低硒小鼠病毒性心肌炎与细胞凋亡关系的研究

目的　探讨低硒小鼠病毒性心肌炎与细胞凋亡的关系。方法　应用低硒和常硒合成饲料喂养 5周龄BALB/C雄性小鼠 5周后 ,经腹腔接种柯萨奇B3m病毒 (CVB3m) 10 3 TCD50 0 .1ml,建立小鼠心肌炎模型。对照组腹腔注射PRM164 0。通过此模型 ,采用原位末端标记法 (TUNEL法 )测定心肌凋亡细胞 ,并采用免疫组化法测定凋亡相关基因C -myc、Bcl -2及相关因子TGF -β1的表达。结果　光镜下低硒病毒组 (Ⅰ组 ) ,常硒病毒组 (Ⅱ组 )病变检出率分别为 75 %和 3 5 % ,常硒对照组 (Ⅲ组 )为 0 ,经 χ2 检验Ⅰ组显著高于Ⅱ组 (P <0 .0 5 )。对低硒及常硒病毒组的心肌采用TUNEL法检测发现凋亡细胞 ,Ⅰ组小鼠心肌中有凋亡者占 75 % ,Ⅱ组有凋亡者占 5 5 % ,Ⅲ组未见凋亡细胞。采用免疫组化法检测发现 ,Ⅰ组鼠心肌中可见C -myc和TGF -β1的阳性表达 ,分布区域与TUNEL法标记的凋亡细胞一致。Ⅲ组心肌中可见BCL -2基因表达产物 ,而Ⅰ、Ⅱ组中很少见。结论　本实验结果提示低硒能促进病毒感染引起的心肌细胞凋亡 ,并能促进C -myc、TGF -β1的表达 ,抑制BCL -2表达。     

小鼠病毒性心肌炎与心肌细胞凋亡的关系

实验通过给５周龄ＢＡＬＢ／Ｃ小鼠腹腔接种Ｃｏｘｓａｃｋｉｅ Ｂ３ｍ病毒（ＣＶＢ３ｍ），诱发小鼠急性病毒性心肌炎（ＶＭＣ），感染病毒９天后处死小鼠，利用光镜、电镜及原位末端标记法对心肌组织进行检测，结果显示：ＶＭＣ检出率为９３．３３％，心肌细胞凋亡阳性率为８０．００％，凋亡细胞多分布于心内膜下、心外膜下心肌组织，血管内皮细胞和坏死病灶周围，提示细胞凋亡可能是小鼠ＶＭＣ的发病机制之一。     

细胞因子和病毒性心肌炎的关系

本文着重阐述了细胞因子在病毒性心肌炎发生，发展中的作用。     

急性病毒性心肌炎小鼠相关基因差异表达的研究

目的应用基因表达谱芯片筛选急性病毒性心肌炎相关基因,并对这些基因的功能进行初步分析。方法将8 464条小鼠PCR产物按微聚阵排列点样于化学涂层的载玻片上,制成基因芯片;用柯萨奇B3 (CVB3)病毒感染BALBc小鼠,在感染后的第4、8、21天,分别将正常和病毒性心肌炎心肌中mRNA逆转录,合成荧光分子(cy3／cy5)掺入的cDNA链制备表达谱探针,芯片杂交和严格洗片后,用ScanArray3000荧光扫描芯片荧光信号图像,利用计算机分析病毒性心肌炎心肌与正常心肌中丝氨酸苏氨酸磷酸化酶基因(PIM-3)、血管形成因子基因(Ang)-1、组织相容性基因-2的表达,并对获得的基因进行生物信息学分析。结果小鼠在CVB3感染后的第4、8、21天,心肌中分别发现了1 684、284、98条差异表达基因:PIM-3、Ang-1、组织相容性基因-2的表达被持续抑制。结论应用基因表达谱芯片可筛选出差异表达基因,PIM-3、Ang-1、组织相容性基因-2的差异表达可能与病毒性心肌炎发生和发展有关。     

