黄芪皂甙对CVB3病毒引起Vero 细胞凋亡的研究

目的: 细胞水平上建立病毒性心肌炎( Viral Myocarditis,VMC) 模型,通过相关实验得出黄芪皂甙
( total extract astragalus,TEA) 具有抗病毒能力,进一步明确VMC 发病过程中是否存在细胞凋亡以及黄芪皂甙是
否能够抑制细胞凋亡。方法: 体外培养Vero 细胞和出生1 ～ 3 d 乳鼠心肌细胞,通过细胞病变抑制实验、病毒繁
殖抑制实验测定黄芪皂甙抗病毒能力,采用AnnexinV－FITC 染色观察VMC 发病过程中心肌细胞是否有凋亡,采
用流式细胞仪检测TEA 是否抑制细胞凋亡。结果: 通过MTT 染色分析发现TEA 可有效保护细胞免受CVB3
病毒侵袭,病毒繁殖抑制实验显示中、高剂量组TCID50比病毒组相差一个以上对数值,且TEA 对细胞凋亡有抑制
作用。结论: 实验证实黄芪皂甙对心肌细胞有一定的保护作用,并且在VMC 发病过程中存在细胞凋亡,且TEA
能抑制心肌细胞凋亡。     

THE INHIBITORY EFFECT OF ASTRAGALUS MEMBRANACEUS ON COXSACKIE B-3 VIRUS RNA REPLICATION

Using mice infected with coxsackie B-3 virus (CVB3) as a viral myocarditis model,we observed the inhibitory effect of Astragalus membranaceus (AM) on CVB3-RNA replication in myocardial tissue of mice by RNA-RNA in situ hybridization with negative-strand RNA probes labelled with ^35S and quantitative imaging analysis of positive signals.The mechanism of its effect on CVB3-RNA replication has been inves-tigated by detection of beta-interferon (β-IFN) as well.Results showed that the copy numbers of CVB3-RNA as well as the histologic scores (necrosis) in myocardial tissues of infected-AM treated mice were sig-nificantly lower than those in infected and normal saline treated mice,suggesting that AM could inhibit the replication of CVB3-RNA,but its effect on CVB3-RNA replication had no correlation with induction of β-IFN.     

柯萨奇B_3病毒感染体外培养神经元和星形胶质细胞的研究

目的 :研究体外纯培养的神经元和星形胶质细胞对柯萨奇B3 病毒 (CoxsackievirusB3 ,CVB3 )的敏感性。方法 :CVB3 分别感染纯培养新生Balb/c鼠脑的神经元和星形胶质细胞 ,收集感染后 6、2 4、48、72h的培养上清。显微镜下连续动态观察病变及检测感染后不同时间的培养上清接种于Hela细胞上的病毒滴度。结果 :CVB3 在培养的神经元和星形胶质细胞内均有增殖 ,但两者发生病变的时间及程度不同。结论 :Balb/c鼠脑神经元上有CVB3 的受体 ,而星形胶质细胞上CVB3 的受体情况尚待进一步研究     

基于CMV启动子构建CVB3感染性克隆载体的初步研究

目的利用CMV启动子构建CVB3感染性克隆载体。方法将质粒pcDNA3.1（＋）的CMV启动子下游区域通过PCR技术替换成设计好的多克隆位点区,分别插入PCR合成的HdvRz核心序列和polyA尾片段,形成框架载体pc-H-A,通过瞬时转染HeLa细胞初步鉴定pc-H-A的可用性;用本室保存的CVB3（nancy株）感染HeLa细胞,取感染液上清提取病毒RNA,利用长链RT-PCR和长链PCR技术反复优化条件扩增病毒基因组全长片段,将此全长片段克隆至设计好的框架载体pc-H-A中,筛选阳性克隆,并对其进行测序鉴定。结果与结论构建了带有CMV启动子的框架载体pc-H-A并初步鉴定了该载体可用,利用此框架载体构建了与基因库中序列相似性为99%的CVB3全长cDNA克隆pc-CVB3-H-A。该研究为进一步鉴定所构建的CVB3全长克隆的感染性提供了基础,并为后续在病毒学研究和基因治疗方面的研究提供了手段。     

