病毒性心肌炎所致小鼠心力衰竭心肌组织内质网应激相关的凋亡关系的研究

目的：探讨病毒性心肌炎心力衰竭小鼠心肌组织内质网应激介导的凋亡途径。方法：40只雄性Balb／c小鼠分为病毒性心肌炎组和正常对照组（n=20）,病毒性心肌炎组应用柯萨奇B3病毒制作BALB／c小鼠病毒性心肌炎模型,观察小鼠的一般情况,7d行血流动力学检查后处死取心脏标本,用TUNEL法检测心肌细胞凋亡,RT-PCR检测心肌细胞内质网伴侣蛋白葡萄糖调节蛋白（GAP）78和GRP04的mRNA表达水平。结果：①与正常对照组相比,病毒性心肌炎组小鼠血流动力学指标明显降低（P〈0．01）;②TUNEL染色显示病毒性心肌炎心力衰竭小鼠心肌组织凋亡明显增多（P〈0．01）;③病毒性心肌炎组小鼠内质网伴侣蛋白GRP78和GRP94的mRNA表达水平均明显高于对照组（P〈0．01）。结论：病毒性心肌炎心力衰竭小鼠内质网应激可能介导了心肌细胞凋亡。     

C-myc蛋白的表达与小鼠病毒性心肌炎心肌细胞凋亡的关系

细胞凋亡（apoPthei一是指在活组织中,单个细胞受其内在基因的编程调控,通过节动的生化过程而‘咱杀”死亡的现象,是机体正常生理过程。近年来研究发现,细胞凋C参与多种疾病的发生与发展。实验通过对病毒性心肌炎（VMC）小鼠心肌细胞调C及凋亡相关基因C－myC蛋白进行检测,旨在探讨VMC、细胞凋亡和C一mp蛋白三者Z间关系。给出几出C／J‘鼠腹腔接种啥鼠心肌100TCIqo柯萨奇B3病毒（CWhm）0．Ind诱发急性病毒性心肌炎,感染病毒gd后,断脊处死小鼠,取心肌组织分别进行石蜡包理及相关检查。光学显微镜检查,发现小鼠心肌组织有广…     

雷公藤甲素对病毒性心肌炎细胞凋亡及Fas/FasL 蛋白表达的研究

目的:研究雷公藤甲素对柯萨奇病毒B3病毒(CVB3)感染的病毒性心肌炎小鼠心肌细胞凋亡和Fas/FasL蛋白表达的抑制作用,探讨TP治疗病毒性心肌炎的作用机制。方法:将Balb/c小鼠随机分成4组作为动物模型,分别为对照组、模型组、利巴韦林组和TP组。对照组腹腔注射生理盐水,其余三组腹腔注射CVB3,利巴韦林组和TP组小鼠分别予以相应的药物治疗后,测定各组小鼠存活率及心肌病变积分,采用末端转移酶标记技术(TUNEL法)检测小鼠心肌细胞凋亡,免疫组化法检测Fas/FasL蛋白阳性表达。结果:空白对照组心肌无病变,利巴韦林组、TP组与模型组相比有显著性差异(P<0.01)。正常组鲜见心肌细胞凋亡,模型组细胞凋亡率较正常组显著增加(P<0.01),治疗组利巴韦林组和TP组凋亡率比模型组明显降低(P<0.05,P<0.01)。模型组Fas/FasL表达比正常组显著增多(P<0.01),治疗组利巴韦林组和TP组较模型组显著降低(P<0.01)。结论:雷公藤甲素具有通过抑制Fas/FasL蛋白的表达,减缓心肌细胞凋亡,达到抑制病毒性心肌炎从而保护心肌细胞的作用。     

氧化应激与病毒性心肌炎

病毒性心肌炎是指由柯萨奇病毒、埃可(ECHO)、脊髓灰质炎、腺病毒,流感病毒等病毒感染引起的心肌局限性或弥漫性的急性或慢性炎症病变,属于感染性心肌疾病。重症易发生恶性心率失常、急性心衰、心源性猝死等,在临床及法医尸检中常常得到证实。在病毒所致的心肌损伤中包括病毒的直接损伤、免疫应答反应、炎细胞的浸润等。近年对于氧化应激与急性病毒性心肌炎的相关性研究越来越深入,已证实活性氧和细胞抗氧化防御机制之间的失衡在病毒性心肌炎的心肌损伤过程中起到了重要作用。本文将综述氧化应激的来源及其在病毒性心肌炎发病机制中的作用和当前抗氧化治疗的现状。     

