【作者】 马红梅；

【导师】 肖洪广；

【作者基本信息】 广州医学院 ， 临床检验诊断学， 2011， 硕士

【摘要】 流感病毒感染是严重威胁人类健康的最常见的原因之一。1930年R.E.Shope成功的用病猪呼吸道过滤液感染雪貂,分离出了猪流感病毒[1]。1933年Smith等人将首次从人体中分离到的病毒称甲型流感病毒[2]。历史上第一次甲型流感大流行在1918年-1919年造成2000-4000万人死亡[3-4],本世纪初2009年的这次H1N1型流感病毒侵袭人类并造成世界范围内的广泛流行。流感病毒致病机制的研究一直都是医学研究的热点。2010年,刘彦明等[5]用免疫组织化学染色法检测病毒感染狗肾转化细胞(MDCK)之后细胞DNA鸟嘌呤的氧化损伤产物8-氧鸟嘌呤脱氧核苷(8-oxo-7,8-dihydroguanine,8-oxo-dG)的表达,实验结果证实甲型H1N1 (Influenza A)流感病毒感染能造成宿主细胞DNA氧化损伤,氧化应激的后果不仅是造成蛋白质、脂质的氧化,还会造成核酸的氧化。另一研究表明H1N1型流感病毒能明显诱导宿主MDCK细胞凋亡,凋亡率随着检测时间推移升高[41]。在各种碱基中,鸟嘌呤最易被氧化成8-氧鸟嘌呤脱氧鸟苷(8-oxo-dG)[6]。DNA上的8-oxo-dG既可以由于原位氧化,也可由复制过程8-oxo-dGTP的错误掺入而致。人类细胞内DNA的低氧化状态维持依赖多种基因修复酶,其中8_羟基鸟嘌呤核苷酸酶(MutT homologue 1,MTH1)可以水解核苷酸代谢池中的8-oxo-dGTP为8-oxo-dGMP,阻断错误掺入过程[7];而8-羟基鸟嘌呤DNA糖苷酶(8-oxo-guanine DNA glycosylase1,OGG1)能够清除DNA上无论是由于原位氧化还是错误掺入而形成的8-oxo-dG[8]。两种酶在不同阶段的共同作用预防DNA的氧化或减轻DNA的氧化损伤,从而使DNA的正常低氧化状态得以维持。2008年2月份DNA Repair杂志刊登Hill的文章,作者发现氧化应激所致的培养细胞DNA过度氧化与OGG1的降解有相关性[9]。同时,我们的前期研究结果表明流感病毒感染靶细胞后,会导致靶细胞内氧化损伤标志物8-oxo-dG表达量增加,提示了细胞发生氧化应激损伤,最终引起靶细胞凋亡,根据上述研究进展,我们认为氧化损伤修复酶参与DNA过度氧化修复过程,可能在流感病毒致宿主细胞凋亡发生发展的过程中起到一定的作用。目前国际上对流感病毒感染后宿主细胞DNA氧化损伤机制的研究鲜有报道,本研究将从DNA氧化损伤修复方面探讨流感病毒致病机制。目的本课题在流感病毒感染宿主细胞可导致脂质氧化损伤和DNA碱基氧化损伤的基础上,进一步探讨其可能的作用机制。揭示流感病毒致宿主细胞的氧化损伤在宿主细胞凋亡中的作用,即DNA的过度氧化损伤而氧化修复酶的表达不足以对抗过高的氧化损伤,最终导致宿主细胞的凋亡和症状的发生发展。本研究旨在加强对流感病毒致宿主细胞氧化应激机制的认识,为该病毒致病机理研究提供了新的思路,为抗流感药物的开发提供新的靶点,也为高致病性人禽流感的预防、防控提供了新的视角和依据。方法第一部分流感病毒感染细胞模型的建立采用细胞培养技术培养MDCK细胞。鸡胚尿囊腔接种H1N1型流感病毒,经病毒复制、扩增、收获病毒,通过检测MDCK细胞的半数组织细胞感染剂量(TCID50),确定该毒株实验用毒剂量。第二部分流感病毒H1N1感染细胞后MTH1基因表达的研究细胞培养后提取总RNA,经优化RT-PCR反应检测细胞内MTH1基因的表达量。条带经Scion Immage软件分析并与内参基因GAPDH相比,对体系的准确性、重复性和灵敏度进行分析。MDCK细胞培养后,H1N1型流感病毒感染0 h、1 h、3 h、6 h、12 h、24 h、48 h,在各时间点进行检测:经RT-PCR反应检测细胞内MTH1基因的相对表达量。第三部分流感病毒H1N1感染细胞后OGG1基因的表达的研究MDCK培养后提取RNA,反转录成cDNA,设计引物进行聚合酶链式反应对目的基因OGG1进行扩增同时条带经Image J软件分析并与扩增的内参基因GAPDH相对比,检测体系的准确性、重复性。MDCK细胞培养后,流感病毒感染0 h、1 h、3 h、6 h、12 h、24 h、48 h,在各时间点进行检测:采用PCR仪检测细胞OGG1基因的表达量变化。结果1.成功培养MDCK细胞,同时培养H1N1流感病毒,测得该毒株感染MDCK细胞的TCID50为10-3.91/0.1ml,实验用毒剂量为100 TCID50。2.(1)扩增产物经测序分析证实与GenBank中MTH1基因序列一致;(2)灵敏度检测发现最低可从50 ng总RNA中检出目的基因的表达。(3)重复性测定发现,MDCK细胞MTH1与GAPDH灰度值比值的均值为2.02±0.09 (n=10),CV值为4.31%。甲型H1N1流感病毒感染后0 h、6 h、12 h,MTH1基因的表达量与正常对照组无显著性差别,感染后1 h、3 h小时MTH1基因的表达量显著高于正常对照组,感染后24 h、48 h表达量显著低于正常对照组。3.引物设计合理,能够扩增出内参基因和目的基因,扩增产物经测序分析证实与GenBank中OGG1基因序列一致。重复性测定发现,MDCK细胞OGG1与GAPDH表达灰度值比值的均值为0.84±0.025 (n=6),CV值为2.99%。甲型H1N1流感病毒感染后0 h、48 h OGG1基因的基因的表达量与正常对照组无显著性差别,感染后1 h、3 h、6 h、12 h、24 h、OGG1基因的表达量显著高于正常对照组。结论经研究发现:MTH1基因表达的半定量RT-PCR检测方法的特异性扩增条带的片段大小不仅与预期一致,其碱基序列也与GenBanK序列完全匹配,而阴性对照无阳性扩增,证实本方法具有极好的特异性,其阳性完全为MTH1 mRNA的RT-PCR产物。对其重复性检测则证明本体系具有较好的重复性。综上,本体系具有极好的准确性、较高的灵敏度和重复性,而且对设备要求低、操作简便。这一体系的建立对开展细胞内MTH1 mRNA表达研究、尤其是流感病毒感染相关研究奠定良好基础。RT-PCR检测流感病毒H1N1感染细胞后MTH1基因的表达量的变化研究发现,与对照组比较氧化损伤修复基因MTH1在换病毒液一小时后表达量有所上调持续到更换维持液后3h,到6h后开始下降到正常水平持续至12h,到实验监测点的24h及48h MTH1的表达显著低于对照组。MTH1可以保护DNA避免受到氧化损害,说明在病毒感染初期细胞发生氧化损伤后及时启动MTH1的上调来增加抗氧化能力,而到病毒感染细胞的后期阶段细胞凋亡的增加,细胞MTH1的表达受到抑制,DNA氧化修复能力下降,随着感染所至损伤的不断加深,氧化应激产物的增加形成恶性循环从而促使细胞死亡。方法学研究发现OGG1基因的扩增片段其大小与预期一致,测序结果也与GenBanK序列完全匹配, H1N1流感病毒感染细胞的OGG1基因mRNA表达水平从换病毒维持液1h到24h持续增加,OGG1基因的上调可增加细胞对DNA氧化损伤的修复能力,到48小时降低,可能是此时凋亡因素占据上峰OGG1基因表达下降,在流感发生和防御机制中起到作用。而此前, 2009年柯跃斌等,利用H2O2作用于Ⅱ型人类肺泡上皮细胞A549发现:hOGG1 mRNA的表达水平也因H2O2暴露而24h内均呈上升趋势。本实验研究从氧化应激角度探索流感病毒,加深对该病毒致宿主细胞氧化应激机制的认识。更多还原

