發(fā)布時(shí)間：2018-01-09 12:12

  本文關(guān)鍵詞：還原型谷胱甘肽在登革病毒增殖中的作用研究　出處：《第三軍醫(yī)大學(xué)》2010年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 登革病毒(DV) 氧化還原狀態(tài) GSH NF-κB

【摘要】： 登革病毒(dengue virus,DV)是黃病毒屬的單股正鏈RNA病毒,根據(jù)E蛋白抗原性的不同,分為四個(gè)血清型(DV1-4)。廣泛流行于熱帶和亞熱帶地區(qū),主要通過(guò)埃及伊蚊和白紋伊蚊傳播。其基因組只有一個(gè)開(kāi)放閱讀框(open reading frame, ORF),編碼三種結(jié)構(gòu)蛋白和七種非結(jié)構(gòu)蛋白,從5’端到3’端依次為5’-C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3’。DV感染可引起人類(lèi)登革熱(classical dengue fever,DF)和登革出血熱/登革休克綜合征(dengue hemorrhagic fever/dengue shock syndrome,DHF/DSS)。近年來(lái),隨著地球溫暖化和流動(dòng)人口的增加,DF和DHF/DSS流行、暴發(fā)越來(lái)越頻繁,流行區(qū)域也在不斷擴(kuò)大,登革病毒感染已是嚴(yán)重的公共衛(wèi)生問(wèn)題。但到目前為止,尚無(wú)安全有效的疫苗和特效藥用于DV感染的防治。 病毒由于缺乏能量代謝系統(tǒng)和必要的酶類(lèi)而不能獨(dú)立生存,嚴(yán)格依賴(lài)于宿主細(xì)胞。進(jìn)入細(xì)胞后病毒利用細(xì)胞的合成代謝系統(tǒng)進(jìn)行大分子合成,因而擾亂了宿主細(xì)胞的自身代謝和生理功能,導(dǎo)致細(xì)胞的氧化還原平衡被打破,處于氧化應(yīng)激狀態(tài)。新近研究表明細(xì)胞的氧化還原狀態(tài)(redox state)參與病毒復(fù)制和致病過(guò)程。細(xì)胞通過(guò)產(chǎn)生谷胱苷肽(GSH),超氧化物歧化酶(SOD),硫氧還蛋白,觸酶等抗氧化分子,保持它的還原狀態(tài)。其中含三個(gè)半胱氨酸的GSH在真核細(xì)胞抗氧化方面最為重要。GSH是細(xì)胞內(nèi)最主要的抗氧化物質(zhì)之一。它是細(xì)胞內(nèi)一種含巰基的小分子抗氧化劑,是由谷氨酸、半胱氨酸、甘氨酸組成的天然小分子三肽,是體內(nèi)重要的氧化還原緩沖系統(tǒng),維持機(jī)體的氧化-抗氧化平衡。GSH水平的下降有利于病毒的復(fù)制,同時(shí)影響宿主的防御反應(yīng)等。研究表明許多病毒感染引起宿主細(xì)胞氧化應(yīng)激,病毒可通過(guò)增加細(xì)胞內(nèi)氧自由基,使細(xì)胞內(nèi)GSH減少。包括1型單純皰疹病毒、仙臺(tái)病毒、HIV、流感病毒、丙型肝炎病毒等。在病毒與宿主細(xì)胞相互作用過(guò)程中,病毒感染所致的細(xì)胞促氧化狀態(tài)可能觸發(fā)了某些轉(zhuǎn)錄因子,如核因子κB (NF-κB),進(jìn)而誘導(dǎo)細(xì)胞凋亡或細(xì)胞過(guò)度增殖等病理過(guò)程。NF-κB是一個(gè)氧化應(yīng)激敏感的轉(zhuǎn)錄因子,能與多種細(xì)胞基因啟動(dòng)子或增強(qiáng)子序列特定位點(diǎn)發(fā)生特異性結(jié)合而促進(jìn)轉(zhuǎn)錄和表達(dá),與炎癥反應(yīng)、免疫應(yīng)答以及細(xì)胞的增生、轉(zhuǎn)化和凋亡等重要的病理生理過(guò)程密切相關(guān)。作為一個(gè)常見(jiàn)的信號(hào)轉(zhuǎn)導(dǎo)通路,氧化應(yīng)激反應(yīng)途徑可以激活NF-κB信號(hào)通路。細(xì)胞內(nèi)巰基在調(diào)節(jié)NF-κB活化方面起重要作用。低水平巰基促進(jìn)NF-κB的活化;高水平巰基抑制NF-κB的活化。目前關(guān)于DV感染引起宿主細(xì)胞氧化還原狀態(tài)改變的研究尚未見(jiàn)文獻(xiàn)報(bào)道。 據(jù)文獻(xiàn)報(bào)道,DV可以直接感染肝細(xì)胞,引起肝臟損傷,換言之,肝臟可能是DV重要的靶器官之一。最近臨床研究發(fā)現(xiàn)登革熱病人體內(nèi)出現(xiàn)氧化損傷。因此,本研究通過(guò)研究DV感染過(guò)程中GSH的變化及其作用,初步探討DV2感染與細(xì)胞內(nèi)氧化還原狀態(tài)的關(guān)系。期望本研究結(jié)果為深入闡明DHF/DSS的發(fā)病機(jī)制及防治措施提供理論依據(jù)。 本研究主要結(jié)果與結(jié)論如下: 1. DV2感染對(duì)HepG2細(xì)胞內(nèi)外GSH水平的影響為了研究DV2感染對(duì)細(xì)胞GSH水平的影響,我們檢測(cè)了DV2感染后,不同時(shí)相點(diǎn)HepG2細(xì)胞內(nèi)外的GSH的水平。以DV2 (MOI=10)感染HepG2細(xì)胞,模擬感染組則加入56℃滅活30min的病毒液,同等條件置于37℃,以病毒吸附起始記為感染0時(shí),在感染10min、20min、30min、40min、60min/1h、2h、6h、12h、24h、48h (后5個(gè)時(shí)相點(diǎn)吸附后1h,更換病毒維持液)的時(shí)相點(diǎn),分別用PBS充分洗滌細(xì)胞,胰蛋白酶消化,取出細(xì)胞。