黄芪三萜皂苷对CVB3诱导病毒性心肌炎的保护作用研究

目的：研究黄芪三萜皂苷（Astragalus saponins,AST）对CVB3病毒诱导病毒性心肌炎的保护作用及机制。方法：利用CVB3病毒、原代心肌细胞及小鼠构建病毒性心肌炎模型并使用AST进行干预,在实验过程中记录小鼠的生存率和体质量变化,体外超声评价小鼠的心脏功能及检测心肌蛋白中LDH和CK-MB水平。心肌组织天狼星红染色评价纤维化水平及TUNNEL染色检测心肌凋亡情况。Western Blot检测心肌蛋白中Caspase-3和Fas表达情况以研究AST保护病毒性心肌炎的机制。结果：AST能够显著增加CVB3注射后的小鼠生存率、缓解CVB3诱导小鼠的体质量减轻。体外超声结果显示,AST能够显著改善CVB3诱导小鼠心脏的收缩功能障碍。CBV3诱导组小鼠心肌蛋白中LDH和CM-KB水平较正常小鼠显著增加,而AST对此有显著的抑制作用。心肌组织病理染色结果显示,AST对CVB3诱导的心肌扩张和纤维化具有显著的保护作用。CVB3诱导组小鼠心肌组织的凋亡水平显著高于正常小鼠,而AST对此有显著的抑制作用。在CVB3感染的心肌组织中,Caspase-3和Fas表达水平显著的升高,而AST能够显著的抑制CVB3诱导的二者在心肌组织中的表达。结论：AST通过提高小鼠的生存率、抑制心肌扩张、心肌组织纤维化和心肌细胞凋亡对CVB3诱导的病毒性心肌炎具有显著的保护作用,这种作用可能与抑制CVB3诱导的Caspase-3和Fas在心肌组织中表达有关。     

Modulation of innate immunity in human pancreatic islets infected with enterovirus in vitro

Present knowledge of innate immunity in infected cells relies on studies of cell lines and animal models. In this study, primary human pancreatic islets of Langerhans were used to study virus-host interactions in a model of the possible induction of type 1 diabetes by enterovirus (EV). Human islets were infected with a strain of EV isolated at onset of type 1 diabetes, or exposed to synthetic dsRNA (poly(I:C)), used commonly to mimic viral infection. Induction of innate immunity and the effect of the female sex hormone 17β-estradiol, known to have cell-protective effects, on islet chemokine secretion were investigated. 17β-Estradiol reduced EV-but not poly(I:C)-induced IP-10/CXCL10 secretion from human islets, suggesting that separate signaling pathways of the innate immune response are triggered by EV and poly(I:C), respectively. Infection with EV increased the gene-expression of toll-like receptor 3, interferon-β, and the intracellular helicase MDA5, involved in antiviral innate immunity, multi-fold over time, whereas poly(I:C) increased the expression of these genes transiently. The induced expression pattern was similar in all donors, but the expression levels varied greatly. Pre-exposure to poly(I:C) blocked viral replication in islets from 56% of the donors. These data provide insight on the innate immune responses induced by EV in human islets, and show that this can be modulated by 17β-estradiol, and suggest an important difference between virus- and poly(I:C)-induced signaling.     

穿孔素在慢性心肌损伤中的表达及其意义

目的：探讨穿孔素在阿萨奇病毒B组3型（CVB3m)至T－2毒素染毒小鼠心肌损伤中的表达及其意义,方法：实验组BALB／C小鼠1．0mg/kg体重T－2毒素隔日灌胃,3周后腹腔接种0．1ml内含1000TCID50的CVB3m病毒液,对照组生理盐水灌胃,腹腔接种0．1ml 1640营养液,观察心肌病理改变,同时用RTPCR技术对心肌组织中CVB3m RNA和穿孔素mRNA表达进行检测,结果：CVB3m感染T－2毒素染毒小鼠心肌出现慢性损伤,可见纤维结缔组织增生和坏死灶癜痕修复。心肌组织中持续表达CVB34m RNA和穿孔素mRNA。结论CVB3m RNA和穿孔素mRNA的持续表达可能是CVB3m致T－2毒素染毒小鼠心肌慢性损伤的机制之一。     

