血管内皮细胞TLR4介导汉滩病毒引起IRF-3和NF-ΚB的核移位研究

发布时间：2018-04-18 07:44

  本文选题：汉滩病毒 + TLR4　； 参考：《第四军医大学》2010年硕士论文

【摘要】： 汉滩病毒(Hantaan virus,HTNV)为布尼亚病毒科(family Bunyaviridae)汉坦病毒属(Hantavirus,HV)的一种单股分节段的负链RNA病毒。HTNV虽以啮齿类动物为储存宿主,却不引起宿主发病,然而在人类可引起两类急性传染病:肾综合征出血热(hemorrhagic fever with renal syndrome, HFRS)和汉坦病毒肺综合征(hantavirus pulmonary syndrome, HPS)。全世界每年大约有15万HFRS和HPS患者,其中HFRS病例的90%以上发生在亚洲。我国是世界上HFRS疫情最严重的国家,HTNV是引起我国重症HFRS的主要病原体,因此,HTNV/HFRS严重危害人民的健康。 自1978年首次从宿主动物中成功分离HV以来,国内外学者对HV及其相关疾病的病原学、流行病学、发病机制、诊断及防治等方面进行了大量的研究并取得了一系列的成果,但其发病的分子机制尚未完全明了,也没有特异有效的治疗措施,安全有效的疫苗尚待进一步开发。因此,探讨HV感染致病的分子机理,寻找有效的抗病毒治疗药物及研制新型HV疫苗,一直是相关领域研究的热点。 目前普遍的观点认为,HV相关疾病是由免疫介导的病理反应所致。固有免疫(innate immunity)作为机体抗感染免疫的第一道防线,在汉坦病毒感染及发病机制中可能起重要作用,然而HV诱导固有免疫的分子机制尚不清楚。HV主要通过感染血管内皮细胞从而引起机体的免疫病理反应。TLR4是血管内皮细胞表面的一种重要的膜分子,参与内皮细胞抗感染的固有免疫反应。LPS是TLR4的主要配体,但是近年来发现TLR4在抗病毒免疫中也发挥了重要作用,HTNV可能通过与TLR4的相互作用诱发机体的固有免疫反应。前期我们研究发现,HTNV 76-118株感染人脐静脉内皮细胞系EVC304后可诱导TLR4的表达升高,进一步采用RNAi技术,构建了EVC304 TLR4-细胞系,以此为基础发现TLR4介导HTNV感染EVC304细胞所导致的IFN-β,IL-6和TNF-α的表达升高,TLR4信号转导途径中TRIF的表达在HTNV感染前后变化明显,而MyD88的表达变化不明显。本研究以上述为基础,通过间接免疫荧光实验观察TLR4下游转录因子IRF-3和NF-κB的细胞核移位以及它与HTNV感染的时程关系,同时以RT-PCR和间接免疫荧光方法观察HTNV感染EVC304细胞后,TLR4是否介导细胞间粘附分子-1(ICAM-1)的表达变化,阐明TLR4信号转导通路在HTNV感染EVC304细胞中的作用,为HFRS的发病机理研究和药物研制提供新资料。 本课题研究内容和结果如下: 1. HTNV的培养及其对EVC304细胞的感染 培养Vero细胞,然后用本室所保存的HTNV 76-118株感染Vero细胞,通过Vero细胞的传代进一步扩增HTNV,7-10天后反复冻融Vero细胞并分离上清。然后培养EVC304 TLR4+细胞和EVC304 TLR4-细胞,用制备的HTNV上清分别感染,发现HTNV可以有效地感染这两种细胞。 2. EVC304 TLR4-细胞中的TLR4表达检测 分别培养EVC304TLR4+细胞和EVC304TLR4-细胞,各取1×106个细胞提取总RNA,用半定量RT-PCR来检测两种细胞中TLR4的表达情况。结果表明在EVC304 TLR4-细胞中TLR4表达水平很低(即在该细胞中TLR4能被有效抑制),提示EVC304 TLR4-细胞可以在本实验中用于研究TLR4的基因功能。 3.转录因子IRF-3和NF-kB的细胞核移位及其与HTNV感染的时程关系 HTNV分别感染EVC304 TLR4+细胞和EVC304 TLR4-细胞,以LPS刺激作为阳性对照组,以未进行任何刺激组作为阴性对照组。观察在感染后0.5h、1h、3h、6h、12h后转录因子IRF-3和NF-kB的细胞核移位情况。结果发现在所有组中刺激0.5h后IRF-3和NF-kB都没有发生细胞核移位,在感染EVC304 TLR4+细胞1h、3h、6h后IRF-3和NF-kB发生了细胞核移位,其中以感染6h的细胞核移位最为明显,而在12h时没有观察到细胞核移位现象。在HTNV感染EVC304 TLR4-各组中各个时段均未发现细胞核移位现象。 4.粘附分子ICAM-1在HTNV感染前后的表达 HTNV分别感染EVC304 TLR4+细胞和EVC304 TLR4-细胞6h后,用RT-PCR和间接免疫荧光技术分别检测粘附分子ICAM-1的表达变化,结果显示HTNV感染EVC304前后以及TLR4基因沉默前后ICAM-1变化不明显,且阳性对照组变化亦不明显。 结论:①HTNV感染EVC304细胞后TLR4可能介导转录因子IRF-3和NF-kB的细胞核移位,且在一定时间内(1-6h),核移位随感染时间延长而增加。②HTNV感染EVC304细胞前后,ICAM-1变化不明显,且与TLR4沉默与否无明显关系。本实验初步阐明了在HTNV感染EVC304细胞中TLR4所引起的固有免疫信号通路活化的分子机制,为HFRS发病机理研究提供了新资料。
[Abstract]:Hantaan virus ( HTNV ) is a single - strand RNA virus of Hantaan ( HV ) of family Bunyaviridae . HTNV is not responsible for host disease although rodents are used as storage hosts . However , two types of acute infectious diseases can be caused in humans : hemorrhagic fever with renal syndrome , renal syndrome hemorrhagic fever with renal syndrome and Hantan pulmonary syndrome . HTNV is one of the most serious countries in the world , HTNV is the main pathogen of the disease in China , and HTNV is a serious threat to the health of people .