經(jīng)過(guò)四次快速凍融,離心取上清用于GSH的測(cè)定。取相同數(shù)量的細(xì)胞經(jīng)超聲裂解,用于測(cè)定細(xì)胞蛋白濃度。結(jié)果發(fā)現(xiàn),DV2感染導(dǎo)HepG2細(xì)胞內(nèi)GSH水平的降低。與模擬感染組比較,病毒感染后10min、20min、30min、40min、60min/1h,HepG2細(xì)胞內(nèi)GSH水平呈下降趨勢(shì),其中以30min下降最為顯著,為16.82±0.86 nmol/mg,與模擬感染組的23.14±1.41nmol/mg和感染組的其他時(shí)相點(diǎn)比較,均有顯著差異(P0.01)。在病毒吸附期結(jié)束(感染1 h)時(shí),GSH水平有所提高,但依然低于模擬感染組。病毒感染后2h、6h、12h、24h、48h,HepG2細(xì)胞內(nèi)GSH水平仍呈下降趨勢(shì),其中以2h、24h下降較為明顯,分別為29.51±3.16 nmol/mg和17.75±3.32 nmol/mg,與相應(yīng)時(shí)相點(diǎn)的模擬感染組35.45±3.55 nmol/mg和22.91±4.15 nmol/mg以及感染組的其它時(shí)相點(diǎn)比較,差異顯著(P0.05),其后逐漸恢復(fù),48h時(shí)已接近模擬感染組水平,二者無(wú)顯著差異(P0.05)。 感染后30min上清中的GSH含量為47.86±3.00 nmol/ml (n=4),較模擬感染組升高33.09%,且有顯著差異(P0.05),而模擬感染組與病毒原液則無(wú)顯著差異(P0.05)。感染24h,感染組與模擬感染組細(xì)胞外GSH水平?jīng)]有顯著差異(P0.05)。以上結(jié)果說(shuō)明DV2感染能夠使HepG2細(xì)胞內(nèi)GSH水平下降,改變宿主細(xì)胞的氧化還原狀態(tài),且呈現(xiàn)階段性變化。 2. DV2 E、NS3蛋白對(duì)宿主細(xì)胞內(nèi)外GSH水平的影響以上實(shí)驗(yàn)證實(shí)了DV2感染可影響細(xì)胞內(nèi)外GSH的水平,由此推測(cè)病毒蛋白可能參與了這一過(guò)程。為證實(shí)這一推測(cè),我們首先構(gòu)建了穩(wěn)定表達(dá)E、NS3蛋白的HepG2細(xì)胞株pRe-E/HepG2、pRe-NS3/HepG2,通過(guò)間接免疫熒光法和western blot進(jìn)行了鑒定和檢測(cè),確認(rèn)了細(xì)胞中E、NS3蛋白的表達(dá)。同時(shí)構(gòu)建了穩(wěn)定轉(zhuǎn)染空質(zhì)粒和能表達(dá)綠色熒光蛋白的的細(xì)胞株pRe/HepG2、pCI-GFP/HepG2作為對(duì)照。 在此基礎(chǔ)上,我們測(cè)定了pRe-E/HepG2、pRe-NS3/HepG2以及pRe/HepG2、pCI-GFP/HepG2細(xì)胞內(nèi)外GSH的水平。結(jié)果發(fā)現(xiàn):與pRe/HepG2細(xì)胞對(duì)照組比較,pRe-E/HepG2和pRe-NS3/HepG2細(xì)胞內(nèi)GSH水平均呈下降趨勢(shì),分別為對(duì)照細(xì)胞的79%和77%,而表達(dá)GFP蛋白的pCI-GFP/HepG2細(xì)胞內(nèi)GSH與pRe/HepG2細(xì)胞比較無(wú)明顯變化。取對(duì)數(shù)生長(zhǎng)期細(xì)胞的培養(yǎng)上清0.5ml測(cè)定GSH濃度,發(fā)現(xiàn)pRe-E/HepG2、pRe-NS3/HepG2細(xì)胞外GSH分別為對(duì)照細(xì)胞的64%和65%,兩者差異顯著(P0.05)。以上結(jié)果表明,穩(wěn)定表達(dá)E、NS3蛋白細(xì)胞內(nèi)外GSH濃度均下降顯著,提示E、NS3蛋白在宿主細(xì)胞中的表達(dá)不僅可以改變細(xì)胞內(nèi)的氧化還原狀態(tài),還可以使宿主細(xì)胞外的GSH水平降低,在DV2感染誘使的細(xì)胞氧化還原狀態(tài)改變的過(guò)程中,可能起重要作用。 3. GSH處理對(duì)DV2感染的影響 本實(shí)驗(yàn)首先通過(guò)MTT實(shí)驗(yàn)和形態(tài)學(xué)觀(guān)察,確定了外源性GSH的工作濃度為10mM和20mM。 為證實(shí)GSH對(duì)病毒增殖的影響,我們首先確認(rèn)了向培養(yǎng)上清中分別加入10mM、20mM GSH溶液對(duì)細(xì)胞內(nèi)GSH含量無(wú)明顯影響,與空白對(duì)照組相比均無(wú)顯著差異(P0.05);在此基礎(chǔ)上,實(shí)施DV2感染+GSH處理實(shí)驗(yàn),從感染起始到感染24h維持上清內(nèi)GSH濃度分別為10mM、20mM,空白對(duì)照組不加藥物處理。感染后24h收取培養(yǎng)上清,測(cè)定病毒滴度(PFU/ml)。