小鼠外周血心肌特异性microRNAs 的变化与病毒性心肌炎 相关关系初探

目的:通过识别病毒性心肌炎小鼠和正常小鼠血浆中miR-1,miR-133,miR-206表达量的差异,分析外周血中心肌特异性microRNAs的变化与病毒性心肌炎相关关系,为探索miRNAs作为病毒性心肌炎诊断的生物标志物提供可行性的研究资料。方法:在小鼠病毒性心肌炎模型的基础上,采用荧光定量PCR方法,检测病毒性心肌炎急性期小鼠组和正常小鼠组血浆中相关miRNAs含量,并进行统计学分析。再于病毒注射后1d,3d,5d,7d,9d,11d,分别处死病毒小鼠,观察血浆miRNAs的动态变化规律。同时用Elisa检测心肌肌钙蛋白的变化,对目的 miRNAs与心肌肌钙蛋白进行相关性分析。结果:急性期时三种miRNAs血浆含量显著上调。病毒注射后3d开始上升明显,并持续保持在较高水平到7d,于9d时开始下降。发病期间,血浆miRNAs含量与心肌肌钙蛋白呈现良好的正相关性。结论:小鼠外周血中心肌特异性microRNAs在病毒性心肌炎发病过程中的呈现明显上调并与病程和相应指标存在相关关系,为miRNAs作为病毒性心肌炎诊断的生物标志物提供了重要线索。     

武汉地区小儿心肌炎病毒病因初探

本文介绍武汉地区小儿病毒性心肌炎病原调查结果。33例小儿急性心肌炎患者,取其粪便、咽拭子或心肌组织进行病毒分离,并采集患者急性与恢复期血清,用微量中和试验检测对Coxsackie B组6个型病毒的抗体增长情况。结果从26例分离出7株病毒,同时伴有对同型毒株双份抗体四倍或四倍以上增长者4例。1例为CoxsackieB_3,3例为腺病毒。对22例双份血清抗体检测结果:有13例分别对Coxsackie B组2、4、3型抗体有4倍以上增长,阳性率为59％。表明CoxsackieB组病毒为武汉地区小儿病毒性心肌炎重要病因。但腺病毒在病毒性心肌炎中的病原作用亦不容忽视。     

黄芪总黄酮对病毒性心肌炎小鼠心室肌细胞单通道钙电流的影响

目的:观察黄芪总黄酮对病毒性心肌炎小鼠心室肌细胞单通道钙电流的影响,探讨黄芪总黄酮改善病毒性心肌炎小鼠血流动力学机制。方法:36只雄性Balb/c小鼠分为正常对照组、病毒性心肌炎组和黄芪总黄酮组(n=12),病毒性心肌炎组应用柯萨奇B3病毒制作BALB/c小鼠病毒性心肌炎模型,7 d处死小鼠取心脏Langendorff离体心脏逆向灌流法分离小鼠心室肌细胞,应用细胞贴附式方式记录心室肌细胞钙电流,分析对钙单通道电流的影响。结果:黄芪总黄酮组可使病毒性心肌炎小鼠心室肌细胞钙通道开放时间减少,开放概率明显降低(P0.05),关闭时间明显增高(P0.05)。结论:黄芪总黄酮通过降低病毒性心肌炎小鼠心室肌细胞钙通道开放时间、增加关闭时间、降低开放概率抑制心室肌细胞钙电流,抑制心肌细胞钙超载,改善病毒性心肌炎小鼠血流动力学。     

柯萨奇B3病毒性心肌炎的免疫抗炎症研究

病毒性心肌炎(viral myocarditis,VMC)属感染性心肌疾病,可由多种病毒诱导,其中以柯萨奇B组3型病毒(Coxsackie virus B3,CVB3)最为常见。由CVB3引起的VMC典型表现为心肌细胞炎症反应所导致的心肌损伤和坏死,并最终发展为慢性炎症或扩张性心肌病,在人类有很高的发病率和致死率。目前,柯萨奇B3病毒性心肌炎抗炎症治疗措施仍不完善,且相关免疫抗炎症治疗机理未完全阐明,因此探索其免疫抗炎症治疗机理和作用可能成为治疗柯萨奇B3病毒性心肌炎的重要靶点。现主要从Wnt11基因、巨噬细胞、半乳糖凝集素3、锌指抗病毒蛋白、蜂毒素和丙戊酸6个免疫抗炎症相关方面,对柯萨奇B3病毒性心肌炎的最新免疫抗炎症研究予以综述。     

