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。     

CVB3m病毒致低硒低维生素E鼠心肌损伤的发病机制

目的 为深入研究CVB3m病毒感染导致的低Se低VE鼠心肌病变中氧化损伤的作用。方法 1周龄乳鼠给予低Se低VE饲料喂养，4周后腹腔接种病毒，7d后处死，测定全血谷胱甘肽过氧化物酶（GSH－Px)活性以及肝脏中脂质过氧化物（LPO）含量，光镜下观察心肌的病理改变。结果 Se和VE联合缺乏鼠感染CVB3m病毒7d后，小鼠全血GSH－Px活性下降，肝脏中LPO水平增加，光镜下小鼠心肌病变检出率和严重程度均高于补Se常VE病毒组。结论 氧化应激损伤在病毒性心肌炎实验动物模型的发病机制中起重要作用。     

克山病与病毒感染关系的实验研究

目的为了探讨克山病与肠道病毒特别与柯萨奇病毒(CVB)的关系.方法①选择用CVB3病毒的cDNA探针,应用原位核酸杂交的方法,检测克山病尸检心肌组织;②采用CVB5病毒VP1壳蛋白的单克隆抗体应用免疫组化法检测克山病尸检心肌组织;③用不同配方的低硒合成饲料喂养小鼠,分别感染CVB2、CVB4m'、CVB3m以及先后感染CVB4和CVB3m的病毒.结果①原位杂交法急型克山病、亚急型克山病、慢型克山病的阳性率分别为61.5%、75.8%和68%;②免疫组化法克山病阳性率分别为89.2%,两种方法阳性检出率均显著高于健康人,高于心肌炎及扩张型心肌病,但无差异;③光镜下低硒病毒组心肌病变位于心肌实质、乳头肌等.为多发性灶状坏死伴少量炎性细胞浸润,补硒组心肌病变为间质炎性细胞浸润,病变程度及检出率、低硒病毒组均重/高于补硒病毒组.结论以上实验结果均支持克山病的发生与肠道病毒,特别CVB病毒的感染高度相关.     

柯萨奇B2病毒致低硒乳鼠心肌细胞凋亡及其调控的实验研究

目的 观察低硒条件下野毒株柯萨奇B2病毒(CVB2)感染对乳鼠心肌细胞凋亡的影响及调控。方法 应用低硒合成饲料(含硒0．016mg／kg)喂养昆明鼠5周后交配，取其子代7日龄乳鼠经腹腔注入10^7 TCID50 CVB2 0．1ml，9d后处死，取其心脏。TUNEL法检测心肌细胞凋亡的发生情况，免疫组化方法对凋亡相关基因蛋白的表达情况进行检测。结果 补硒病毒组和低硒对照组乳鼠心肌均可检测到凋亡细胞，而补硒对照组和低硒病毒组未见细胞凋亡发生。各组乳鼠C-myc、Bcl-2、p53以及TGFβ-1蛋白表达异常。结论 低硒和病毒分别作用均可引起乳鼠心肌细胞发生凋亡。低硒条件下感染CVB2(野毒株)不能引起心肌细胞发生凋亡。     

柯萨奇B2病毒致低硒乳鼠心肌损伤的实验研究

目的：探讨柯萨奇B2病毒（CVB2）在低硒足蛋白条件下对乳鼠心肌损伤的作用。方法：应用低硒合成饲料（硒含量0．016mg/kg）喂养昆明鼠5周后交配，取其子代7日龄乳鼠经腹腔注入CVB210^7TCID500.1ml,9d后处死，取其心脏。常规石蜡包埋、切片、HE染色光镜检查；部分心肌做电镜观察。结果：低硒、补硒合成饲料感染病毒组鼠光镜下心肌病检出率分别为44．1％和15．7％，低硒和补硒对照组均未检出病变。结论：足蛋白条件下硒缺乏仍可增强CVB2病毒对昆明乳 鼠的致病作用。     