結(jié)果發(fā)現(xiàn):GSH處理可使細(xì)胞培養(yǎng)上清病毒滴度下降,并呈現(xiàn)劑量依賴(lài)特點(diǎn),10mM與20mM GSH處理組病毒滴度分別下降為對(duì)照組的62%和40%(n=5),兩種濃度GSH處理組均與空白對(duì)照組差異顯著(P0.05),且20mM GSH處理組病毒滴度下降更為明顯(P0.05);以上結(jié)果說(shuō)明不僅DV2可以引起細(xì)胞內(nèi)的氧化還原狀態(tài)改變,細(xì)胞內(nèi)的氧化還原狀態(tài)反過(guò)來(lái)也可以影響DV2的增殖。 4. BSO處理對(duì)DV2感染的影響 本實(shí)驗(yàn)首先通過(guò)MTT實(shí)驗(yàn)和形態(tài)學(xué)觀(guān)察,確定了BSO的工作濃度為0.2mM和1mM。 為進(jìn)一步證實(shí)GSH與病毒在細(xì)胞內(nèi)增殖情況,我們?cè)趯?shí)驗(yàn)中用BSO處理細(xì)胞,一種抑制GSH合成的化合物,觀(guān)察在低水平GSH情況下,DV2的增殖情況。在不感染病毒的情況下,培養(yǎng)上清中加入0.2mM、1mM BSO處理18h,細(xì)胞內(nèi)GSH含量是空白對(duì)照組的58%,但是兩種濃度下降幅度無(wú)顯著差異(P0.05)。在DV2感染+BSO處理組,感染前18h分別用0.2mM、1mM BSO預(yù)處理HepG2細(xì)胞,然后用DV2 (MOI=1)感染HepG2細(xì)胞,并維持BSO濃度至感染24h,空白對(duì)照組不加藥物處理。感染后24h收取培養(yǎng)上清,噬斑試驗(yàn)測(cè)定病毒滴度(PFU/ml)。結(jié)果發(fā)現(xiàn)0.2mM與1mM BSO處理組病毒滴度為空白對(duì)照組的211%和215% (n=5),與之比較差異顯著(P0.05),但兩種濃度BSO處理組的病毒滴度增加幅度無(wú)顯著差異(P0.05)。 5. DV感染以及E、NS3蛋白導(dǎo)致NF-κB轉(zhuǎn)錄活性增加 前面實(shí)驗(yàn)我們證實(shí)高水平的GSH抑制病毒增殖;使GSH水平降低,促進(jìn)病毒增殖,推測(cè)GSH水平降低可能激活了與氧化還原有關(guān)的轉(zhuǎn)錄因子,進(jìn)而促進(jìn)某些細(xì)胞因子的釋放,參與DHF/DSS的發(fā)生。因此,我們檢測(cè)了對(duì)氧化應(yīng)激敏感的核轉(zhuǎn)錄因子NF-κB的活性。利用NF-κB熒光報(bào)告載體檢測(cè)NF-κB活性,發(fā)現(xiàn)病毒感染導(dǎo)致NF-κB熒光報(bào)告載體活性增加,加入GSH抑制報(bào)告載體的活性增加;加入BSO,降低GSH水平,熒光報(bào)告載體活性增加。病毒感染組、病毒感染+GSH處理組和病毒感染+BSO處理組NF-κB活性分別為模擬感染組的184%、124%和271%。轉(zhuǎn)染了DV2 E蛋白、NS3蛋白的細(xì)胞NF-κB轉(zhuǎn)錄活性同樣增加,分別為對(duì)照細(xì)胞的243%和309%。在此基礎(chǔ)上,我們還檢測(cè)了NF-κB目標(biāo)基因的表達(dá),發(fā)現(xiàn)感染后白介素6 ( IL-6 )表達(dá)量逐漸增加,48h達(dá)峰值,而IL-8和TNF-α在感染后所觀(guān)察的時(shí)間內(nèi)(至48h)無(wú)顯著變化(P0.05)。以上結(jié)果說(shuō)明DV2感染引起了NF-κB的活性增加及其目標(biāo)基因的表達(dá)。 綜上所述,DV2感染和登革病毒蛋白E及NS3表達(dá)可使細(xì)胞內(nèi)GSH濃度降低,導(dǎo)致NF-κB活性增加和目標(biāo)基因的表達(dá)。人為增加或減少細(xì)胞內(nèi)GSH的濃度,可改變NF-κB活性和病毒的增殖;可見(jiàn)細(xì)胞內(nèi)GSH濃度與DV2感染關(guān)系密切。病毒感染后細(xì)胞內(nèi)GSH的減少,NF-κB的活性增加和IL-6分泌增加,可能與DHF/DSS發(fā)生有關(guān),因而,GSH對(duì)防治DV感染可能具有潛在的應(yīng)用價(jià)值。
[Abstract]:The dengue virus ( DV ) is a single strand RNA virus of the genus Flavivirus , which is divided into four serotypes ( DV1 - 4 ) according to the antigenicity of the E protein . The genome has only one open reading frame ( ORF ) , encodes three structural proteins and seven non - structural proteins , and is sequentially 5 ' - C - prM ( M ) - E - NS4A - NS4B - NS5 - 3 ' from the 5 ' end to the 3 ' end . DV infection can cause dengue fever ( DF ) and dengue hemorrhagic fever / dengue shock syndrome ( DHF / DSS ) . In recent years , with the warming of the earth and the increase of the