TGF—β1表达与小鼠病毒性心肌炎细胞凋亡的关系

本实验通过给ＢＡＬＢ／Ｃ小鼠腹腔接种柯萨奇Ｂ－３ｍ病毒（ＣＶＢ３ｍ）诱发病毒性心肌炎（ＶＭＣ）感染病毒９ｄ后，ＶＭＣ检出率９３．３３％，应用原位末端标记法（Ｔ／ＶＮＥＬ）及免疫组化技术检测发现，７６．６７％感染病毒鼠心肌中发现凋亡细胞，７３．３３％，有ＴＧＦ－β１的表达，二者之间分布区域一致，提示细胞凋亡可能参与ＣＶＢ３ｍ诱导的ＶＭＣ的发生，发展，而ＴＧＦ－β１可能参与ＶＭＣ细胞凋亡的调控。     

NO与小鼠病毒性心肌炎心肌损伤的关系

目的 嗜鼠心肌Ｃｏｘａｃｋｉｅ Ｂ３病毒（ＣＶＢ３ｍ）可诱导ＢＬＡＢ／Ｃ小鼠病毒性心肌炎（ＶＭＣ）。探讨自由基一氧化氮（ＮＯ）与ＶＭＣ心肌损伤的关系。方法 给ＢＡＬＢ／Ｃ小鼠腹腔接种０．１ｍｌ１００ＴＣＤ５０ＣＶＢ３ｍ,感染后分批断脊处死小鼠。应用双重荧光染色技术、原位末端标记法（ＴＵＮＥＬ）和共聚焦显微镜对小鼠心肿中细胞凋亡、ＮＯ相关合成酶（ＮＯＳ１、ＮＯＳ２和ＮＯＳ３）及其降解产物（Ｎｉｔｒｏ     

NO与小鼠病毒性心肌炎发生发展的关系

目的 探讨ＮＯ在小鼠病毒性心肌炎发生发展中的作用。方法 建立柯萨奇病毒Ｂ３（ＣＶＢ３）病毒性心肌炎ＢＡＬＢ／Ｃ小鼠模型及ＢＡＬＢ／Ｃ小鼠腹腔巨噬细胞（Ｍφ）培养体系。酶法测定小鼠心肌及Ｍφ分泌ＮＯ含量;免疫组化法检测心肌ｉＮＯＳ;微量滴定法测定心肌内感染性病毒;ＲＴ－ＰＣＲ法检测心肌病毒ＲＮＡ;ＴＵＮＥＬ法检测凋亡细胞;光镜、电镜检查心肌损伤状况。结果 （１）小鼠心肌内ＮＯ于ＣＶＢ３感染后７～３０     

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。     

应用嗜鼠心肌CoxsackieB3病毒反复感染致BALB／c小鼠心肌损伤

应用嗜鼠心肌ＣｏｘｓａｃｋｉｅＢ３病毒（ＣＶＢ３ｍ）给ＢＡＬＢ／Ｃ小鼠重复腹腔注射，连续３次，间隔３０天，末次注射后１０天处死小鼠。光学显微镜检查发现，小鼠心肌可见不同时期的病灶（坏死灶、瘢痕），心肌损伤检出率达１００％。应用ＴＵＮＥＬ法检测发现，８２．５０％感染组鼠心肌中可见散在心肌细胞凋亡，与对照组（２３．０８％）比较，Ｐ＜０．０１，有极显著性差异。提示ＣＶＢ３ｍ重复感染可致ＢＡＬＢ／Ｃ小鼠慢性心肌损伤，心肌细胞凋亡参与心肌损伤的发生与发展。     

小鼠病毒性心肌炎与细胞凋亡及氧自由基的关系

采用电镜技术及原位末端标记法（ＴＵＮＥＬ）对柯萨奇病毒Ｂ３（ＣＶＢ３）心肌炎小鼠不同时期心肌组织中细胞凋亡进行检测,并采用硫代巴比妥酸法检测外周血丙二醛（ＭＤＡ）。ＣＶＢ３感染１００只雄性Ｂａｌｂ／ｃ小鼠,ＶＭＣ发病率为８６％。各期病鼠心肌中均见到凋亡细胞,包括心肌细胞、血管内皮细胞及间质细胞。ＴＵＮＥＬ法检出率为７８．９４％,中、重度ＶＭＣ小鼠中阳性检出率明显高于轻度ＶＭＣ小鼠（Ｐ〈０．０５）。     

