一步證實在特定位點I-SceⅠ識別序列處發(fā)生的DSB。HBV血清接種細胞后,ELISA檢測細胞培養(yǎng)上清中HBsAg和HBeAg的水平:接種后最初兩天,L-02和HepG2細胞上清中均有較高濃度的HBsAg和HBeAg表達;隨后,HBsAg和HBeAg表達明顯下降,至接種后第四天,L-02細胞上清中HBsAg和HBeAg檢測結(jié)果呈陰性(P/N 2.1),HepG2上清中HBsAg仍呈陽性表達,并以較低濃度維持相當長的一段時間。感染后第八天,巢式PCR產(chǎn)物直接測序結(jié)果經(jīng)BLAST分析后獲得HBV整合入HepG2細胞位點特異性DSB的直接證據(jù)。psiRNA1～psiRNA7經(jīng)酶切與測序鑒定后成功導入HepG2細胞,RT-PCR結(jié)果經(jīng)UVP凝膠成像分析系統(tǒng)分析結(jié)果顯示:psiRNA1和psiRNA2作用后,HepG2細胞中Rad52 mRNA分別下降83.75%和56.50%;psiRNA3和psiRNA4對Ku70 mRNA的抑制率分別為62.45%和71.92%;psiRNA5和psiRNA6對Ku80 mRNA的抑制率為77.59%和60.41%。psiRNAs在翻譯水平干預(yù)靶基因效果顯示,psiRNA1和psiRNA2作用HepG2細胞后, Rad52蛋白分別下調(diào)70.92%和51.65%; psiRNA3和psiRNA4對Ku70蛋白表達的抑制率分別為54.02%和65.24%;psiRNA5和psiRNA6對Ku80蛋白的抑制率分別為67.14%和66.83%。由此可見,psiRNA1～6均可不同程度地干擾靶基因的表達。相對而言,psiRNA1、psiRNA4和psiRNA5分別較psiRNA2、psiRNA3和psiRNA6對靶基因的干預(yù)效果更佳。熒光顯微鏡及流式細胞儀結(jié)果顯示:psiRNA1作用細胞后,EGFP表達明顯低于未處理組,預(yù)示HR途徑修復(fù)位點特異性DSB比例下調(diào),而靶向Kus基因的串聯(lián)shRNAs表達系統(tǒng)psiRNAkus(能同時表達針對Ku70的siRNA4和針對Ku80的siRNA5)處理組EGFP表達顯著上調(diào),這就意味著更多的DSB是經(jīng)過HR來修復(fù)的。Real-time PCR結(jié)果觀察到經(jīng)psiRNAkus作用的細胞中的病毒整合量明顯減少或完全缺如;反之,經(jīng)psiRNA1作用的細胞中的乙肝病毒整合較未處理組明顯增多。 結(jié)論DSB是一個潛在的、優(yōu)勢的HBV DNA整合靶位點;針對門控基因的siRNA能有效調(diào)控HR和NHEJ的修復(fù)比例; HBV DNA整合入DSB與NHEJ密切相關(guān),通過對門控蛋白Ku70/Ku80和Rad52的調(diào)控,可達到調(diào)控HBV位點特異性整合的目的。本研究一定程度上豐富了HBV整合的分子機制,并為干預(yù)乙肝病毒整合、預(yù)防HBV DNA整合引發(fā)的基因組不穩(wěn)定性及肝癌的發(fā)生提供一種全新的策略。
[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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R373
本文編號：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.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R373
【參考文獻】
相關(guān)期刊論文 前4條
1 杜荔菁,林菊生,李紹白,陳春蓮;肝細胞癌p53蛋白過表達的免疫組織化學研究[J];中國組織化學與細胞化學雜志;1999年04期
2 甘人寶,儲美瑾,沈綠萍,錢蘇雯,李載平;克隆的adr亞型乙型肝炎病毒(pADR-1)DNA的全順序[J];中國科學(B輯 化學 生物學 農(nóng)學 醫(yī)學 地學);1986年01期
3 李雨,趙芳,鄭秀龍,羅成基,蔡建明;WT對輻射致DNA損傷修復(fù)的影響[J];中華放射醫(yī)學與防護雜志;2000年03期
4 顧健人,胡利富,萬大方,田培坤,郭嬋,黃樂泓;人原發(fā)性肝癌轉(zhuǎn)化基因——N-ras的研究[J];腫瘤;1985年02期
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