Since the first successful separation of HV from host animals in 1978 , scholars and scholars have carried out a series of researches on the etiology , epidemiology , pathogenesis , diagnosis and control of HV and related diseases , but the molecular mechanism of the disease has not been fully understood , and the safe and effective vaccine has yet to be further developed . Therefore , the molecular mechanism of HV infection is discussed . Therefore , it is a hot spot in the relevant field to study the molecular mechanism of HV infection , find effective antiviral therapy drugs and develop new HV vaccines .


In recent years , we have found that the expression of IL - 6 and TNF - 伪 in human umbilical vein endothelial cells infected by HTNV is higher than that of HTNV infection .


The contents and results of this subject are as follows :


1 . Culture of HTNV and Its Infection to EVC304 Cells


Vero cells were infected with HTNV 76 - 118 strain preserved in the chamber , HTNV was further amplified by passage of Vero cells , Vero cells were frozen and thawed repeatedly for 7 - 10 days , then the supernatant was isolated .


2 . Detection of Toll - 4 Expression in EVC304 Toll - like Cells


EVC304 4 + cells and EVC304 4 - cells were cultured respectively . The total RNA was extracted from 1 脳 106 cells . The expression of TL4 in both cells was detected by semi - quantitative RT - PCR . The results showed that the level of TL4 was very low in EVC304 - like cells ( i.e . , it could be effectively inhibited in the cells ) , suggesting that the EVC304 - 4 - cells could be used to study the gene function in the experiment .


3 . Nuclear translocation of transcription factor IRF - 3 and NF - kB and its duration relationship with HTNV infection


The nuclear translocation of IRF - 3 and NF - kB was observed at 0 . 5 h , 1 h , 3 h , 6 h , 12 h after infection , and no nuclear translocation was observed in IRF - 3 and NF - kB after h , 3h , 6h , 6h after infection .


4 . Expression of ICAM - 1 in HTNV


The expression of ICAM - 1 was detected by RT - PCR and indirect immunofluorescence in the presence of HTNV , and the expression of ICAM - 1 was detected by RT - PCR and indirect immunofluorescence respectively . The results showed that the changes of ICAM - 1 before and after the expression of HTNV infection EVC304 and before and after silencing were not obvious , and the changes of the positive control group were not obvious .


Conclusion : 鈶，

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