三七总黄酮通过miR-223-3p/FOXO1分子轴缓解病毒性心肌炎炎症反应和细胞损伤

病毒性心肌炎严重影响患者身体健康,中药材中黄酮类物质被证实对病毒性心肌炎有治疗作用,但其中三七总黄酮对柯萨奇B3病毒导致的心肌炎发挥治疗作用的分子机制尚不明确.以探讨三七总黄酮缓解病毒性心肌炎炎症反应及细胞损伤的作用机制.采用RT-qPCR检测心肌细胞中miR-223-3p的表达水平;Western blotting检测心肌细胞中转录因子叉头框蛋白O1(Forkhead box O1,FOXO1)蛋白表达水平;MTT实验检测心肌细胞存活率;流式细胞术检测心肌细胞凋亡率;ELISA检测炎症因子肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)和白细胞介素1β(Interleukin-1β,IL-1β)、心肌酶谱磷酸肌酸激酶(Creative kinase,CK)和乳酸脱氢酶(Lactic dehydrogenase,LDH)、心肌损伤标志物心肌肌钙蛋白T(Cardiac troponin T,cTnT)和B型尿钠肽(Brain natriuretic peptide,BNP)的水平;双荧光素酶报告基因检验miR-223-3p和FOXO1之间的靶向关系.实验结果显示,三七总黄酮能够缓解病毒性心肌炎模型细胞的炎症反应及细胞损伤,并显著上调模型细胞中miR-223-3p的水平.敲除模型细胞中的miR-223-3p能够逆转三七总黄酮对病毒性心肌炎的治疗作用.通过双荧光素酶报告基因实验验证miR-223-3p靶向负调控FOXO1蛋白的表达.进一步研究发现,过表达FOXO1可抑制三七总黄酮对病毒性心肌炎的治疗作用;但同时过表达miR-223-3p后,过表达FOXO1对三七总黄酮疗效的抑制作用被逆转.由此得出结论,三七总黄酮可缓解病毒性心肌炎模型细胞的炎症反应及细胞损伤,其作用机制是通过调控miR-223-3p/FOXO1分子轴实现的.     

心肌肌钙蛋白T在病毒性心肌炎心肌组织中的表达及意义

目的:了解病毒性心肌炎心肌组织中肌钙蛋白T的表达情况,探讨病毒性心肌炎时心肌结构蛋白损伤的机制及意义。方法:运用免疫组化和计算机图像分析技术,观察13例明确性病毒性心肌炎和17例界限性病毒性心肌炎尸检心脏标本中心肌肌钙蛋白T的表达与分布。结果:在正常对照的心肌组织中,蛋白成强阳性表达,分布均匀,未见缺染。在明确性心肌炎及14例界限性心肌炎心肌组织中都存在着不同程度的蛋白表达缺染或脱失。缺染的范围及分布与病毒性心肌炎病变特点基本一致,但其范围往往小于炎症细胞浸润范围。计算机图像分析和数据统计结果显示缺染区域的心肌肌钙蛋白T表达量要明显小于其周边区域和正常心肌细胞(P＜0．01)。结论:病毒性心肌炎患者的心肌损害要早于炎症细胞的浸润,病毒的作用可能是心肌肌钙蛋白T脱失的主要因素。心肌肌钙蛋白T的免疫组化检查可以作为一种有效的手段,来辅助病毒性心肌炎的病理学诊断。     