低硒与Coxsackie B3m病毒感染对BALB／c小鼠肝脏脂质过氧化物？…

用国产低硒食用酵母合成低硒饲料（硒０．０１３ｍｇ／ｋｇ，ＶＥ含量为２０ｍ／１００ｇ）喂养断奶后ＢＡＬＢ／Ｃ雄性小鼠，５周后腹腔接种嗜鼠心肌病毒ＣＶＢ３ｍ１０＾３ＴＣＤ５００．１ｍｌ７天后处死建立低硒状态下病毒性心肌炎模型，测定肝脏组织中脂质过氧化物（ＬＰＯ）含量及全血中谷胱甘肽过氧化物酶（ＧＳＨ－Ｐｘ）活性。结果表明低硒感染ＣＶＢ３ｍ小鼠肝组织中ＬＰＯ含量明显高于常硒病毒感染组及常硒对照组（Ｐ〈０     

细胞凋亡与小鼠病毒性心肌炎发生发展的关系

通过对ＢＡＬＢ／Ｃ小鼠腹腔接种ＣＶＢ３ｍ后３，６，９，１２，１５，３０天批随机处死小鼠，取心脏进行检测发现，感染后９－１５天，病毒性心肌炎检出率可达９２．３０％。感染后３－９天，心肌中可分离出病毒，而病毒核酸可持续存在于感染后３个。应用电镜，原位末端标记法及免疫组化法检测发现：ＶＭＣ鼠心肌中可见心肌细胞和血管内皮细胞凋主ＴＧＦ－β１和Ｃ－ｍｙｃ蛋白的表达。     

低硒与Coxsackie B_(3m)病毒感染对BALB/c小鼠肝脏脂质过氧化物代谢的影响

用国产低硒食用酵母合成低硒饲料（硒０．０１３ｍｇ／ｋｇ，ＶＥ含量为２０ｍｇ／１００ｇ）喂养断奶后ＢＡＬＢ／Ｃ雄性小鼠，５周后腹腔接种嗜鼠心肌病毒ＣＶＢ３ｍ１０３ＴＣＤ５００．１ｍｌ，７天后处死建立低硒状态下病毒性心肌炎模型，测定肝脏组织中脂质过氧化物（ＬＰＯ）含量及全血中谷胱甘肽过氧化物酶（ＧＳＨ－Ｐｘ）活性。结果表明低硒感染ＣＶＢ３ｍ小鼠肝组织中ＬＰＯ含量明显高于常硒病毒感染组及常硒对照组（Ｐ＜０．０１）；低硒感染ＣＶＢ３ｍ病毒组小鼠全血ＧＳＨ－Ｐｘ活力也最低。结果提示：低硒因素加重病毒感染引起ＧＳＨ－Ｐｘ活力降低，ＬＰＯ堆积，降低机体的抗氧化能力     

柯萨奇B3m病毒致低硒BALB/C成年鼠心肌损伤特点实验研究

应用低硒和补硒合成饲料喂养５周龄ＢＡＬＢ／Ｃ雄性小鼠５周后，经腹腔接种柯萨奇Ｂ３ｍ病毒（ＣＶＢ３ｍ）１０＾３ＴＣＩＯＤ５００．１ｍｌ对照组腹腔注射ＰＲＭ１６４０，光镜下低硒病毒组（１组），补硒病毒组（２组）病变检出率分别为７５％和３５％，经Ｘ＾２检验１组显高于２组（Ｐ〈０．０５）。且病变部位不同，低硒病毒１组病变主要位于左心室中层，大体标本未见心肌外膜白斑。２组见于心外膜及心外膜下心肌，大体标本     