钙在CVB3诱导大鼠心肌细胞凋亡中的作用

目的 探讨细胞内游离钙〔Ｃａ＾２＋〕ｉ在柯萨奇病毒Ｂ３（ＣＶＢ３）诱导培养心肌细胞凋亡中的作用。方法 ＤＮＡ裂点检测法（３’－末端标记）及透射电镜检测细胞凋亡。Ｆｌｕｏ３－ＡＭ负主肌细胞,共聚焦显微镜观察〔Ｃａ＾２＋〕ｉ荧光强度变化。结果感染２４ｈ心肌细胞内ＣＶＢ３滴度达峰值。感染１０ｈ未见凋亡的心肌细胞,１７、２４和３６ｈ凋亡细胞分别为５％、６０％和９０％,感染１７ｈ心肌〔Ｃａ＾２＋〕ｉ浓度达     

病毒性心肌炎细胞感染模型的建立及实验研究

目的：建立病毒性心肌炎细胞感染模型。方法：以柯萨奇B1病毒感染原代培养的乳鼠心肌细胞，镜下观察心肌细胞搏动次数、细胞病变、测定DNA含量及MTT活细胞染色。结果：采用差速贴壁法获得的活心肌细胞数〉90％（台盼蓝染色），心肌细胞多核、多形性特征明显（HE染色）；感染CVB324小时后，心肌搏动频率紊乱，感染48小时至一周后心肌搏动次数明显减少（P〈0．01），随后肌丝断裂，细胞圆缩、脱落，细胞病变程度与MTT染色OD值呈明显负相关。细胞周期分析显示细胞受阻于G1期，有凋亡峰出现。结论：乳鼠心肌细胞原代培养及CVB3感染方法的建立，可作为研究病毒性心肌炎药物筛选和治疗的重要体外模型。     

信号转导和转录激活子3与病毒性心肌炎的关系

目的:探讨信号转导和转录激活子3(STAT3)在病毒性心肌炎(VMC)发病机制中的作用.方法:以柯萨奇病毒B3感染balb/c小鼠,建立急慢性VMC的系列动物模型;经组织病理学方法证实模型后,采用免疫印迹法(Western blot)和免疫组化检测心肌组织细胞质和细胞核中STAT3和磷酸化STAT3(p-STAT3)蛋白.结果:Western-blot和免疫组化法显示柯萨奇病毒B3感染小鼠心肌细胞核STAT3和p-STAT3蛋白含量较对照组明显增高(P＜0.05).细胞质STAT3和p-STAT3蛋白含量与对照组比较差异无统计学意义(P＞0.05).结论:VMC时心肌细胞核STAT3和p-STAT3蛋白明显增高,与其发病密切相关.     

黄芪注射液治疗Coxsackie B病毒性心肌炎的临床疗效

<正> 1993午1月—1996年12月,我们用黄芪注射液治疗Coxsackie B病毒性心肌炎32例,并与单用西药组36例作比较,观察临床疗效及探讨其作用机理.现报告如下.     

盐酸阿比朵尔对病毒性心肌炎小鼠的治疗作用

目的探讨盐酸阿比朵尔对病毒性心肌炎的治疗作用。方法以柯萨奇B3病毒构建小鼠病毒性心肌炎模型,灌胃给予盐酸阿比朵尔药液(高、中、低)3个剂量治疗7 d。然后观察各组小鼠生存率、血清心肌酶活性、心脏匀浆的病毒滴度以及心脏切片的病理变化并计算病理积分。结果与病毒对照组比较,治疗组的小鼠生存率明显增加,心肌病毒繁殖量明显降低,心肌酶活性明显下降,心肌炎病灶明显减轻。结论盐酸阿比朵尔对病毒性心肌炎具有一定的治疗作用。     