floating population , DF and DHF / DSS have become more and more frequent , and the epidemic area is growing more and more frequently . The epidemic area is also expanding , and dengue virus infection has been a serious public health problem . So far , there is no safe and effective vaccine and special medicine for the prevention and treatment of DV infection . In the process of interaction of virus and host cell , it is an important oxidation - reduction buffer system , which can promote transcription and expression . It is one of the most important antioxidant substances in cells . It is reported that DV can directly infect hepatocytes and cause liver injury , in other words , liver may be one of the important target organs of DV . Recent clinical studies have found that oxidative damage occurs in dengue fever patients . The main results and conclusions of this study are as follows : In order to study the effect of DV2 infection on GSH in HepG2 cells , the levels of GSH in HepG2 cells were detected after the infection of DV2 . The results showed that the levels of GSH in HepG2 cells were significantly lower than those in simulated infection group ( 16.82 鹵 0.86 nmol / mg ) . The levels of GSH in HepG2 cells decreased significantly after 24 h , 48 h and 48 h , respectively 29.51 鹵 3.16 nmol / mg and 17.75 鹵 3.32 nmol / mg , respectively , which were significantly higher than those in the simulated infection group ( 35.45 鹵 3.55 nmol / mg and 22.91 鹵 4.15 nmol / mg and 22.91 鹵 4.15 nmol / mg , respectively ) . The GSH content was 47.86 鹵 3.00 nmol / ml ( n = 4 ) in the supernatant of 30 min after infection , 33.09 % higher in the simulated infection group and no significant difference in the level of GSH in the infected group ( P0.05 ) . 2 . The effects of DV2 E and NS3 protein on GSH levels inside and outside the host cell showed that the infection of DV2 could affect the level of GSH in both extracellular and extracellular GSH . In order to confirm the hypothesis , we constructed HepG2 cell line pRe - E / HepG2 and pRe - NS3 / HepG2 which stably expressed E and NS3 protein . The expression of E and NS3 protein was confirmed by indirect immunofluorescence and western blot . The stable transfected null plasmid and the cell line pRe / HepG2 and pCI - GFP / HepG2 expressing green fluorescent protein were constructed as control . The levels of GSH in pRe - E / HepG2 , pRe - NS3 / HepG2 and pRe / HepG2 and pCI - GFP / HepG2 cells were determined . 