柯萨奇B3m病毒致低硒低维生素E鼠心肌损伤的实验研究

目的 探讨柯萨奇（CV）B3m感染低Se低VE鼠心肌损伤的发病特点。方法 1周龄乳鼠分别给予低Se低VE和补Se常VE饲料喂养,4周后腹腔接种病毒,对照组接种等量PRMI1640培养液,7d后处死,光镜下观察心肌的病理改变。结果 CVB3m感染的Se和VE均缺乏鼠的心肌病变检出率为93．3％,病变为多发性大面积灶状坏死,融合成片,炎性细胞较少浸润,与急性克山病的病理改变类似;而补Se常VE组仅见散发间质炎性灶,面积较小,伴有大量的炎细胞,检出率为16．7％。结论 低硒低维生素E状态下,柯萨奇病毒B3m可显著加重小鼠的心肌损伤。     

酵母双杂交筛选与CVB3 3A相互作用的人心脏蛋白

目的 以CVB3非结构蛋白3A为诱饵,从人心脏cDNA文库中筛选与其相互作用的宿主蛋白,对阳性cDNA克隆进行初步分析和鉴定。方法 构建pGBKT7-3A重组质粒, Western Blot检测3A融合蛋白的表达;利用酵母双杂交技术筛选人心脏cDNA文库,经PCR扩增cDNA插入片段和Alu I酶切等实验将阳性克隆归类,并通过测序、相似性比对分析和回返配合实验去除假阳性蛋白。结果 成功构建诱饵质粒pGBKT7-3A,Western Blot结果表明,pGBKT7-3A重组质粒在酵母菌中成功表达3A融合蛋白;经双杂交筛选和回返配合实验,获得了7个相互作用的蛋白,分别为:兰尼碱受体2(Ryanodine Receptor 2,RyR2),信号肽酶 2(Signal peptidase complex subunit 2 ,SPCS2),跨膜蛋白emp24(Transmembrane emp24 protein transport domain containing 4,TMED4),细胞色素c氧化酶亚基I(Cytochrome c oxidase subunit I ,COX1),细胞色素c氧化酶亚基III(Cytochrome c oxidase subunit III,COX3),肌球蛋白重链(Myosin heavy chain,MYH7)和琥珀酸脱氢酶亚基D(Succinate dehydrogenase complex subunit D,SDHD)。结论 应用酵母双杂交技术,以CVB3 3A为诱饵蛋白,从人心脏cDNA文库中获得7种不同的阳性基因: RyR2 、SPCS2 、TMED4、COX1、COX3、MYH7和 SDHD,在分子水平上探索3A蛋白的新功能。     

CAR在柯萨奇B3病毒感染心肌细胞中作用

目的探讨柯萨奇病毒和腺病毒受体(coxsackievirusandadenovirusreceptor,CAR)在嗜心性柯萨奇B3病毒(myocarditiccoxsaekievirusB3,CVB3m)感染心肌细胞中的作用。方法采用体外培养新生小鼠心肌细胞方法,建立CVB3m感染心肌细胞模型,将培养48h的心肌细胞分为对照组、CVB3m组和CAR抗体 CVB3m组,并作相应处理,继续培养48h后,分别观察各组心肌细胞病变效应(cytopathiceffect,CPE),并用四唑盐(MTT)比色法测定各组心肌细胞存活力。结果CAR抗体 CVB3m组心肌细胞CPE明显减轻,存活力显著增强,与CVB3m组比较差异显著(P<0.01),与对照组比较差异无显著性(P>0.05)。结论CAR抗体对CVB3m感染心肌细胞具有保护作用,提示CAR在CVB3m感染心肌细胞中可能发挥重要的介导作用。     