Intracardiac thrombus in murine coxsackievirus B3 myocarditis

Summary We studied the appearance of intracardiac mural thrombi with time and the relationship between thrombosis and congestive heart failure (CHF) in murine coxsackievirus B3 (CB3) myocarditis. Fourto six-week-old C₃H/He mice were inoculated intraperitoneally with CB3 and were observed for 90 days. Mice were sacrificed periodically on days 4, 8, 14, 30, and 90. Among 129 mice with myocarditis, 35 (27.1%) developed CHF and 40 (31.0%) demonstrated thrombi after day 8. The total incidence of thrombosis was significantly higher in mice with CHF (71.4%; 25/35) than in those without CHF (16.0%; 15/94) (P < 0.001). The present study suggests that CB3 myocarditis carries a significant risk of thromboembolism, and that CHF is a risk factor for the appearance of thrombi.This work was supported by research grants of the Japanese Heart Foundation, Education Fellowship of Toyama Medical and Pharmaceutical University, Iwaki Fellowship, Japanese Education of Science and Welfare (Nos. 01570478 and 03670445), and the Uehara Memorial Foundation     

Lymphokine activated killer cells in murine coxsackievirus B3 myocarditis

The purpose of the present study was to determine whether lymphokine activated killer (LAK) cells were involved in the development of coxsackievirus B3 (CB3) myocarditis in both the acute viremic (Experiment I) and the subacute aviremic (Experiment II) stages. To induce LAK cells, recombinant human interleukin-2 (IL-2) was administered to CB3-infected mice subcutaneously daily, starting on day 0 in Experiment I and on day 7 in Experiment II for 7 days, respectively. The treated groups were compared to infected controls. Splenic lymphocytes of IL-2 treated mice were further cultured in vitro in IL-2 containing medium for 7 days, and LAK cell activity, i.e., cytotoxic activity of the lymphocytes against EL-4 tumor cells and against cultured fetal myocytes, was assayed by⁵¹Cr-release method. In Experiment I, histologic scores, myocardial virus titers, and LAK cell activity did not differ significantly between IL-2 treated and untreated groups. In contrast, in Experiment II, there were more cellular infiltration associated with severe necrosis and higher LAK cell activity against EL-4 cells and cultured myocytes in IL-2 treated than in untreated groups. The presence of LAK cells was demonstrated in the subacute stage of murine CB3 myocarditis. Thus, the behavior of LAK cell activity may vary with the course of myocarditis, and enhanced LAK cell activity may be involved in the development of the disease.This work was supported by research grants from the Conference on Coronary Artery Disease, Japanese Education of Science and Walfare (Nos. 08877110 and 09470164), Kanae Shinyaku Foundation, and Japan Cardiovascular Research Foundation.     

Effect of metoprolol in acute coxsackievirus B3 murine myocarditis

Recent studies suggest that beta-adrenergic blocking agents show promise in the management of cardiomyopathies; however, their role in acute myocarditis is unknown. One hundred 3 week old mice were infected with coxsackievirus B3 and were given either metoprolol (n = 50) or normal saline solution (n = 50) intraperitoneally for 10 days. Twenty mice from each group were observed for mortality for 30 days. Of the remaining 60 mice, 10 from each group were killed on day 3, 6 or 10 and examined for heart viral titers and pathologic changes. Mortality rate in the metoprolol group was 60% compared with 0% in the saline group (p less than 0.005). Viral titers on day 10 of infection were 10(2.6 +/- 0.2) median tissue culture infective dose for the metoprolol group versus 10(2.1 +/- 0.1) for the saline group (p less than 0.05). Whereas pathologic changes at days 3, 6 and 10 of infection were similar in both groups, on day 30 of infection, inflammation, necrosis and mineralization scores (mean +/- SEM) were 1.1 +/- 0.3, 2.1 +/- 0.4, 2.2 +/- 0.5 for the metoprolol group versus 0.3 +/- 0.1, 0.4 +/- 0.3, 0.4 +/- 0.3 for the saline group, respectively (p less than 0.01). Six noninfected mice received metoprolol intraperitoneally for 10 days; there was no mortality during 30 days of observation. In conclusion, metoprolol administration exerts deleterious effects in acute coxsackievirus B3 murine myocarditis.     

