还原型谷胱甘肽在登革病毒增殖中的作用研究

发布时间：2018-01-09 12:12

本文关键词：还原型谷胱甘肽在登革病毒增殖中的作用研究　出处：《第三军医大学》2010年博士论文　论文类型：学位论文

【摘要】： 登革病毒(dengue virus,DV)是黄病毒属的单股正链RNA病毒,根据E蛋白抗原性的不同,分为四个血清型(DV1-4)。广泛流行于热带和亚热带地区,主要通过埃及伊蚊和白纹伊蚊传播。其基因组只有一个开放阅读框(open reading frame, ORF),编码三种结构蛋白和七种非结构蛋白,从5’端到3’端依次为5’-C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3’。DV感染可引起人类登革热(classical dengue fever,DF)和登革出血热/登革休克综合征(dengue hemorrhagic fever/dengue shock syndrome,DHF/DSS)。近年来,随着地球温暖化和流动人口的增加,DF和DHF/DSS流行、暴发越来越频繁,流行区域也在不断扩大,登革病毒感染已是严重的公共卫生问题。但到目前为止,尚无安全有效的疫苗和特效药用于DV感染的防治。病毒由于缺乏能量代谢系统和必要的酶类而不能独立生存,严格依赖于宿主细胞。进入细胞后病毒利用细胞的合成代谢系统进行大分子合成,因而扰乱了宿主细胞的自身代谢和生理功能,导致细胞的氧化还原平衡被打破,处于氧化应激状态。新近研究表明细胞的氧化还原状态(redox state)参与病毒复制和致病过程。细胞通过产生谷胱苷肽(GSH),超氧化物歧化酶(SOD),硫氧还蛋白,触酶等抗氧化分子,保持它的还原状态。其中含三个半胱氨酸的GSH在真核细胞抗氧化方面最为重要。GSH是细胞内最主要的抗氧化物质之一。它是细胞内一种含巯基的小分子抗氧化剂,是由谷氨酸、半胱氨酸、甘氨酸组成的天然小分子三肽,是体内重要的氧化还原缓冲系统,维持机体的氧化-抗氧化平衡。GSH水平的下降有利于病毒的复制,同时影响宿主的防御反应等。研究表明许多病毒感染引起宿主细胞氧化应激,病毒可通过增加细胞内氧自由基,使细胞内GSH减少。包括1型单纯疱疹病毒、仙台病毒、HIV、流感病毒、丙型肝炎病毒等。在病毒与宿主细胞相互作用过程中,病毒感染所致的细胞促氧化状态可能触发了某些转录因子,如核因子κB (NF-κB),进而诱导细胞凋亡或细胞过度增殖等病理过程。NF-κB是一个氧化应激敏感的转录因子,能与多种细胞基因启动子或增强子序列特定位点发生特异性结合而促进转录和表达,与炎症反应、免疫应答以及细胞的增生、转化和凋亡等重要的病理生理过程密切相关。作为一个常见的信号转导通路,氧化应激反应途径可以激活NF-κB信号通路。细胞内巯基在调节NF-κB活化方面起重要作用。低水平巯基促进NF-κB的活化;高水平巯基抑制NF-κB的活化。目前关于DV感染引起宿主细胞氧化还原状态改变的研究尚未见文献报道。据文献报道,DV可以直接感染肝细胞,引起肝脏损伤,换言之,肝脏可能是DV重要的靶器官之一。最近临床研究发现登革热病人体内出现氧化损伤。因此,本研究通过研究DV感染过程中GSH的变化及其作用,初步探讨DV2感染与细胞内氧化还原状态的关系。期望本研究结果为深入阐明DHF/DSS的发病机制及防治措施提供理论依据。本研究主要结果与结论如下: 1. DV2感染对HepG2细胞内外GSH水平的影响为了研究DV2感染对细胞GSH水平的影响,我们检测了DV2感染后,不同时相点HepG2细胞内外的GSH的水平。以DV2 (MOI=10)感染HepG2细胞,模拟感染组则加入56℃灭活30min的病毒液,同等条件置于37℃,以病毒吸附起始记为感染0时,在感染10min、20min、30min、40min、60min/1h、2h、6h、12h、24h、48h (后5个时相点吸附后1h,更换病毒维持液)的时相点,分别用PBS充分洗涤细胞,胰蛋白酶消化,取出细胞。经过四次快速冻融,离心取上清用于GSH的测定。