CVB3-VP1基因免疫诱导特异性抗病毒免疫应答及保护作用

目的 :构建表达柯萨奇病毒B3(CVB3)主要包膜蛋白VP1的基因疫苗 ,并研究该疫苗诱导CVB3特异性免疫应答及免疫保护的作用。方法 :抽提CVB3RNA ,以RT PCR扩增VP1基因 ,克隆于真核表达载体 pcDNA3中 ,构建质粒pcDNA3 VP1。将该质粒转染Hela细胞 ,观察其表达情况 ;以 5 0 μgpcDNA3 VP1质粒DNA肌注免疫BALB/c小鼠 3次 ,检测CVB3特异性体液和细胞免疫应答。间隔 4wk以 5×LD50 的CVB3攻击小鼠 ,观察攻击后小鼠的存活情况。结果 :构建了重组质粒 pcDNA3 VP1,并在体外获得有效表达。以该质粒肌肉免疫BALB/c小鼠 ,可诱生高水平的IgM和IgG ,VP1多肽特异性淋巴细胞增殖反应及CTL活性均显著高于 pcDNA3免疫的对照组。病毒攻击试验表明 ,pcDNA3 VP1免疫组33.3%小鼠可长期存活 ,其心肌组织未见明显的病理学改变 ;而对照小鼠平均仅存活 6 .7d ,心肌显示大量的局灶性坏死和炎性细胞浸润。结论 :pcDNA3 VP1免疫可诱生CVB3特异性体液及细胞免疫应答 ,保护免疫小鼠抵抗CVB3的致死性攻击     

柯萨奇B4病毒非结构蛋白P2C重组质粒的构建

目的：构建柯萨奇B4病毒非结构蛋白P2C重组质粒。方法：用RT-PCR技术，从柯萨奇B4病毒中钓取非结构蛋白P2C cDNA片段，克隆入PUCan-T载体，转化大肠杆菌DH5α，构建重组质粒。结果：以柯萨奇B4病毒的总RNA为模板，扩增出987bp大小的片段，克隆入PUCan-T载体，用BamHⅠ、HindⅢ双酶切鉴定，与非结构蛋白P2C的PCR扩增产物片段大小一致。结论：成功地构建了柯萨奇B4病毒非结构蛋白P2C重组质粒。     

A coxsackievirus B5‐associated aseptic meningitis outbreak in Shandong Province,China in 2009

In 2009, a major outbreak of aseptic meningitis was noted in Linyi city, Shandong province, China. From June to September 2009, a total of 2,104 cases were involved in this outbreak, and 98.6% of patients were <16 years of age. To determine the pathogen of the outbreak, 42 cerebrospinal fluid specimens collected from aseptic meningitis cases were tested for cell culture, and 17 (40.5%) enteroviruses were isolated and identified as Coxsackievirus B5 (CVB5). Homologous comparison indicated that these isolates had 0–7.7% nucleotide divergence with each other. Phylogenetic reconstruction showed global CVB5 could be separated into four genogroups, and all Linyi CVB5 isolates belonged to the genogroup C which had been circulating for recent 27 years in Asia and Europe. Interestingly, two distinct lineages were observed for the 17 isolates in the phylogenetic tree, indicating that at least two different transmission chains of CVB5 were responsible for this outbreak. This study showed that CVB5‐associated aseptic meningitis is an emerging concern in China. J. Med. Virol. 85:483–489, 2013. © 2012 Wiley Periodicals, Inc.     

