【作者】 徐芳玲；

【导师】 杨占秋；

【作者基本信息】 武汉大学 ， 病原生物学， 2004， 博士

【摘要】 汉滩病毒(Hantaan Virus, HTNV)是肾综合征出血热(HFRS)的病原体，属于布尼亚病毒科汉坦病毒属，它能在原代和传代细胞中增殖，但一般不引起细胞病变效应。研究表明整合素可能充当了汉坦病毒细胞膜受体，与病毒侵入宿主细胞有关，而且汉坦病毒感染会引起宿主细胞形态结构、能量代谢等改变而影响感染细胞的生长，甚至能诱导传代人胚肾细胞和淋巴细胞凋亡，但其作用机制仍不明了。探讨汉坦病毒感染对人胚肺细胞的影响，能为阐明汉坦病毒与宿主细胞相互作用的分子机制提供依据。基因芯片技术的发展为研究病原体与宿主细胞的相互作用开辟了新的领域，表达谱基因芯片通过全面监测病原体感染机体后宿主及病原体基因表达的改变，可回答宿主细胞或器官是如何认识病原体，而且可揭示在何种水平上宿主能对不同因子进行辨别，以及宿主与病原体之间的交谈、潜在的治疗靶位和疾病转归的预后标志。因此，联合应用基因芯片技术、流式细胞术等方法和mRNA合成抑制剂对汉坦病毒感染细胞进行基因及相关蛋白表达水平和细胞周期等进行动态分析，将对研究汉坦病毒与细胞的相互作用添上新的一页。 目的：动态观察汉坦病毒感染原代人胚肺细胞(HEPFs)基因表达谱，进一步分析细胞凋亡关键蛋白的表达变化、细胞膜磷脂酰丝氨酸翻转情况和细胞周期改变，以了解汉坦病毒对HEPFs的作用以及诱导细胞凋亡的情况，为阐明汉坦病毒对宿主细胞的作用机制提供新的理论依据。 方法：1．实验分组及病毒接种 将HEPFs随机分为三组，即病毒感染组，病毒感染及mRNA合成抑制剂3’-脱氧腺苷处理组和对照组。前两组接种HTNV76-118病毒悬液，感染量为5-10 M.O.I．，37℃吸附2小时后接种病毒的细胞和正常对照细胞同时补加5％DMEM维持液继续培养，但病毒感染加3’-脱氧腺苷处理组所加维持液中含有200μM 3’-脱氧腺苷。 2．汉坦病毒感染细胞观察 分别在汉坦病毒感染6、24和96小时及有无3’-脱氧腺苷处理时观察HEPFs，并进行细胞涂片，采用间接免疫荧光法(IFA)对各组细胞进行病毒抗原检测。 3.各组细胞总RNA提取分别在汉坦病毒感染6、24及%小时收集不同时间点及有无3’一脱氧腺普处理的细胞，改良一步法提取总RNA，并分析RNA质量。 4.病毒核酸检测采用逆转录一聚合酶链反应(RT一PCR)对各组HEPFs总RNA进行汉坦病毒特异性核酸片段进行检测。 5.基因芯片筛选差异表达基因将各组细胞完整总RNA逆转录并进行荧光标记，带有Cys荧光标记的实验组和带有cy3荧光标记的对照组探针同时与所用芯片杂交，然后进行扫描和荧光信号分析筛选出差异表达基因。 6.半肤氨酸蛋白酶(caspase3)蛋白活性检测设立汉坦病毒感染组、汉坦病毒感染及3’一脱氧腺昔处理组、阴性对照组、汉坦病毒感染加抑制剂组、汉坦病毒感染及3’一脱氧腺普处理加casPase3抑制剂组、汉坦病毒感染不加casPase3底物组和汉坦病毒感染及3’一脱氧腺普处理不加底物组，并且每组细胞样本设立二重复。分别在不同时间点收集各组细胞，经裂解液裂解细胞后，收集各样本上清液，根据Caspase3检测试剂盒说明进行后续操作，经光谱分析测定仪读取数据，并建立标准曲线计算出caspase3的活性单位。 7.细胞膜磷脂酞丝氨酸(PS)翻转测定分别在不同时间点收集各组细胞，设立二重复。按照APoAlert钙依赖磷脂结合蛋白v(AnnexinV)试剂盒操作步骤通过流式细胞分析仪对各样本细胞膜上PS的情况进行分析，并用WlnMID 2 .8软件对数据进行整理和分析。 8.细胞周期分析分别在不同时间点收集各组细胞，设立二重复。细胞经乙醇固定后，用碘化丙锭(Pl)溶液(Pl 509/ml，0.1%柠檬酸钠，0.2%Np一40，RNA酶0.25mg/ml)避光染色后通过流式细胞分析仪对细胞周期进行分析。根据细胞周期分析程序(B eekinen Coulter)计算GO/GI，GZ舰，S和sub一GI各期的细胞数目。 结果:1.病毒感染细胞形态及病毒抗原表达在汉坦病毒感染6、24和%小时不同时间点及有无3’一脱氧腺昔处理细胞时，显微镜下观察各组细胞的形态没有明显差异，病毒抗原也没有检测到。 2.细胞样本总RNA质量紫外分光光度法分析表明汉坦病毒感染组、汉坦病毒感染并给予3’一脱氧腺昔处理组和对照组总RNA OD26夕O场8。比值均在2，0左右，这和电泳结果共同表明RNA质量较高，符合实验要求。 3.病毒核酸检测汉坦病毒感染人胚肺细胞6、24和%小时以及给予3’一脱氧腺昔处理时，都能检测到特异性汉坦病毒核酸片段。 4.基因差异表达汉坦病毒76一118株感染HEPFs6小时时，有134项基因出现差异表达，给予RNA合成抑制剂3’一脱氧腺普处理时有“项基因出现差异表达;在感染后24小时有27项基因出现差异表达，给予3’一脱氧腺昔处理时，有195项基因出现差异表达;当感染%小时，有7项基因出现差异表达，给予3’一脱氧腺昔处理时，有130项基因出现差异表达。分析不同时间点、不同处理细胞差异表达基因及其Ratio值显示表达上调的基因共有292项:其中感染6小时有97项，感染并给予3’一脱氧腺昔有25项;感染24小时有10项，感染并给予3’一脱氧腺昔有109项;感染96小时有3项，感染并给予3’一?更多还原

