本文選題：漢灘病毒 + TLR4�。� 參考：《第四軍醫(yī)大學》2010年碩士論文

【摘要】： 漢灘病毒(Hantaan virus,HTNV)為布尼亞病毒科(family Bunyaviridae)漢坦病毒屬(Hantavirus,HV)的一種單股分節(jié)段的負鏈RNA病毒。HTNV雖以嚙齒類動物為儲存宿主,卻不引起宿主發(fā)病,然而在人類可引起兩類急性傳染病:腎綜合征出血熱(hemorrhagic fever with renal syndrome, HFRS)和漢坦病毒肺綜合征(hantavirus pulmonary syndrome, HPS)。全世界每年大約有15萬HFRS和HPS患者,其中HFRS病例的90%以上發(fā)生在亞洲。我國是世界上HFRS疫情最嚴重的國家,HTNV是引起我國重癥HFRS的主要病原體,因此,HTNV/HFRS嚴重危害人民的健康。 自1978年首次從宿主動物中成功分離HV以來,國內(nèi)外學者對HV及其相關疾病的病原學、流行病學、發(fā)病機制、診斷及防治等方面進行了大量的研究并取得了一系列的成果,但其發(fā)病的分子機制尚未完全明了,也沒有特異有效的治療措施,安全有效的疫苗尚待進一步開發(fā)。因此,探討HV感染致病的分子機理,尋找有效的抗病毒治療藥物及研制新型HV疫苗,一直是相關領域研究的熱點。 目前普遍的觀點認為,HV相關疾病是由免疫介導的病理反應所致。固有免疫(innate immunity)作為機體抗感染免疫的第一道防線,在漢坦病毒感染及發(fā)病機制中可能起重要作用,然而HV誘導固有免疫的分子機制尚不清楚。HV主要通過感染血管內(nèi)皮細胞從而引起機體的免疫病理反應。TLR4是血管內(nèi)皮細胞表面的一種重要的膜分子,參與內(nèi)皮細胞抗感染的固有免疫反應。LPS是TLR4的主要配體,但是近年來發(fā)現(xiàn)TLR4在抗病毒免疫中也發(fā)揮了重要作用,HTNV可能通過與TLR4的相互作用誘發(fā)機體的固有免疫反應。前期我們研究發(fā)現(xiàn),HTNV 76-118株感染人臍靜脈內(nèi)皮細胞系EVC304后可誘導TLR4的表達升高,進一步采用RNAi技術,構建了EVC304 TLR4-細胞系,以此為基礎發(fā)現(xiàn)TLR4介導HTNV感染EVC304細胞所導致的IFN-β,IL-6和TNF-α的表達升高,TLR4信號轉導途徑中TRIF的表達在HTNV感染前后變化明顯,而MyD88的表達變化不明顯。本研究以上述為基礎,通過間接免疫熒光實驗觀察TLR4下游轉錄因子IRF-3和NF-κB的細胞核移位以及它與HTNV感染的時程關系,同時以RT-PCR和間接免疫熒光方法觀察HTNV感染EVC304細胞后,TLR4是否介導細胞間粘附分子-1(ICAM-1)的表達變化,闡明TLR4信號轉導通路在HTNV感染EVC304細胞中的作用,為HFRS的發(fā)病機理研究和藥物研制提供新資料。 本課題研究內(nèi)容和結果如下: 1. HTNV的培養(yǎng)及其對EVC304細胞的感染 培養(yǎng)Vero細胞,然后用本室所保存的HTNV 76-118株感染Vero細胞,通過Vero細胞的傳代進一步擴增HTNV,7-10天后反復凍融Vero細胞并分離上清。然后培養(yǎng)EVC304 TLR4+細胞和EVC304 TLR4-細胞,用制備的HTNV上清分別感染,發(fā)現(xiàn)HTNV可以有效地感染這兩種細胞。 2. EVC304 TLR4-細胞中的TLR4表達檢測 分別培養(yǎng)EVC304TLR4+細胞和EVC304TLR4-細胞,各取1×106個細胞提取總RNA,用半定量RT-PCR來檢測兩種細胞中TLR4的表達情況。結果表明在EVC304 TLR4-細胞中TLR4表達水平很低(即在該細胞中TLR4能被有效抑制),提示EVC304 TLR4-細胞可以在本實驗中用于研究TLR4的基因功能。 3.轉錄因子IRF-3和NF-kB的細胞核移位及其與HTNV感染的時程關系 HTNV分別感染EVC304 TLR4+細胞和EVC304 TLR4-細胞,以LPS刺激作為陽性對照組,以未進行任何刺激組作為陰性對照組。觀察在感染后0.5h、1h、3h、6h、12h后轉錄因子IRF-3和NF-kB的細胞核移位情況。結果發(fā)現(xiàn)在所有組中刺激0.5h后IRF-3和NF-kB都沒有發(fā)生細胞核移位,在感染EVC304 TLR4+細胞1h、3h、6h后IRF-3和NF-kB發(fā)生了細胞核移位,其中以感染6h的細胞核移位最為明顯,而在12h時沒有觀察到細胞核移位現(xiàn)象。在HTNV感染EVC304 TLR4-各組中各個時段均未發(fā)現(xiàn)細胞核移位現(xiàn)象。 4.粘附分子ICAM-1在HTNV感染前后的表達 HTNV分別感染EVC304 TLR4+細胞和EVC304 TLR4-細胞6h后,用RT-PCR和間接免疫熒光技術分別檢測粘附分子ICAM-1的表達變化,結果顯示HTNV感染EVC304前后以及TLR4基因沉默前后ICAM-1變化不明顯,且陽性對照組變化亦不明顯。 結論:①HTNV感染EVC304細胞后TLR4可能介導轉錄因子IRF-3和NF-kB的細胞核移位,且在一定時間內(nèi)(1-6h),核移位隨感染時間延長而增加。②HTNV感染EVC304細胞前后,ICAM-1變化不明顯,且與TLR4沉默與否無明顯關系。本實驗初步闡明了在HTNV感染EVC304細胞中TLR4所引起的固有免疫信號通路活化的分子機制,為HFRS發(fā)病機理研究提供了新資料。
[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 - 偽 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，

本文編號：1767453