基于CXCL12/CXCR4生物轴探讨清心饮抑制CVB3感染后CMVECs发生EndMT的作用

[目的] 探讨趋化因子12(chemokine 12,CXCL12)/趋化因子受体4(chemokine receptor 4,CXCR4)生物轴在清心饮含药血清抑制柯萨奇B3病毒(Coxsackie virus B3,CVB3)感染后心脏微血管内皮细胞(cardiac microvascular endothelial cells,CMVECs)发生内皮间充质转分化(endothelial-mesenchymal transition,EndMT)过程中的作用。[方法] 取生长状态良好的CMVECs设计为正常对照组、模型组、CXCR4拮抗剂组、清心饮组和清心饮+CXCR4拮抗剂组。除正常对照组用含10%胎牛血清(fetal bovine serum,FBS)的杜尔伯克改良伊格尔培养基(Dulbecco‘s modification of Eaglews medium,DMEM)培养外,其余各组CMVECs先用CVB3培养基感染2 h,后弃去CVB3培养基,改为普通培养。通过细胞计数试剂(cell counting kit-8,CCK8)检测各组细胞活力,免疫荧光(immunofluorescence,IF)、免疫印迹和实时荧光定量聚合酶链式反应(Real-time quantitative polymerase chain reaction,Real-time qPCR)等实验方法检测内皮细胞标志物血小板-内皮细胞黏附分子(platelet endothelial cell adhesion molecule-1,PECAM-1/CD31)、间充质细胞标志物α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、CXCL12和CXCR4的蛋白及基因表达。[结果] 与正常对照组比较,模型组CD31表达下降(P＜0.01),α-SMA、CXCL12和CXCR4表达增强(P＜0.01)。与模型组比较,CXCR4拮抗剂组、清心饮组CD31表达增强(P＜0.01),α-SMA和CXCR4表达下降(P＜0.01),CXCR4拮抗剂组CXCL12表达差异无统计学意义(P＞0.05),清心饮组CXCL12表达下降(P＜0.05)。与清心饮组比较,清心饮+CXCR4拮抗剂组CD31的表达增强(P＜0.05),α-SMA、CXCL12和CXCR4表达下降(P＜0.01,P＜0.05)。[结论] CXCL12/CXCR4生物轴参与了CVB3感染后CMVECs发生EndMT的过程,拮抗CXCL12/CXCR4生物轴能够明显增强清心饮对该过程的抑制。     

Inhibition of IL-2 inducible T-cell kinase alleviates T-cell activation and murine myocardial inflammation associated with CVB3 infection

Background

Coxsackievirus B3 (CVB3) infection causes myocarditis, pancreatitis, and aseptic meningitis. Targeting antigen-specific T cell reactions might be a promising way to alleviate the inflammatory response induced by CVB3 infection. IL-2-inducible T-cell kinase (ITK), a member of Tec kinase family expressed mainly in T cells, plays an important role in the activation of T cells. The role of ITK in viral myocarditis induced by CVB3 has not been documented.

Methodology

In this study, we inhibited the ITK expression in Jurkat cells, primary human peripheral blood mononuclear cells (PBMC), and mouse splenocytes by ITK-specific siRNA. The inhibition efficiently suppressed cell proliferation (P < 0.05) and T-cell related cytokine secretion (P < 0.05). In order to inhibit ITK in vivo, the pGCSIL plasmid containing short hairpin RNAs targeting ITK was constructed and transduced into mice infected with CVB3. ITK-inhibited mice showed reduced cell proliferation (3, 5, and 7 days post-challenge, P < 0.05) as well as CD4+ and CD8+ T cells (5 days post-challenge, P < 0.05). The altered production of inflammatory cytokines alleviated pathologic heart damage and improved mice survival rate (P < 0.05).

Conclusion

ITK played an important role in the T cell development and represented a new target for the modulation of T-cell-mediated inflammatory response by CVB3 infection.     

特异性的VP1-siRNA对CVB3复制抑制作用的研究

目的 研究特异性的CVB3-VP1 siRNA对CVB3体外复制的抑制作用的量效关系与时效关系。方法 CVB3感染HeLa细胞，利用脂质体介导将CVB3-VP1siRNA转染HeLa细胞，用RT-PCR法检测CVB3-VP1 RNA水平，免疫荧光检测CVB3-VP1蛋白的表达水平。结果 60pmol／LCVB3-VP1 siRNA是抑制CVB3 RNA及VP1表达的最佳剂量；在转染后的48h，siRNA对CVB3-VP1的抑制作用达到最佳状态。结论 特异性的CVB3-VP1 siRNA对CVB3-VP1蛋白表达水平及CVB3-RNA复制水平呈明显的剂量依赖关系和时效关系，转染后48h呈现出完全抑制作用。     