【Abstract】 Hantaan virus is the pathogeny of hemorrhagic fever with renal syndrome (HFRS).It multiplicates in primary and passaged cells, but there is few cellular pathogenic effect in these infected cells.Researches show that integrin may be the cellular membrane receptor for hantaan virus and it is related to hantaan virus’s entry. In addition, hantaan virus causes changes of morphology and metabolism et al in infected cells. Furthermore, it can induce apoptosis in human embryonic kidney cell and lymphocyte, but the mechanism is not clear. Study the effect of hantavirus on primary human embryonic pulmonary fibroblasts (HEPFs) will get evidence for the molecular mechanism of interaction between hantavirus and host cells. The development of gene chip brings forth new area for investigating the interaction between pathogens and host cells, and expression microarray can answer how host cells or organ to recognize pathogens, in which level the host can distinguish different pathogenic factors and indicate the cross talk between host and pathogens, potential therapeutic target and the prognosis mark for disease. In thus, dynamic analysis of the level of gene and related protein expression and cell cycle in HEPFs infected with hantavirus through techniques of gene chip and flow cytometry and by using inhibitor of mRNA-cordycepin, will add a new page into the study of interaction between hantavirus and cells.Objective: To analyze the gene expression profile, activity of caspase 3, phosphatidylserine (PS) translocation and change of cell cycle of HEPFs infected with Hantaan virus (HTNV) 76-118 dynamically,and to learn the effect of hantavirus on HEPFs and apoptosis induced by hantavirus.Methods: 1. Experiment groups and virus inoculation HEPFs were classified as three groups randomly. They were virus infection group, virus infection with cordycepin treatment group and normal control group. Cells in former two groups were infected with hantaan virus (M.O.I. 5-10),after inocubated in 37C for 2 hours, medium of infection and control group were changed with 5% maintaining DMEM, and medium of infection withcordycepin treatment group were changed with 5% maintaining DMEM with 200 u M cordycepin.2. Observing HEPFs infected with hantavirus HEPFs were investigated directly by phase-microscope after hantavirus infection with or without cordycepin treatment at 6 hour (h), 24h and 96h post infection (p.i.) respectively. Furthermore, cells smears were prepared for the detection of viral antigen of different cells in different group by indirect fluorescent assay (IFA).3. Total RNA extraction from HEPFs in different group HEPFs were harvested at 6 h, 24h and 96h p.i. with or without cordycepin treatment. Then total RNA were extracted by modified one-step method and the quality of RNA was also analyzed.4. Detection of viral nuclear acid Total RNA of each group was analyzed by reverse transcription-polymerase chain reaction(RT-PCR) for detecting the nuclear acid of HTNV.5. Choosing the differently expressed genes by gene chip The intact cellular RNAs of each group were used for reverse transcription as well as labeling by fluorescence. Then experimental groups were labeled by Cy5 and control groups were labeled by Cy3. All these labeled cDNAs were hybridized with chips which were used in our study. After hybridization, chips were scanned and fluorescent signals were analyzed for choosing variational genes.6. Detection of the activity of caspase 3 Duplicate cell plates were set up for the following samples: hantavirus infection group, hantavirus infection with cordycepin treatment group, negative control group, hantavirus infection with inhibitor for caspase 3 and hantavirus infection without substrate for caspase 3. Cells of each group were harvested and were resuspended in chilled cell lysis buffer. Then the supernatants of each group were harvested and following processes were done according to BD ApoAlert Caspase 3 assay’s user handbook. The data were read at 405 nm in a microplate reader and units of caspase更多还原