柯萨奇3m病毒和T-2毒素致小鼠慢性心肌损伤细胞免疫功能改变

目的 观察慢性心肌损伤过程中小鼠细胞免疫功能的改变。方法 BALB／C小鼠随机分成4组，病毒（柯萨奇3m，CVB3m）组、毒素（T-2）＋病毒组、毒素组和对照组。观察各组小鼠脾淋巴细胞亚群及其在刀豆蛋白A（CoA）刺激下增殖及白细胞介素-2（IL-2）的分泌水平。结果 毒素＋病毒组小鼠心肌形成慢性损伤，感染病毒7d时，毒素＋病毒组脾淋巴细胞亚群、淋巴细胞增殖反应及IL-2的分泌水平均低于病毒组，14d后虽有不同程度的提高，但低于病毒组最高值。结论 T-2毒素染毒小鼠感染CVB3m后，心肌出现慢性损伤，这可能是由于淋巴细胞数量、功能和活性异常，细胞免疫功能紊乱，导致发病高峰延迟，阻碍早期清除病毒及杀伤受染细胞的作用有直接关系。     

柯萨奇B3m病毒致T-2毒素染毒鼠心肌损伤的病理观察

目的：观察T－2毒素染毒小鼠感染柯萨奇B3m病毒（CVB3m）后心肌损伤的特点。方法：BALB／C小鼠随机分成4组，病毒组腹腔接种病毒液；毒素＋病毒组接种病毒前T－2毒素隔日灌胃，3周后腹腔接种与病毒组同等剂量的病毒液;毒素组单纯T－2毒素隔日灌胃；对照组生理盐水灌胃，腹腔接种1640营养液。观察各组小鼠心肌光镜、普通电镜、酶学电镜改变以及心肌病毒核酸持续感染情况。结果：毒素＋病毒组心肌损伤明显重于病毒组和毒素组，病灶纤维化明显，可见结缔组织增生、瘢痕形成。线粒体空泡变明显，酶活性产物严重脱失。结论：T－2毒素梁毒小鼠感染CVB3m后，心肌病变加重，心肌出现慢性损伤。     

柯萨奇病毒B4''致低硒鼠心肌损伤及细胞凋亡机制的研究

目的 为了探讨柯萨奇病毒B4’致低硒鼠心肌损伤及细胞凋亡的机理。方法 用低硒和补硒饲料分别喂养昆明鼠，4周后交配，给其子代4周雄性鼠腹腔接种10—7TCID50柯萨奇病毒B4’0．1ml，对照组接种等量RPMI1640培养掖。7天后处死，取心脏制片，光镜观察病变，并采用TUNEL法和免疫组化法技术检测凋亡细胞及其相关基因。结果 低硒和补硒饲料病毒组的心肌病变检出率分别为78．1％和16．7％。低硒和补硒饲料对照组的心肌未见病变。低硒和补硒病毒组及低硒对照组的心肌均发生不同程度的细胞凋亡。补硒对照组未见心肌细胞凋亡。结论 柯萨奇病毒B4’感染低硒昆明鼠引起的心肌损伤有细胞凋亡机制参与。     

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。     

柯萨奇B_4和B_(3M)病毒重叠感染对低硒昆明鼠T细胞亚群的影响

目的：观察低硒,补硒鼠先后感染ＣＶＢ４,ＣＶＢ３Ｍ两种病毒七天后,小鼠脾脏Ｔ细胞亚群的变化。方法：小鼠先后感染ＣＶＢ４,ＣＶＢ３Ｍ两种病毒七天后,检测脾脏Ｔ细胞亚群细胞数,血清中和抗体及心肌组织病毒滴度。结果：在低硒感染组ＣＤ４,ＣＤ８,ＣＤ３均显著下降,而补硒感染组ＣＤ３,ＣＤ４,ＣＤ８Ｔ淋巴细胞亚群都明显升高,心肌组织病毒,病变程序及检出率低硒病毒组显著重于补硒组。血清中和抗体效价低硒组显著低于补硒组。结论：硒缺乏可以导致机体免疫功能的低下,而ＣＶＢ４,ＣＶＢ３Ｍ病毒的二重感染导致低硒鼠的免疫功能进一步低下。     