取相同数量的细胞经超声裂解,用于测定细胞蛋白浓度。结果发现,DV2感染导HepG2细胞内GSH水平的降低。与模拟感染组比较,病毒感染后10min、20min、30min、40min、60min/1h,HepG2细胞内GSH水平呈下降趋势,其中以30min下降最为显著,为16.82±0.86 nmol/mg,与模拟感染组的23.14±1.41nmol/mg和感染组的其他时相点比较,均有显著差异(P0.01)。在病毒吸附期结束(感染1 h)时,GSH水平有所提高,但依然低于模拟感染组。病毒感染后2h、6h、12h、24h、48h,HepG2细胞内GSH水平仍呈下降趋势,其中以2h、24h下降较为明显,分别为29.51±3.16 nmol/mg和17.75±3.32 nmol/mg,与相应时相点的模拟感染组35.45±3.55 nmol/mg和22.91±4.15 nmol/mg以及感染组的其它时相点比较,差异显著(P0.05),其后逐渐恢复,48h时已接近模拟感染组水平,二者无显著差异(P0.05)。感染后30min上清中的GSH含量为47.86±3.00 nmol/ml (n=4),较模拟感染组升高33.09%,且有显著差异(P0.05),而模拟感染组与病毒原液则无显著差异(P0.05)。感染24h,感染组与模拟感染组细胞外GSH水平没有显著差异(P0.05)。以上结果说明DV2感染能够使HepG2细胞内GSH水平下降,改变宿主细胞的氧化还原状态,且呈现阶段性变化。 2. DV2 E、NS3蛋白对宿主细胞内外GSH水平的影响以上实验证实了DV2感染可影响细胞内外GSH的水平,由此推测病毒蛋白可能参与了这一过程。为证实这一推测,我们首先构建了稳定表达E、NS3蛋白的HepG2细胞株pRe-E/HepG2、pRe-NS3/HepG2,通过间接免疫荧光法和western blot进行了鉴定和检测,确认了细胞中E、NS3蛋白的表达。同时构建了稳定转染空质粒和能表达绿色荧光蛋白的的细胞株pRe/HepG2、pCI-GFP/HepG2作为对照。在此基础上,我们测定了pRe-E/HepG2、pRe-NS3/HepG2以及pRe/HepG2、pCI-GFP/HepG2细胞内外GSH的水平。结果发现:与pRe/HepG2细胞对照组比较,pRe-E/HepG2和pRe-NS3/HepG2细胞内GSH水平均呈下降趋势,分别为对照细胞的79%和77%,而表达GFP蛋白的pCI-GFP/HepG2细胞内GSH与pRe/HepG2细胞比较无明显变化。取对数生长期细胞的培养上清0.5ml测定GSH浓度,发现pRe-E/HepG2、pRe-NS3/HepG2细胞外GSH分别为对照细胞的64%和65%,两者差异显著(P0.05)。以上结果表明,稳定表达E、NS3蛋白细胞内外GSH浓度均下降显著,提示E、NS3蛋白在宿主细胞中的表达不仅可以改变细胞内的氧化还原状态,还可以使宿主细胞外的GSH水平降低,在DV2感染诱使的细胞氧化还原状态改变的过程中,可能起重要作用。 3. GSH处理对DV2感染的影响本实验首先通过MTT实验和形态学观察,确定了外源性GSH的工作浓度为10mM和20mM。为证实GSH对病毒增殖的影响,我们首先确认了向培养上清中分别加入10mM、20mM GSH溶液对细胞内GSH含量无明显影响,与空白对照组相比均无显著差异(P0.05);在此基础上,实施DV2感染+GSH处理实验,从感染起始到感染24h维持上清内GSH浓度分别为10mM、20mM,空白对照组不加药物处理。感染后24h收取培养上清,测定病毒滴度(PFU/ml)。结果发现:GSH处理可使细胞培养上清病毒滴度下降,并呈现剂量依赖特点,10mM与20mM GSH处理组病毒滴度分别下降为对照组的62%和40%(n=5),两种浓度GSH处理组均与空白对照组差异显著(P0.05),且20mM GSH处理组病毒滴度下降更为明显(P0.05);以上结果说明不仅DV2可以引起细胞内的氧化还原状态改变,细胞内的氧化还原状态反过来也可以影响DV2的增殖。 