【Abstract】 BackgroundInfluenza infection is one common factor that seriously impacts human health. 1930 R.E.Shope successfully isolated flu virus by infecting ferrets with the swine respiratory filtered liquid[1]. 1933 Smith first named the virus isolated from the human body influenza A virus [2]. The first influenza pandemics caused 2000-4000 million deaths in 1918 -1919 [3,4]。In the beginning of this century, the 2009 H1N1 influenza viruses attacked human and caused a world-wide pandemic. The pathogenesis of influenza virus infection has been the focus of medical research.In 2010,LiuYan-ming [5] use Immunohistochemistry method to investigate expression of biochemical marker 8-hydroxy-2′-desoxyguanosine (8-OXO-dG) of DNA damage in MDCK cell after infected with H1N1 influenza virus.Results indicated that H1N1 influenza virus infection can induce significant DNA oxidative damage.Oxidative stress not only caused oxidation of protein and lipid, but also made nucleic acids be oxidated. In another study, Results showed that H1N1 influenza virus can significantly induce apoptosis of the host MDCK and with the time going, the apoptosis rates increased apparently [41]. The guanine can be oxidized in situ, or replaced into genome in the form of 8-oxo-dG during DNA replication. In human cells, the maintenance of a low oxidation state depend on multiple gene repair enzymes. For example, MTH1 can hydrolyze 8-oxo-dGTP to 8-oxo-dGMP, and block error incorporation process [7]; OGG1 can remove 8-oxo-dG that is formed in situ or incorporated during DNA replication from genome [8]. The two enzymes cooperate in different stages to prevent or reduce oxidative damage of DNA, so that the normal genome DNA can be maintained in a low oxidation state. Published in DNA Repair in February 2008, Hill found that over-oxidation of DNA in cultured cells was related to the degradation of OGG1[9]. Our preliminary results indicate that the quantity of 8-oxo-dG within target cells would increase after infected with influenza viru, which suggested that oxidative stress ultimately lead to apoptosis of target cells. According to the research progress, we believe that Oxidative damage repair enzymes participate over-oxidation DNA repair processe, this may play a role during the apoptosis of host cells infected by influenza virus. It has been rarely reported that the mechanisms of DNA oxidative damage of host cell infected with influenza in the current international. This research will explore influenza virus pathogenesis from view ofDNA oxidative damage repair.ObjectiveThe subject aims to further explore the possible pathogenic mechanism of H1NI virus, on the base that influenza virus infection can cause lipid oxidation and DNA oxidative damage of