miRNA378*表达对柯萨奇B3病毒感染乳鼠心肌细胞凋亡、网腔钙结合蛋白、内质网应激伴侣蛋白表达的影响

目的：研究沉默miRNA378^*表达对柯萨奇B3病毒（CVB3）感染心肌细胞凋亡、内质网应激、网腔钙结合蛋白（calumenin）影响。方法：原代培养乳鼠心肌细胞分为：对照组（正常细胞）、柯萨奇病毒感染组（正常细胞+柯萨奇B3病毒）、miRNA378^*沉默对照组（正常细胞+柯萨奇B3病毒+转染miRNA378^*空质粒）、miRNA378^*沉默组（正常细胞+柯萨奇B3病毒+转染miRNA378^*沉默质粒）,各组细胞分别转染和感染处理后置37℃、CO₂培养箱中培养3 d。检测细胞α-平滑肌肌动蛋白（α-SMA）、细胞凋亡率、网腔钙结合蛋白、葡萄糖调节蛋白78（GRP78）及内质网应激信号通路因子激活转录因子6（ATF6）、转录因子C/EBP同源蛋白（CHOP）的表达。结果：通过检测ɑ-SMA蛋白,证实分离乳鼠细胞为心室肌细胞。TUNEL法检测不同组心室细胞凋亡情况发现,柯萨奇病毒感染组心室肌细胞凋亡明显,与柯萨奇病毒感染组心肌细胞相比较,miRNA378^*沉默组心肌细胞凋亡细胞量明显减少。与柯萨奇病毒感染组比较,Calumenin表达减少（P<0.01）,而GRP78、ATF6、CHOP表达增加（P<0.01）。结论：CVB3病毒感染心肌细胞作用与miRNA378^*,引发内质网应激并激活信号通路因子,心肌细胞凋亡增加。     

Interferon treatment reduced adherence, invasiveness and intracellular multiplication of Shigella flexneri in coxsackie B1 virus-infected cells.

The effect of interferon treatment on interaction of Shigella flexneri with in vitro cultured cells was investigated. Pretreatment of HEp-2 cells with human interferons had no effect on the susceptibility of cells to S. flexneri, measured by invasiveness and adhesiveness. Human leukocyte interferon and human recombinant interferon-alpha-A reduced adhesiveness, intracellular multiplication and invasiveness of S. flexneri in HEp-2 cells preinfected with coxsackie B1 virus. Also non-receptor mediated-phagocytosis was reduced by interferon treatment in virus infected cells. The interferon effects were dependent on continuous protein synthesis, because they were not expressed when cycloheximide or abrin was added to the virus infected cell cultures. No effect of interferon was detected on intracellular content of Na+ or K+, Na(+)-K+ activated ATPase activity or cytoplasma membrane polarity, in virus infected or control cell cultures. The interferon effect on bacterial invasiveness seems to be dependent on an interferon receptor interaction on cytoplasma membrane level because directly microinjected interferon showed no effect.     

柯萨奇B组病毒感染与胰岛素依赖型糖尿病关系的研究

探讨柯萨奇B组病毒(CBV)感染与胰岛素依赖型糖尿病之间的关系。用PT-PCR方法检测38例IDDM患者外周血中CBV-RNA,同时用ELISA法测定血清中CBV特异性IgM。结果显示,病例组38例患者外周血CBV-RNA阳性率为42.10％,血清中CBV-IgM阳性率为36.84％。对照组的阳性率分别为7.5％和5％。两组比较有显著性差别(P<0.05)。说明胰岛素依赖型糖尿病与柯萨奇B组病毒感染有关。     

Roles for the E4 orf6, orf3, and E1B 55-Kilodalton Proteins in Cell Cycle-Independent Adenovirus Replication

Adenoviruses bearing lesions in the E1B 55-kDa protein (E1B 55-kDa) gene are restricted by the cell cycle such that mutant virus growth is most impaired in cells infected during G(1) and least restricted in cells infected during S phase (F. D. Goodrum and D. A. Ornelles, J. Virol. 71:548-561, 1997). A similar defect is reported here for E4 orf6-mutant viruses. An E4 orf3-mutant virus was not restricted for growth by the cell cycle. However, orf3 was required for enhanced growth of an E4 orf6-mutant virus in cells infected during S phase. The cell cycle restriction may be linked to virus-mediated mRNA transport because both E1B 55-kDa- and E4 orf6-mutant viruses are defective at regulating mRNA transport at late times of infection. Accordingly, the cytoplasmic-to-nuclear ratio of late viral mRNA was reduced in G(1) cells infected with the mutant viruses compared to that in G(1) cells infected with the wild-type virus. By contrast, this ratio was equivalent among cells infected during S phase with the wild-type or mutant viruses. Furthermore, cells infected during S phase with the E1B 55-kDa- or E4 orf6-mutant viruses synthesized more late viral protein than did cells infected during G(1). However, the total amount of cytoplasmic late viral mRNA was greater in cells infected during G(1) than in cells infected during S phase with either the wild-type or mutant viruses, indicating that enhanced transport of viral mRNA in cells infected during S phase cannot account for the difference in yields in cells infected during S phase and in cells infected during G(1). Thus, additional factors affect the cell cycle restriction. These results indicate that the E4 orf6 and orf3 proteins, in addition to the E1B 55-kDa protein, may cooperate to promote cell cycle-independent adenovirus growth.     