MCP-1与CVB3相互作用参与病毒性心肌炎的发生

目的　研究单核细胞趋化蛋白 - 1(MCP- 1)在 B3型柯萨奇病毒 (CVB3)诱导病毒性心肌炎 (VMC)发病中的作用。方法　构建小鼠 MCP- 1真核表达质粒 p VM。 CVB3腹腔注射雄性 BAL B/c小鼠 ,随机分四组 :单独 CVB3感染 VMC组 (A组 )、10 μg过表达 MCP- 1组 (B组 )、4 0 μg过表达 MCP- 1组 (C)和空质粒对照组 (D组 )。并设未感染生理盐水对照组 (E组 )和未感染过表达组 (F组 )。比较各组小鼠心脏重量和体重的比值 (HW/BW)、心肌组织病理学积分 (PS)、心肌组织病毒载量、血清 CK- MB水平。结果　与 E组相比 ,A组的 HW/BW、PS、血清 CK- MB和心肌组织病毒载量均有明显改变。表明 CVB3感染后小鼠出现一系列的体征改变。与 A组相比 B组的 HW/BW、PS、血清 CK- MB和心肌组织病毒载量均未呈明显改变。但 C组 CVB3载量明显降低 (P<0 .0 5 ) ,血清 CK- MB水平升高 (P<0 .0 1) ,而 HW虽然升高(P<0 .0 5 ) ,但是 HW/BW未呈明显的改变 (P>0 .0 5 ) ,PS升高 (P<0 .0 5 )。 D组与 A组相比 ,CVB3载量、血清 CK-MB水平、HW/BW和 PS均未呈明显的改变 (P>0 .0 5 )。 F组的 HW/BW和血清 CK- MB水平与 E组相比未呈现显著性差异 ,而 PS升高 ,但低于 A组 (P<0 .0 1)。结论　单独在心肌组织过表达 MCP- 1仅引起心肌组织学改变 ,不能     

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。     

利用直接原位PCR法探讨柯萨奇B3病毒与心肌炎的病原学关系

目的探讨柯萨奇B3病毒(CVB3)感染与急性心肌炎的病原学关系。方法应用生物素标记的核苷酸直接掺入法，建立了直接法原位逆转录-聚合酶链式反应(RT-PCR)技术，分别对感染的HeLa细胞、病毒性心肌炎鼠心肌样本及临床心肌炎的心肌活检标本中的CVB3进行检测。结果病毒感染的HeLa细胞可见阳性信号，细胞呈蓝紫色，而未感染的HeLa细胞无阳性信号，细胞不着色。在CVB3感染的病毒性心肌炎小鼠心肌组织中可检测到病毒RNA的存在。16例病理学及临床诊断为心肌炎的患者心肌组织中，7例检测到CVB3 RNA，而16例正常心肌标本中均为阴性。结论CVB3感染与心肌炎发病有密切关系。     

NK-derived IFN-γ/IL-4 triggers the sexually disparate polarization of macrophages in CVB3-induced myocarditis

Coxsackievirus B3 (CVB3) is a common etiology of myocarditis with an increased morbidity and mortality in males. We previously reported that differential polarization of macrophages contributed to sexually dimorphic susceptibility of mice to CVB3-induced myocarditis. However, the underlying kinetics, impetus as well as the molecular mechanism remain unclear. Here, we demonstrated that myocardial macrophages started to polarize at as early as day 5 post CVB3 infection in both genders of BALB/c mice, with M1 phenotype detected in males and M2a phenotype in females, and this trend was further amplified at day 7 when myocarditis reached peak. In addition, we identified that prevailed IFN-γ in males and dominant IL-4 in females were critical myocardial cytokines for the disparate macrophage polarization, which respectively activated JAK1–STAT1 and JAK3–STAT6 pathways. Strikingly, we found that the main source of IFN-γ and IL-4 cytokines in both genders were myocardial infiltrating NK cells, which differentially secreted cytokines in various microenvironments manifested synergistically by sex hormones and CVB3 infection. Consistently, depletion of NK cells significantly impeded the myocardial macrophage polarization in both genders of CVB3-infected mice. Collectively, these data indicated that myocardial NK-derived IFN-γ/IL-4 was critical for the differential polarization of macrophages in CVB3-induced myocarditis via activating JAK1–STAT1 and JAK3–STAT6 pathways respectively. Our study may help understand the mechanism of sexually differential polarization of macrophages and provide clues for the gender bias in CVB3-induced myocarditis.     