the host cells. Evaluate the role of oxidative damage in the process of host cell apoptosis caused by influenza virus. The expression of DNA repair enzyme is not enough to fight the excessive oxidative damage, eventually which leadto host cell apoptosis and the occurrence, development of symptoms. The aim of this study is to clarify oxidative stress mechanism of the infected host cell; provide a new idea for influenza virus pathogenesis mechanism research; new targets for the research of drugs against influenza; and a new perspective for the prevention and control of highly pathogenic avian influenza.MethodsPart one Set up Influenza virus infection cell modelMDCK cell were cultured using cell culture technology. Inoculate influenza virus in chicken embryos allantoic cavity and harvest virus. Test 50% tissue culture infective dose of MDCK cells and determine the experiment using dose of H1N1 influenza virus. Part two Study on the expression of MTH1 gene in MDCK cells infected by influenza A virusEstablishment of a semi-quantitative RT-PCR system for detection of MTH1 expression:Total RNA within cultured MDCK cells were extracted and used for RT-PCR. The expression quantity of MTH1 and control gene GAPDH were analyzed using Scion Image software and then the ratio of MTH1/ GAPDH were used for the accuracy, repeatability and sensitivity analysis of the method. MDCK cells were cultured and infected with H1N1.The relative expression of MTH1 gene of the host cells were detected by RT-PCR after infected for 1,3,6,12,24 and 48 hours.Part three the expression of OGG1 gene in MDCK cells infected by influenza A virusRNA was extracted from cultured MDCK cells, then reverse-transcripted into cDNA, and design primers for polymerase chain reaction amplification of the target gene OGG1. The expression quantity of MTH1 and control gene GAPDH were analyzed with Image J software and then the ratio of OGG1/ GAPDH were used to analyse teh the accuracy and repeatability of this method. MDCK cells were cultured and infected with H1N1.Then, the relative expression of OGG1 gene of the host cells were detected by RT-PCR after infected for 1,3,6,12,24 and 48 hours.Results1.MDCK cells and H1N1 influenza virus were successfully cultured. H1N1 influenza virus 50% tissue culture infective dose was 10-3.91/0.1ml. The experiment using dose of H1N1 influenza virus was 100 TCID50/0.1ml.2.(1) Sequencing results of the RT-PCR products show identical with that of the GenBank. (2) Sensitivity detection revealed this system could detect the positive expression of MTH1 from only 50ng total RNA. (3) Repeatability detection showed that the averaged ratio of MTH1/ GAPDH was 2.02±0.09 (n=10), and the CV was 4.31%. There were no significant differences of MTH1 expression between the experimental groups and the control groups at 6 and 12 hours after H1N1 infection,but the MTH1 expression was significantly higher than that of the control group at 1 h and 3 h, and was significantly lower than that of the control group at 24 h and 48 h after H1N1 infection. 