化学合成siRNA体外对柯萨奇B3病毒的影响

目的RNA干扰是近年来研究基因功能的重要工具。本研究是在培养HELA细胞中，体外化学合成siRNA（small interfering RNA）对柯萨奇B3病毒（Coxsackievirus B3，CVB3）的影响，探讨RNA干扰的抗病毒效应与其应用前景。方法根据siRNA靶序列设计原则，设计了一段针对编码CVB3病毒VP4蛋白基因组的siRNA，并在体外通过化学合成形成siRNA。同时合成一段与CVB基因组序列无关的阴性对照siRNA。用CVB3感染培养HELA细胞，将其分为3组，即siRNA组（n=8），阴性siRNA组（n=8），病毒对照组（n=8），分别观察转染后第1天到第5天的细胞病变，并与未感染CVB3的空白对照组进行比较；采用RT-PCR技术检测感染CVB3各组的病毒RNA片断，观察病毒RNA与内参GAPDH的OD比值；用免疫荧光技术检测各组CVB3蛋白的表达。结果RT-PCR检测各组CVB3病毒5’端非编码区（5’UTR）RNA，siRNA组与阴性siRNA组和病毒对照组比较无明显变化（P〉0．05）；免疫荧光检测各组CVB3病毒蛋白也未见显著差异；细胞病理效应（cytophatic effects，CPE）各组问未见明显差异。结论本文选择针对编码CVB3病毒VP4蛋白基因组的siRNA未能抑制CVB3病毒复制。     

Once-monthly radiotherapy for the palliation of pelvic gynecological malignancy

Twenty-seven patients with advanced pelvic gynecological malignancies were treated using a once-monthly fractionation scheme of 10 Gy to a total dose of 30 Gy to the whole pelvis. This course was well tolerated and provided effective palliation with a minimum of hospitalization. The results obtained suggest this course merits consideration for the palliation of other pelvic malignancies.     

硝酸甘油和硝酸异山梨酯抗柯萨奇病毒的体外实验研究

目的研究硝酸甘油(GTN)和硝酸异山梨酯(ISDN)对柯萨奇B组3型病毒(CVB3)的体外抑制作用。方法用Hela细胞扩增并收获柯萨奇B组3型病毒(CVB3)后,测定CVB3的半数组织培养感染剂量(TCID50)和空斑形成单位(PFU),应用MTT法测定实验所用药物的细胞毒性,用细胞病变效应(CPE)抑制实验和空斑减少实验,观察和分析研究GTN、ISDN对CVB3的抑制作用。结果GTN、ISDN可以明显抑制病毒的细胞病变效应以及空斑形成(P<0.05)。结论GTN、ISDN等临床常用于抗心绞痛的硝酸酯类药物具有明确的抗CVB3感染Hela细胞的作用。     

环维黄杨星干预高血压大鼠脑组织NPY与超微结构的研究

目的观察中药单体环维黄杨星(CVB)对易卒中型肾血管性高血压大鼠(RHRSP)脑缺血-复流不同时间脑组织神经肽Y(NPY)表达与细胞超微结构的干预作用。方法采用环形银夹使SD大鼠的双侧肾动脉狭窄,制成RHRSP模型,再用线栓法制成一侧大脑中动脉闭塞(MCAO)脑缺血-复流实验组;观察CVB对脑缺血6h后再灌注6、72、168h不同时间点大鼠脑组织NPY表达、含水量、梗死面积百分率、行为学评分及细胞超微结构的干预作用(治疗组),并与生理盐水处理者(对照组)及假手术组对照。结果对照组脑缺血-复流6h后脑组织NPY、含水量与假手术组相比均明显升高,72h上升至高峰,脑梗死面积百分率亦急剧增加,168h开始下降,但仍高于假手术组。电镜显示,对照组脑缺血-复流上述不同时间分别出现不同程度神经元和血管壁的超微结构损害,脑缺血-复流72h后治疗组较对照组大鼠脑NPY、含水量及梗死面积显著降低,受损脑组织神经元和血管壁的超微结构亦明显改善。结论NPY可能参与了RHRSP急性缺血-复流性脑损伤病理过程,CVB对脑缺血脑损伤RHRSP大鼠NPY、水肿与超微结构有一定的干预作用。