4. BSO处理对DV2感染的影响本实验首先通过MTT实验和形态学观察,确定了BSO的工作浓度为0.2mM和1mM。为进一步证实GSH与病毒在细胞内增殖情况,我们在实验中用BSO处理细胞,一种抑制GSH合成的化合物,观察在低水平GSH情况下,DV2的增殖情况。在不感染病毒的情况下,培养上清中加入0.2mM、1mM BSO处理18h,细胞内GSH含量是空白对照组的58%,但是两种浓度下降幅度无显著差异(P0.05)。在DV2感染+BSO处理组,感染前18h分别用0.2mM、1mM BSO预处理HepG2细胞,然后用DV2 (MOI=1)感染HepG2细胞,并维持BSO浓度至感染24h,空白对照组不加药物处理。感染后24h收取培养上清,噬斑试验测定病毒滴度(PFU/ml)。结果发现0.2mM与1mM BSO处理组病毒滴度为空白对照组的211%和215% (n=5),与之比较差异显著(P0.05),但两种浓度BSO处理组的病毒滴度增加幅度无显著差异(P0.05)。 5. DV感染以及E、NS3蛋白导致NF-κB转录活性增加前面实验我们证实高水平的GSH抑制病毒增殖;使GSH水平降低,促进病毒增殖,推测GSH水平降低可能激活了与氧化还原有关的转录因子,进而促进某些细胞因子的释放,参与DHF/DSS的发生。因此,我们检测了对氧化应激敏感的核转录因子NF-κB的活性。利用NF-κB荧光报告载体检测NF-κB活性,发现病毒感染导致NF-κB荧光报告载体活性增加,加入GSH抑制报告载体的活性增加;加入BSO,降低GSH水平,荧光报告载体活性增加。病毒感染组、病毒感染+GSH处理组和病毒感染+BSO处理组NF-κB活性分别为模拟感染组的184%、124%和271%。转染了DV2 E蛋白、NS3蛋白的细胞NF-κB转录活性同样增加,分别为对照细胞的243%和309%。在此基础上,我们还检测了NF-κB目标基因的表达,发现感染后白介素6 ( IL-6 )表达量逐渐增加,48h达峰值,而IL-8和TNF-α在感染后所观察的时间内(至48h)无显著变化(P0.05)。以上结果说明DV2感染引起了NF-κB的活性增加及其目标基因的表达。综上所述,DV2感染和登革病毒蛋白E及NS3表达可使细胞内GSH浓度降低,导致NF-κB活性增加和目标基因的表达。人为增加或减少细胞内GSH的浓度,可改变NF-κB活性和病毒的增殖;可见细胞内GSH浓度与DV2感染关系密切。病毒感染后细胞内GSH的减少,NF-κB的活性增加和IL-6分泌增加,可能与DHF/DSS发生有关,因而,GSH对防治DV感染可能具有潜在的应用价值。
[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 卤 0.86 nmol / mg ) . The levels of GSH in HepG2 cells decreased significantly after 24 h , 48 h and 48 h , respectively 29.51 卤 3.16 nmol / mg and 17.75 卤 3.32 nmol / mg , respectively , which were significantly higher than those in the simulated infection group ( 35.45 卤 3.55 nmol / mg and 22.91 卤 4.15 nmol / mg and 22.91 卤 4.15 nmol / mg , respectively ) . The GSH content was 47.86 卤 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 .

【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R373

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