猪流行性腹泻病毒经其附属蛋白抑制细胞凋亡

【背景】orf3位于猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV) s基因与e基因之间,是目前发现的PEDV唯一一个附属基因,编码附属蛋白(ORF3蛋白)。我们前期研究初步发现ORF3蛋白对PEDV诱导的细胞凋亡有影响。【目的】研究ORF3蛋白在PEDV侵染复制过程中的毒力作用机制。【方法】实验用3种PEDV:rDR13att-?ORF3 (orf3基因全部敲除)、DR13-ORF3att (携带有C端截短orf3)、rDR13att-ORF3wt(携带全长orf3基因)感染Vero细胞,观察病变情况,再用活细胞成像仪、流式细胞仪、 DNA断裂的原位末端标记法[terminaldeoxynucleotidyltransferase(TDT)-mediated dUTP nick end labeling,TUNEL]等方法检测不同感染时间点的细胞凋亡情况,然后用蛋白质印迹方法分析PEDV感染宿主细胞中主要凋亡相关蛋白(如Caspase-3)的活化或裂解,最后进行转录组测序研究病毒感染细胞中差异基因的表达情况,再用荧光定量PCR验证转录组结果。【结果】rDR13att-?ORF3引起较多的细胞病变,活细胞成像仪的动态观察结果显示,3种病毒侵染的细胞凋亡水平随着时间的延长均高于正常阴性细胞,但敲除orf3的病毒感染细胞后细胞凋亡率比其他两种病毒更高;敲除orf3病毒感染细胞凋亡率显著高于其他两种病毒;病毒rDR13att-?ORF3感染细胞后TUNEL阳性细胞数比DR13-ORF3att和rDR13att-ORF3wt更多;表达ORF3蛋白的重组PEDV可以抑制Caspase-3的活化;ORF3蛋白对受感染细胞Heat shock 70 kD protein 1B (HSP70)基因转录有促进作用,荧光定量PCR结果表明rDR13att-ORF3wt感染细胞的HSP70表达量高于rDR13att-?ORF3感染细胞。【结论】PEDV通过ORF3蛋白抑制细胞凋亡,而且这种作用可能是通过抑制Caspase-3的活化或增加HSP70的产生来完成的。     

Interaction with decay-accelerating factor facilitates coxsackievirus B infection of polarized epithelial cells

All coxsackie B (CB) viruses can initiate infection by attaching to the coxsackievirus and adenovirus receptor (CAR). Although some CB isolates also bind to decay-accelerating factor (DAF), the role of DAF interaction during infection remains uncertain. We recently observed that CAR in polarized epithelial cells is concentrated at tight junctions, where it is relatively inaccessible to virus. In the experiments reported here we found that, unlike CAR, DAF was present on the apical surface of polarized cells and that DAF-binding isolates of CB3 and CB5 infected polarized epithelial cells more efficiently than did isolates incapable of attaching to DAF. Virus attachment and subsequent infection of polarized cells by DAF-binding isolates were prevented in the presence of anti-DAF antibody. Serial passage on polarized cell monolayers selected for DAF-binding virus variants. Taken together, these results indicate that interaction with DAF on the apical surface of polarized epithelial cells facilitates infection by a subset of CB virus isolates. The results suggest a possible role for DAF in infection of epithelial cells at mucosal surfaces.     