Curcumin inhibits the replication of enterovirus 71 in vitro

Human enterovirus 71 (EV71) is the main causative pathogen of hand, foot, and mouth disease (HFMD) in children. The epidemic of HFMD has been a public health problem in Asia-Pacific region for decades, and no vaccine and effective antiviral medicine are available. Curcumin has been used as a traditional medicine for centuries to treat a diversity of disorders including viral infections. In this study, we demonstrated that curcumin showed potent antiviral effect again EV71. In Vero cells infected with EV71, the addition of curcumin significantly suppressed the synthesis of viral RNA, the expression of viral protein, and the overall production of viral progeny. Similar with the previous reports, curcumin reduced the production of ROS induced by viral infection. However, the antioxidant property of curcumin did not contribute to its antiviral activity, since N-acetyl-l-cysteine, the potent antioxidant failed to suppress viral replication. This study also showed that extracellular signal-regulated kinase (ERK) was activated by either viral infection or curcumin treatment, but the activated ERK did not interfere with the antiviral effect of curcumin, indicating ERK is not involved in the antiviral mechanism of curcumin. Unlike the previous reports that curcumin inhibited protein degradation through ubiquitin–proteasome system (UPS), we found that curcumin had no impact on UPS in control cells. However, curcumin did reduce the activity of proteasomes which was increased by viral infection. In addition, the accumulation of the short-lived proteins, p53 and p21, was increased by the treatment of curcumin in EV71-infected cells. We further probed the antiviral mechanism of curcumin by examining the expression of GBF1 and PI4KB, both of which are required for the formation of viral replication complex. We found that curcumin significantly reduced the level of both proteins. Moreover, the decreased expression of either GBF1 or PI4KB by the application of siRNAs was sufficient to suppress viral replication. We also demonstrated that curcumin showed anti-apoptotic activity at the early stage of viral infection. The results of this study provide solid evidence that curcumin has potent anti-EV71 activity. Whether or not the down-regulated GBF1 and PI4KB by curcumin contribute to its antiviral effect needs further studies.KEY WORDS: Curcumin, Enterovirus 71, Viral replication, GBF1, PI4KB, Ubiquitin–proteasome system, ApoptosisAbbreviations: CVB, coxsackieviurs B; DCFH-DA, dichloro-dihydro-fluorescein diacetate; ERK, extracellular signal-regulated kinase; EV71, enterovirus 71; GBF1, Golgi brefeldin A resistant guanine nucleotide exchange factor 1; GEF, guanine nucleotide exchange factor; HBV, hepatitis B virus; HCV, hepatitis C virus; HFMD, hand, foot, and mouth disease; HIV, human immunodeficiency virus; HPV, human papillomavirus; NAC, N-acetyl-l-cysteine; PARP-1, poly(ADP-ribose) polymerase; PGC-1α, peroxisome proliferator-activated receptor-gamma co-activator 1 alpha; p.i., post-infection; PI4KB, phosphatidylinositol 4-kinase class III catalytic subunit β; PI4P, phosphatidylinositol 4-phosphate; ROS, reactive oxygen species; siRNA, small interfering RNA; SLLVY-AMC, succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin; UPS, ubiquitin–proteasome system     

苦瓜蛋白的分离纯化及其体外抗CoxB3型病毒作用的研究

目的 探讨苦瓜蛋白的分离纯化方法。研究其体外抗病毒作用。为开发这一新型抗病毒药物打下前期工作基础。方法 离心、超滤，阳离子交换色谱，反相高效液相色谱等分离纯化方法，体外抗病毒实验方法。结果 得到了色谱纯的抗3型科萨奇病毒的苦瓜蛋白，其抗病毒作用有明显的剂量-效应关系，在浓度为215μg/mL时具有最大的抗病毒作用，与对照组相比，苦瓜蛋白对CoxB3感染的HepG2的保护作用十分明显。结论 体外抗病毒实验表明一定范围内的苦瓜蛋白对CoxB3感染的HepG2有明显的保护作用。     

柯萨奇B组病毒感染的血清流行病学分析

目的：探讨柯萨奇病毒Ｂ组１至６型和青少年病毒性心肌炎的血清流行病学关系。方法：采用间接ＥＬＩＳＡ法检测３１例病毒性心肌炎患者和４７名义务献血员血清。结果：病毒性心肌炎患者血清中ＣＶＢ－ＩｇＭ抗体阳性率明显高于义务献血人员,ＣＶＢ１—６的ＩｇＭ抗体阳性率依次为：ＣＶＢ３、ＣＶＢ４、ＣＶＢ１、ＣＶＢ２、ＣＶＢ５、ＣＶＢ６。结论：从血清学诊断上提示了ＣＶＢ（尤其是ＣＶＢ３）是病毒性心肌炎的重要病原体。     

苦参总碱体外抗柯萨奇B病毒3型作用测定及其机理的初步研究

先用酸水浸泡、氯仿萃取等处理提取苦参总碱，然后通过微量细胞培养法，病毒蛋白质Ｗｅｓｔｅｒｎｂｌｏｔ，分析了不同浓度苦参总碱对病毒增殖和衣壳蛋白含量的影响，表明苦参总碱在体外试验中能抑制柯萨奇Ｂ病毒３型（ＣＶＢ３）的增殖，并呈一定的剂量依赖性。通过病毒吸附和穿入细胞前后苦参总碱作用的比较，显示苦参总碱能进入细胞内发挥抗病毒作用，它可能不影响ＣＶＢ３吸附、穿入等环节，而是影响ＣＶＢ３侵入细胞后某环节，特别是病毒的生物合成。     

脂筏在柯萨奇B3病毒感染心肌细胞中作用

目的 探讨脂筏在柯萨奇B3病毒(CVB3)感染心肌细胞中的作用,旨在为阐明CVB3致病的分子机理及寻找新的抗病毒靶点提供依据。方法 用甲基-β-环糊精(MβCD)去除细胞膜维持脂筏稳定性的胆固醇分子后感染CVB3,用Western blot法检测CVB3 VP1蛋白的表达并测定病毒的滴度,同时观察补充外源性胆固醇恢复MβCD对CVB3感染的抑制作用。结果 用MβCD去除胆固醇分子破坏细胞膜脂筏结构,可抑制CVB3感染细胞;1.2、5.5和10 mmol/L MβCD去除细胞膜胆固醇CVB3滴度分别为(4.2±0.06)、(3.5±1.05)、(3.0±0.15)和(2.0±0.15)lgPFU/mL,均低于无MβCD处理对照组的(5.7±0.06)lgPFU/mL(P<0.001);补充外源性胆固醇可恢复MβCD对CVB3感染的抑制作用。结论 细胞膜脂筏在CVB3感染心肌细胞中起重要作用,是病毒进入细胞的关键因素。