The Outcome of Coxsackievirus B3-(CVB3-) Induced Myocarditis is Influenced by the Cellular Immune Status

Mice develop a marked age-related susceptibility to myocardial coxsackievirus B3 (CVB3) infections. The lesions observed in mice resemble closely those seen in the human disease. Experimental murine models of CVB3-induced myocarditis have shown that both, host and viral genetic factors, can influence susceptibility to the infection as well as the persistence and progression of the disease. Recently, we have shown that CD4 T cell-deficient MHC Class II knockout mice develop a strong fibrosis with virus persistence in the heart tissue and without production of neutralizing antibodies. To examine the role of CD4+ T cells and especially the role of the T helper 1 cell response for the outcome and pathogenesis of CVB3-induced myocarditis in more detail, 2 different mouse strains with identical genetic background (H-2b) were infected with CVB3-Mü/J (Nancy strain). Immunocompetent C57BL/6 mice and mice with targeted disruption of interleukin (IL-)4 gene (IL-4-/- mice) developed a severe acute myocarditis on day 7 post infection (p.i.). The CVB3-induced inflammation was cured until the 21st day p.i. in hearts of C57BL/6 mice. IL-4-/- mice with insufficient T helper-2 cell immune response developed a severe myocardial damage between day 7 and 21 p.i. with prolonged virus persistence in the heart tissue. Therefore, we suggest that despite an obvious normal T helper-1 cell cytokine pattern, IL-4-/- mice are more susceptible to long-term heart muscle injuries after infection with CVB3.     

Effects of selenium deficiency on tissue selenium content, deiodinase activity, and thyroid hormone economy in the rat during development

The iodothyronine deiodinases, D1, D2, and D3, all contain selenium (Se) in the form of selenocysteine at their active sites, and they play crucial roles in determining the circulating and intracellular levels of the active thyroid hormone (TH), T3. However, not only are serum T3 levels normal in Se-deficient rats but phenotypic and reproductive abnormalities are minimal, and it has been suggested that regulatory mechanisms exist to conserve Se in critical tissues. The present study was designed to determine, in rats: 1) whether the effects of Se-deficiency are greater in the fetus and neonate than in the adult; 2) whether there are tissues other than brain and thyroid in which deiodinase activities are maintained; 3) whether the maintenance of deiodinase activity in a specific tissue is associated with a concomitant preservation of Se level in that tissue; and 4) whether TH economy and general health is maintained over several generations. The tissues studied included liver, cerebrum, thyroid, pituitary, skin, brown adipose tissue, uterus, ovary, testis, placenta, and the implantation site (uterus plus contents) at E9. The results have revealed that, with the exception of liver, skin, and nonpregnant uterus, all of the tissues studied maintained substantial deiodinase activity (>50%) during prolonged Se-deficiency. Second, although the ability of a tissue to maintain deiodinase activity in the face of dietary Se deprivation was associated in some tissues with a concomitant local preservation of Se concentration, this was not the case for all tissues. Only when Se levels were decreased by more than 80% was deiodinase activity markedly decreased. Third, the effects of Se-deficiency were no greater in the fetus than in the adult; and fourth, at the level of Se-deficiency employed in this study, TH economy and general health were successfully maintained over six generations of Se-deficient rats. How Se levels are maintained in specific tissues, whether Se is sequestered in specific cells of a tissue or organ during dietary Se deprivation, and the precise mechanisms by which plasma T3 levels are maintained in Se-deficient animals remain unanswered. Further insights may be gained by using diets that are even lower in Se than those that were used herein and/or by conducting studies using radioactive forms of Se and thyroid hormones.