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。     

黄芪对心肌炎小鼠心肌穿孔素mRNA和CVB3m mRNA表达的影响

目的探讨黄芪注射液治疗小鼠柯萨奇B3(CVB3)病毒性心肌炎(VMC)的疗效及其机制。方法BALB/c小鼠24只腹腔感染柯萨奇病毒B3亲心肌细胞株(CVB3m)后,随机等分为两组:黄芪治疗组(黄芪注射液每天10g/kg腹腔注射)和对照组。实验第8天留取心肌,分别进行心肌病理检查,心肌穿孔素(perforin,PFP)mRNA和CVB3m mRNA逆转录多聚酶链反应(RT-PCR)分析,并对心肌组织PFP mRNA和CVB3m mRNA表达水平进行相关性分析。结果(1)心肌组织病理改变:黄芪治疗组明显轻于对照组;(2)心肌PFP mRNA表达水平RT-PCR半定量:黄芪治疗组心肌PFP mRNA的表达明显低于对照组(1.10±0.07与1.31±0.12,P<0.01);(3)心肌CVB3m mRNA表达水平RT-PCR半定量:黄芪治疗组心肌CVB3m mRNA表达明显低于对照组(1.07±0.04与1.18±0.02,P<0.01);(4)心肌PFP mRNA水平与CVB3mmRNA水平呈显著正相关(r=0.68,P<0.01)。结论PFP介导的细胞毒性作用在病毒性心肌炎的发病中起重要作用。黄芪对小鼠急性心肌炎模型中心肌组织CVB3m病毒复制有明显的抑制作用,心肌组织中PFP mRNA表达水平与心肌组织CVB3m mRNA的表达水平的变化具有同步性,黄芪对CVB3m诱导的小鼠急性心肌炎模型有显著的治疗作用。     

CVB3感染通过MCP-1介导病毒性心肌炎小鼠单个核细胞迁移

目的:研究B3型柯萨奇病毒(CVB3)感染与病毒性心肌炎(VMC)炎症细胞迁移的关系及机制。方法:采用差异贴壁法分离小鼠心肌细胞;趋化试验分析心肌细胞培养上清对VMC小鼠外周血单个核细胞(PMNC)的趋化性;实时定量RT PCR分析心肌细胞MCP 1的表达。结果:( 1)CVB3感染心肌细胞培养上清对VMC小鼠PMNC的趋化性明显增强。( 2 )CVB3感染2小时后MCP 1的表达开始升高,至6小时达到高峰,8小时下降;随着CVB3感染量的增加,MCP 1的表达明显升高。( 3)2 5 μg/ml抗MCP 1抗体处理后,CVB3感染心肌细胞培养上清对VMC小鼠PMNC的趋化性下降5 4 % (P <0 0 1) ;5 μg/ml抗MCP 1抗体处理后,趋化性下降的幅度与2 5 μg/ml抗MCP 1抗体处理的相比未见明显差异(P >0 0 5 )。结论:CVB3感染通过MCP 1介导VMC小鼠单个核细胞迁移,这可能是CVB3感染诱发心肌组织炎症细胞浸润的重要机制之一     

Experimental CVB3-induced chronic myocarditis in two murine strains: Evidence of interrelationships between virus replication and myocardial damage in persistent cardiac infection

In order to analyse the relationships between enteroviral replication and the myocardial damage at the onset of chronic cardiac infection, 2 mouse strains with different degrees of immunological competence (NMRI nu/nu, DBA/2) were infected by a myocarditic Coxsackie virus B3 (CVB3-M1) variant. At 31 days post-inoculation, plaque-forming assay, polymerase chain reaction (RT-PCR), and immunohistochemistry were carried out for detecting viruses and viral components in the myocardium. The virological findings were related to histopathological changes in the myocardium as well to the dilatation of both cardiac ventricles. Chronic myocardial lesions characterized by large fibrosis areas and interstitial inflammatory infiltrates were detected together with cardiomegalia in 52.6% (10/19) of athymic mice and in 9% (2/22) of euthymic mice. Viral replication foci were located and were found only in myocarditic cells adjacent to myocardial inflammatory lesions by immunostaining myocardial tissue sections with anti-serum to VP1 virus capsid protein. Using PCR followed by microwell capture hybridization assay, a large excess of viral positive strand RNA over negative strand was semiquantified in heart tissue from mice with chronic myocarditis, whereas approximately equal amounts of plus and minus strand RNA were detected in cases of persistent cardiac infection without chronic myocardial injuries. These findings provide evidence of the major role of viral replication in the pathogenesis of chronic murine CVB3-induced cardiomyopathy. The results indicate that the cardiac persistence of enteroviral RNAs can be observed without chronic cardiomyopathy, which could be explained by a defective viral positive RNA replication. J. Med. Virol 52:206–214, 1997. © 1997 Wiley-Liss, Inc.