病毒性心肌炎心肌超微结构及细胞凋亡的电镜观察

目的研究病毒性心肌炎(VMC)小鼠心肌超微结构改变及细胞凋亡的形态学变化。方法本实验在接种柯萨奇病毒B3(CVB3)建立VMC动物模型的基础上,用光学显微镜及电子显微镜观察心肌细胞的病变及心肌细胞凋亡。结果研究发现实验组小鼠在接种病毒5 d后光镜或电镜下可见心肌病变及炎细胞浸润,7~9 d病变达高峰,35 d时病变基本恢复。VMC小鼠在接种病毒后7~9d,电镜下可见心肌细胞呈凋亡样改变,并可见凋亡小体。结论实验组小鼠在接种CVB3后可引起VMC,VMC中存在异常的心肌细胞凋亡现象。     

犬弓首线虫感染小鼠脑组织细胞凋亡及凋亡基因bcl-2、bax的表达

目的研究犬弓首线虫(Toxocaracanis)感染小鼠脑组织细胞凋亡及凋亡基因bcl-2、baxmRNA表达情况,探讨幼虫移行症对脑组织细胞影响可能机理。方法取狗小肠内犬弓首线虫成虫进行解剖,取子宫段的虫卵培养至感染期,感染小鼠后不同时间段取脑组织采用流式细胞仪检测小鼠脑组织细胞凋亡;应用原位杂交技术检测bcl-2、baxmRNA表达情况。结果1.犬弓首线虫子宫段虫卵经培养有98%达感染期。2.病理切片HE染色均见感染小鼠脑组织有犬弓首线虫幼虫。3.流式细胞仪检测小鼠脑组织细胞凋亡10d、15d、20d、25d对照组与实验组比较,具统计学意义差别(P<0.05);各时间段原位杂交方法显示bcl-2、baxmRNA均无显著表达,与对照组比较无统计学意义(P>0.05)。结论1.犬弓首线虫感染小鼠早期,脑组织细胞有不同程度细胞凋亡出现。2.其细胞凋亡与凋亡基因bcl-2、bax无明显关系。     

The effects of cyclosporine on acute murine Coxsackie B3 myocarditis

The effects of the immunosuppressant drug cyclosporine were studied in the murine model of Coxsackie B3 myocarditis. Ten BALB/c mice, given daily cyclosporine (15 mg/kg) intraperitoneally but not infected, were normal in all respects after 2 weeks. All 32 BALB/c mice infected, but given no cyclosporine, survived and had moderate myocardial mononuclear infiltrates and minimal necrosis at 7 and 14 days. In contrast, 24 mice concurrently infected and given cyclosporine had a high mortality rate (75%) and a significantly attenuated mononuclear infiltrate in the presence of enhanced necrosis when compared with control infected mice. Sixteen mice started on the drug 1 week after infection had a lower mortality rate (55%), but very similar histologic abnormalities. In contrast to negligible or no virus in the hearts of infected mice that were not given cyclosporine, drug treated, infected groups had easily detectable virus in their hearts 14 days after infection. An identical study in Swiss ICR mice yielded similar results. Cyclosporine, when given early during acute murine Coxsackie B3 myocarditis, causes a significant increase in myocardial necrosis and mortality, possibly secondary to enhanced viral survival.     

Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD8+ T cells in influenza virus-infected mice

Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforin-dependent CD8(+) CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-gamma mRNAs in CD8(+) populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-gamma in activated CD8(+) cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD8(+) cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-gamma mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD8(+) T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung.     

心肌中穿孔素和颗粒酶B基因表达水平与心脏移植急性排斥反应的关系

目的 研究穿孔素和颗粒酶B基因在移植心的心肌组织中表达水平与心脏移植急性排斥反应的关系。方法 应用大鼠腹腔同种异位心脏移植模型，实验组为同种移植，SD大鼠为受体，Wistar大鼠为供体；对照组为同系移植，供受体均为SD大鼠。观察术后1，3，5，7，9，11d的移植心病理改变同时采用RT－PCR法测定的移植心心肌组织中穿孔素和颗粒酶B基因表达水平。用免疫组化方法观察穿孔素及颗粒酶B的表达情况。结果 术后3d起移植心心肌组织中穿孔素和颗粒酶B基因表达水平即明显升高（术后3，5，7，9，11d与对照组相比均为P＜0.01），其中穿孔素基因表达在7－9d达高峰，而颗粒酶B基因表达在11d达高峰，此两种基因表达与心脏的病理学变化有明显的相关性。免疫组化显示术后5d浸润细胞胞浆中穿孔素和颗粒酶B表达明显增多。结论 移植心心肌组织中穿 纱和颗粒酶B基因表达水平与心脏移植急性排斥反应的经过相平等，可作为判定排斥反应的诊断指标。     