3 . Effect of GSH Treatment on DV2 Infection In this experiment , the activity of exogenous GSH was determined to be 10 mM and 20 mM by MTT assay and morphological observation . In order to confirm the effect of GSH on the proliferation of the virus , we first confirmed that the GSH content in the culture supernatant was not significantly affected by the addition of 10 mM and 20 mM GSH solution to the culture supernatant ( P0.05 ) . 4 . Effect of BSO Treatment on DV2 Infection The concentration of BSO was determined to be 0.2 mM and 1 mM by MTT assay and morphological observation . In order to further confirm the proliferation of GSH and virus in the cells , we used BSO to treat the cells in the experiment , a kind of compound inhibiting GSH synthesis , and observed the proliferation of DV2 at low level of GSH . In the case of non - infectious virus , the cells were pretreated with 0.2 mM , 1 mM BSO for 18 h , and the concentration of BSO was maintained for 24 h . The concentration of BSO was determined to be 211 % and 215 % ( n = 5 ) of the blank control group . 5 . DV infection and E , NS3 protein result in increased NF - 魏B transcription activity It was found that the activity of NF - 魏B was increased , the activity of NF - 魏B was increased , and the activity of NF - 魏B was increased . In conclusion , the expression of the DV2 infection and dengue virus protein E and NS3 can decrease the concentration of GSH in the cells , which leads to the increase of the activity of NF - 魏B and the expression of the target gene . The activity of NF - 魏B and the proliferation of the virus can be changed by artificially increasing or decreasing the concentration of GSH in the cells . The GSH content in the cells after virus infection is closely related to the infection of DV2 . The decrease of GSH in the cells after virus infection , the increase of the activity of NF - 魏B and the increase of IL - 6 secretion may be related to DHF / DSS , and therefore , GSH has potential application value for preventing and treating DV infections .

【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2010
【分類(lèi)號(hào)】：R373

【引證文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

1 王吉旭;RNAi抑制G6PD表達(dá)對(duì)胃癌SGC-7901細(xì)胞的影響及相關(guān)機(jī)制研究[D];中南大學(xué);2012年

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本文編號(hào)：1401340