辛伐他汀对柯萨奇B3病毒感染小鼠心肌早期的保护作用（英文）

目的探讨辛伐他汀对急性柯萨奇B3病毒感染小鼠心肌早期的作用。方法将柯萨奇B3病毒感染小鼠随机分为两组:对照组（n=15）及辛伐他汀治疗组（n=15）,分别给予生理盐水及辛伐他汀14 d。第6天时,各组分别随机处死6只。结果辛伐他汀治疗组其心脏质量（69±13mg）较对照组（96±16mg）减轻（P<0.01）;心肌炎症及坏死程度减轻,心肌病理积分（8.0%±7.3%vs22.9%±10.6%）显著降低（P<0.01）;14 d生存率明显升高（P<0.05）。结论辛伐他汀治疗可减轻心肌病理损害,对早期感染柯萨奇B3病毒小鼠心肌有保护作用。     

Beneficial effects of captopril in acute coxsackievirus B3 murine myocarditis

To date, there is no universally accepted therapy for viral myocarditis. We investigated the effect of the angiotensin converting enzyme inhibitor captopril on both early and late phases of coxsackievirus murine myocarditis. Mice were infected with coxsackievirus B3 and were divided into two main protocols. Mice in the early treatment protocol (n = 30) were treated on day 1 after infection with either captopril or saline through day 6 of infection and euthanized on day 6 of infection. In the late treatment protocol, mice (n = 60) were treated starting on day 10 of infection through day 30 of infection with either captopril or saline. Mice were killed on days 20 and 30 of infection. In the early treatment protocol, heart weight was 67 +/- 14 mg in the captopril-treated group versus 98 +/- 17 mg in the control group (p less than 0.0001). The degree of inflammation, necrosis, and dystrophic calcification assessed with a semiquantitative histological score was significantly less in the captopril-treated group. The degree of pathological involvement determined by planimetry of histological sections was 8.1 +/- 7.2% for the captopril-treated group versus 22.5 +/- 10.0% for the saline-treated group (p less than 0.0001). In the late treatment protocol, captopril also caused a reduction in heart weight as compared with controls at day 20 (116 +/- 21 mg in captopril-treated group vs. 166 +/- 34 mg in controls, p less than 0.0001) and also at day 30 (136 +/- 23 mg in captopril-treated group vs. 185 +/- 48 mg in controls, p less than 0.004). On days 20 and 30 of infection, the degree of inflammation, necrosis, and dystrophic calcification was similar in both groups. We conclude that captopril is beneficial in acute coxsackievirus B3 murine myocarditis because it reduces heart weight and necrosis when administered early and reduces heart weight when administered in a delayed manner.     

Expression of perforin in infiltrating cells in murine hearts with acute myocarditis caused by coxsackievirus B3.

BACKGROUND. Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of viral myocarditis. METHODS AND RESULTS. Using a murine model of acute myocarditis caused by coxsackievirus B3, we analyzed the phenotypes and morphology of the infiltrating cells in the hearts by immunofluorescence and electron microscopy. We also examined the expression of a cytolytic factor, perforin, in the infiltrating cells by immunoperoxidase and in situ hybridization. We found that the dominant population of the infiltrating cells were asialo GM1 positive, were negative for T-cell markers, and had electron-dense cytoplasmic granules, which is consistent with a morphology of large granular lymphocytes. Perforin was found in the cytoplasmic granules of the infiltrating cells expressing perforin messenger RNA. These findings provide for the first time the direct evidence that the first wave of cell infiltration in hearts mainly consists of killer cells and strongly suggests that perforin plays, in part, an important role in myocardial cell damage involved in acute viral myocarditis. T-helper cells and cytotoxic T lymphocytes made up the second wave of cell infiltration. CONCLUSIONS. As we previously reported, the expression of major histocompatibility complex class I antigen on cardiac myocytes induced by the infiltrating cells, such as killer cells, may facilitate the interaction between cardiac myocytes and cytotoxic T lymphocytes, and may lead to further myocardial cell damage in a later phase.     

CTLA4-Ig relieves inflammation in murine models of coxsackievirus B3-induced myocarditis

Background

T-cell–mediated cellular immunity is one of the most important factors in viral myocarditis. As an important costimulatory molecule, cytotoxic T-lymphocyte–associated antigen 4 (CTLA4) alleviates autoimmunity by influencing the balance of helper T cell (T_H) subtype 1 (T_H1) to T_H2 in autoimmune diseases. The effects and mechanisms of CTLA4 fusion protein (CTLA4-Ig) in mice with coxsackievirus B3 (CVB₃)–induced myocarditis were investigated.