Perforin and granzyme B of cytotoxic T lymphocyte mediate apoptosis irrespective of Helicobacter pylori infection: possible act as a trigger of peptic ulcer formation

BACKGROUND/AIMS: The perforin/granzyme and Fas/Fas ligand pathways are two known major pathways of cytotoxic T lymphocyte-mediated apoptosis. We designed a clinical study to examine whether cytotoxic T lymphocyte-mediated apoptosis associated with peptic ulcer formation may occur via either or both of these two pathways. METHODOLOGY: Mucosal biopsy specimens were obtained endoscopically from the marginal zone of active stage gastric and duodenal ulcers in patients with or without Helicobacter pylori infection. RT-PCR, immunoblotting and immunohistochemistry were used to study the samples for the expression of apoptotic cells, perforin, granzyme B, Fas, Fas ligand and caspase 3. RESULTS: Apoptotic cells (Tunnel positive cells) appeared in the marginal zone of gastric and duodenal ulcers with and without H. pylori infection. Perforin/granzyme B and caspase 3 were expressed consistently, however Fas ligand was not. Furthermore, the immunohistochemical findings demonstrated apoptotic changes of target cells caused by perforin/granzyme B. CONCLUSIONS: These results suggest that the main pathway of cytotoxic T lymphocyte-mediated apoptosis in peptic ulcer formation is the perforin/granzyme pathway irrespective of H. pylori infection.     

Gene expression analysis of host innate immune responses in the central nervous system following lethal CVS-11 infection in mice

The central nervous system (CNS) tissue of mice infected with the CVS-11 strain of rabies virus (RABV) was subjected to gene expression analysis using microarray and canonical pathway analyses. Genes associated with innate immunity as well as inflammatory responses were significantly up-regulated, corroborating with the previous findings obtained using attenuated viruses that did not induce a fatal outcome in infected mice. Histopathological examination showed that neurons in the cerebellum had undergone apoptosis. Although the extent of Fas ligand up-regulation was not so prominent, perforin and granzyme genes were highly expressed in the CNS of mice infected with CVS-11. The presence of perforin and granzymes both in the Purkinje cells and CD3 T lymphocytes strongly suggested that apoptosis of the former cells was induced by the latter cells.     

Detection of experimental myocarditis by monoclonal antimyosin antibody, Fab fragment

The purpose of this study was to determine whether monoclonal antimyosin Fab (antigen binding fragment) was capable of labeling hearts with experimental coxsackievirus myocarditis, and to determine whether Fab could be used for detecting myocardial damage in either early or chronic phases of the disease. Sixty-five, 3-week-old cesarean-derived 1 (CD 1) mice were divided into two groups: group I (noninfected animals) and group II (infected with coxsackievirus B3). Mice from each group were killed on days 7, 17, 30, or 90 of infection. Forty-eight hours before killing, mice were injected with monoclonal I125 antimyosin, Fab (25 microCi/injection) and radioactivity was counted in the heart. Selected heart sections were also examined by autoradiography. Heart radioactivity, count/m/mg (m +/- SEM) on days 7, 17, 30, and 90 of infection was 10.8 +/- 1.7, 21.3 +/- 1.1, 11.2 +/- 3.4, and 12.4 +/- 1.5 for group I, versus 36.7 +/- 8.0 (p less than 0.01), 50.0 +/- 4.5 (p less than 0.001), 33.4 +/- 16.1 (p = NS), and 40.6 +/- 8.5 (p less than 0.01) for group II, respectively. Autoradiography revealed focal uptake within areas of necrotic myocardium. We conclude that I125 Fab may be useful in detecting myocardial damage in the experimental model of murine myocarditis up to day 90 of infection.