3.Primer can amplify the reference gene and target gene. The sequencing of the RT-PCR PCR products was consistent with the OGG1 gene sequence derived from the Gene Bank. Repeatability detection showed that the averaged ratio of OGG1/ GAPDH was 0.84±0.025 (n=6), and the CV was 2.99%. There were no significant differences of OGG1 expression between the experimental groups and the control groups at 48 hours,but the OGG1 expression was significantly higher than that of the control group after at 1h,3h,6h,12h and 24h of infection.ConclusionIn this study, we found the length of amplified fragment was consistent with the anticipation, and the sequence of that matched with the GenBand sequence. There was no positive amplification in negative control, which confirmed the specificity of this method. Repetitive testing results identified the good repeatability of the system. In summary, the system has not only excellent accuracy, high sensitivity and repeatability, but also low equipment requirement and easy operation. The establishment of this system can lay a good foundation for the research of MTH1 mRNA expression and influenza virus infection related research. RT-PCR detection of MTH1 gene expression indicated that mRNA levels of MTH1 gene continued to increase from 1h to 3h, and the levels reduced to normal from 6h to 12h, after H1N1 influenza virus infected such cells. At the monitoring points of 24h and 48h, MTH1 expression was significantly lower than that of the control group. MTH1 could prevent DNA from oxidative damage, which indicate that target cells timely increase MTH1 expression to strengthen the antioxidant capacity of themselves in early stages that oxidative damage appear. While in the later stages of infection, cells apoptosis increased and then the expression of MTH1 was inhibited, so DNA oxidative repair capacity was decreased. With deepening of viral infections and increasing of oxidative stress products, the death of infected cells also increased correspondingly. Method study results showed the length of amplified fragment is consistent with anticipation, and sequencing of such fragment is also identical with GenBand sequence. After H1N1 influenza virus infected cells, mRNA levels of OGG1 with target cells continued to increase from 1h to 24h, whicn can compensate cells to repair DNA oxidative damage, then reduced from 48 hours, and at this time cells apoptosis factors may happen heavily because of the decreasement of OGG1 expression. The change of expression of OGG1 may play a role in influenza pathogenic mechanism. In 2009, Ke found that the mRNA level of hOGG1 in pulmonary type_II_like epithelial cells A549 also increased dramatically under H2O2 exposure. This study explores the influenza virus from aspect of oxidative stress and deepens the understanding of oxidative stress mechanism of influenza virus-induced host cells.更多还原