Glycoprotein D or J delivered in trans blocks apoptosis in SK-N-SH cells induced by a herpes simplex virus 1 mutant lacking intact genes expressing both glycoproteins

We have made two stocks of a herpes simplex virus 1 mutant lacking intact U(S)5 and U(S)6 open reading frames encoding glycoproteins J (gJ) and D (gD), respectively. The stock designated gD(-/+), made in cells carrying U(S)6 and expressing gD, was capable of productively infecting cells, whereas the stock designated gD(-/-), made in cells lacking viral DNA sequences, was known to attach but not initiate infection. We report the following. (i) Both stocks of virus induced apoptosis in SK-N-SH cells. Thus, annexin V binding to cell surfaces was detected as early as 8 h after infection. (ii) U(S)5 or U(S)6 cloned into the baculovirus under the human cytomegalovirus immediate-early promoter was expressed in SK-N-SH cells and blocked apoptosis in cells infected with either gD(-/+) or gD(-/-) virus, whereas glycoprotein B, infected cell protein 22, or the wild-type baculovirus did not block apoptosis. (iii) In SK-N-SH cells, internalized, partially degraded virus particles were detected at 30 min after exposure to gD(-/-) virus but not at later intervals. (iv) Concurrent infection of cells with baculoviruses did not alter the failure of gD(-/-) virus from expressing its genes or, conversely, the expression of viral genes by gD(-/+) virus. These results underscore the capacity of herpes simplex virus to initiate the apoptotic cascade in the absence of de novo protein synthesis and indicate that both gD and gJ independently, and most likely at different stages in the reproductive cycle, play a key role in blocking the apoptotic cascade leading to cell death.     

改变细胞膜的脂肪酸组成可促进乳腺癌细胞凋亡

目的: 研究n-6脂肪酸脱氢酶 fat-1基因在人乳腺癌细胞内的表达,改变细胞膜脂肪酸组成,对乳腺癌细胞的凋亡作用。方法: 构建含有fat-1 基因的重组腺病毒载体 (Ad.GFP.fat-1),通过包装细胞系（293）产生的腺病毒,感染人乳腺癌细胞MCF-7。提取细胞的总RNA,以fat-1的反义mRNA 作探针,用Northern Blot检测fat-1 基因在MCF-7细胞内的表达。MTT法分析fat-1 基因对MCF-7细胞增殖的影响,凋亡染色试剂盒检测细胞的凋亡。气相色谱仪分析对MCF-7细胞的n-6 PUFAs/n-3 PUFAs含量影响。结果: 通过基因重组技术,得到预期的重组病毒;fat-1 基因在人乳腺癌细胞MCF-7 中能有效异源表达,2天后,可检测到fat-1 mRNA的条带。与对照细胞相比,fat-1基因有效地抑制了MCF-7细胞的增殖（23%,p<0.05）,促进了凋亡（增加35%）;同时降低了人乳腺癌细胞MCF-7细胞膜n-6 PUFAs/n-3 PUFAs的比率。结论: 腺病毒介导的fat-1 基因能在人乳腺癌细胞MCF-7内有效异源表达,且抑制了MCF-7细胞的增殖。机理为降低了细胞膜的n-6 PUFAs/n-3 PUFAs的比率。     

Primary study on the lesions and specific proteins in BEAS-2B cells induced with the 2009 A (H1N1) influenza virus

In order to investigate the lesions and proteins with differential expression in cells infected with the 2009 A (H1N1) virus and to determine the specific proteins involved in cell damage, the present study has been performed. BEAS-2B cells were infected with the 2009 A (H1N1) influenza virus or the seasonal H1N1 influenza virus for 12, 24, 48, and 72 h, and cell cycle and apoptosis were analyzed with flow cytometry. Total cellular proteins were extracted and underwent two-dimensional gel electrophoresis. The differentially expressed proteins underwent mass spectrometry for identification. The results showed that after 12 h, cells infected with the virus strain sourced from severe cases had the highest apoptosis rate (P?P?P?Galectin-1 was specifically observed in BEAS-2B infected with 2009 A (H1N1) influenza viruses, and cofilin-1 was specifically observed in BEAS-2B cells in the late stage of 2009 A (H1N1) influenza virus infection. In conclusion, differential effects of the 2009 A (H1N1) influenza virus and seasonal H1N1 influenza virus were identified on the cell cycle and apoptosis, and galectin-1 may play a role in cell apoptosis induced by 2009 A (H1N1) influenza virus.