Methods

BALB/c mice were randomly divided into a CVB₃ group, an IgG group, a CTLA4-Ig group, and a group of healthy control mice. Mice were humanely killed on day 7 post CVB₃ inoculation, then CVB₃, IFN-γ, mouse IL-4 (mIL-4), and mouse IL-2 (mIL-2) expression in myocardium were examined by real-time quantitative polymerase chain reaction, and the serum concentrations of IFN-γ, mIL-4, and mIL-2 were measured by enzyme-linked immunosorbent assay.

Results

IFN-γ expression was significantly higher and mIL-4 levels in serum were lower in the CVB₃ group when compared with those in the healthy control group (P < 0.01). In the CTLA4-Ig group, the mouse mortality and CVB₃ mRNA in myocardium were reduced compared with those in the CVB₃ group. Furthermore, IFN-γ expression was lower, and mIL-4 was significantly higher compared with those values in the CVB₃ and the IgG groups. The levels of mIL-2 in all groups showed no statistical difference (P > 0.05).

Conclusions

T_H1 cytokines were predominant in the acute phase of viral myocarditis. CTLA4-Ig relieves myocardial inflammation, virus replication, and mouse mortality, probably by influencing the balance of T_H1 to T_H2.     

Nitric oxide and murine coxsackievirus B3 myocarditis: Aggravation of myocarditis by inhibition of nitric oxide synthase

Objectives. This study sought to investigate the effects of nitric oxide inhibition in a murine model of coxsackievirus B3 myocarditis.Background. Little is known about the contribution of nitric oxide to the pathophysiology of myocarditis.Methods. Antiviral activity was tested in vitro using nitric oxide inhibition by treatment with activated macrophages of N^G-nitro-l-arginine methyl ester and N^G-nitro-d-arginine methyl ester (both at 100 μg/ml) were administered to C3H/He mice early (days 0 to 14) and late (days 14 to 35) after infection with coxsackievirus B3.Results. In the in vitro experiments with interferon-gamma-and lipopolysaccharide-induced activated murine macrophages, treatment with the nitric oxide synthase inhibitor N^G-nitro-l-arginine methyl ester, but not its inactive enantiomer N^G-nitro-d-arginine methyl ester, restored coxsackievirus B3 titers. In the in vivo experiments in the early treatment group, myocardial virus titers were higher in N^G-nitro-l-arginine methyl ester-treated than infected untreated animals, and both inflammatory cell infiltration and necrosis were more severe. In the late treatment group, more severe necrosis and more dense myocardial and perivascular fibrosis were observed in N^G-nitro-l-arginine methyl ester-treated than in infected untreated animals. N^G-Nitro-d-arginine methyl ester administration was ineffective.Conclusions. Nitric oxide inhibition increases myocardial virus titers, resulting in the aggravation of cardiac pathology in the early stage of coxsackievirus B3 myocarditis. In the late stage, it induces more severe cardiomyopathic lesions. Nitric oxide plays a defensive role in the pathogenesis of coxsackievirus B3 myocarditis.     

Linkage between elevated PDGF-C expression and myocardial fibrogenesis in coxsackievirus B3-induced chronic myocarditis.

Coxsackievirus B3 (CVB3) is the most common causative agentof myocarditis.¹ Acute myocarditis caused by CVB3 infectioneither recovers completely without any functional and morphologicaldefects or progresses to chronic myocarditis, which is characterizedby chronic inflammation and interstitial hyperplasia that maybe related to progressive intrinsic dysfunction, degeneration,and loss of cardiomyocyte viability.² In later stages of thisdisease, CVB3 persists in the myocardium and results in chronicactivation of fibroblasts and progressive fibrosis of the myocardium,reflected by the accumulation of connective tissue and extracellularmatrix, which is characteristic of dilated cardiomyopathy (DCM).^3,4However, the molecular mechanisms by which acute myocarditisprogresses to chronic myocarditis and DCM still