Herpes Simplex Virus Type 1 ICP27 Protein： Its Expression, Purification and Specific Antiserum Production

Herpes simplex virus type 1 (HSV-1) is the causative agent of cold sores and other more serious diseases. HSV-1 infected-cell protein 27 (ICP27) is an immediate-early regulatory phosphoprotein homologous to gene products identified in all classes of herpesviruses so far. To raise the antiserum to ICP27 for further characterization of its biological function, the ICP27 gene was cloned into the pET-28a (+) vector, then ICP27 protein was expressed in E. Coli and purified by nickel-nitrilotriacetic acid (Ni2+-NTA) affinity resin column,finally the purified protein was used to raise antiserum. Western blot analysis demonstrated that the antiserum recognized the recombinant protein, and the antiserum was able to probe the ICP27 in HSV-1 infected cells with high specificity by immunofluorescence assay (IFA). Therefore, the specific antiserum will provide a valuable tool for further studies investigating ICP27's biological function during HSV-1 infection.     

Executionary pathway for apoptosis：lessons from mutant mice

Apoptosis or programmed cell death(PCD) is an evolutionarily conserved cellular process that is essential for normal development and homeostasis of multicellular organisms.Defects in the apoptosis signaling result in many diseases including autoimmune diseases and cancer.The apoptosis signaling pathway was first described genetically in the nematode Caenorhabditis elegans which serves as a framework for the more complex apoptotic pathways that exist in mammals.In this review,we will discuss the apoptotic pathways that are emerging in mammals as elucidated by studies of gene-targeted mutant mice.     

γδT17细胞活化机制的研究进展

IL-17是一种重要的促炎细胞因子,在炎症性疾病和自身免疫性疾病中发挥重要作用。研究发现,除Th17细胞外,γδT17细胞也是IL-17的重要来源。γδT17细胞参与多种免疫应答过程,与感染性疾病、自身免疫性疾病和肿瘤等疾病的发生发展密切相关,本文综述γδT17细胞活化机制的研究进展,探明γδT17细胞的分化和活化机制将为上述疾病的治疗提供新思路。     

zVAD-fmk对未成熟树突状细胞诱导初始性CD4＋T细胞分化为调节性T细胞影响的体外研究

目的 研究 Caspase 通路在未成熟树突状细胞（imDC）诱导同种异体 CD4＋ T 细胞转化为调节性 T 细胞（Treg）中的作用及探讨免疫耐受机制建立可能的分子机制.方法 将人外周血分离、培养出的imDC 与健康胎儿脐血中分离CD4＋ T 细胞混合培养,同时加入zVAD-fmk,以流式细胞仪检测CD4＋CD25＋ T 细胞（Treg 细胞）转化率.结果 （1）imDC 的鉴定：外周血经诱导后分离的imDC,以流式细胞仪检测细胞表面分子,imDC 表面分子表达的结果：CD80（7.27 ± 0.13）、CD83（3.53 ± 0.35）、CD1a（4.29 ± 0.27）;（2）混合培养后CD4＋CD25＋T细胞的转化率结果为：空白组（1.78 ± 0.11）﹪、对照组（22.23 ± 0.77）﹪、低浓度zVAD-fmk 组（21.63 ± 0.82）﹪、中浓度zVAD-fmk 组（12.24 ± 0.54）﹪、高浓度zVAD-fmk 组（12.20 ± 0.96）﹪,结果对照组和低浓度组、中浓度组和高浓度组间比较,P 〉 0.05,其余各组间比较,P 〈 0.05.加入zVAD-fmk 并与imDC 细胞混合培养的T 细胞转化率相对于未加入阻断剂的T 细胞较低,同时Caspase 信号通路对zVAD-fmk 无浓度依赖性.结论 imDC 可以诱导同种异体初始性CD4＋ T 细胞分化为Treg.Caspase 信号通路特异性的阻断剂zVAD-fmk 可以部分抑制Treg 的转化,说明Caspase 信号通路在诱导免疫耐受中可能起了较为重要的作用.     

From Squalene to Brassinolide： The Steroid Metabolic and Signaling Pathways across the Plant Kingdom

The plant steroid hormones, brassinosteroids （BRs）, and their precursors, phytosterols, play major roles in plant growth, development, and stress tolerance. Here, we review the impressive progress made during recent years in elucidating the components of the sterol and BR metabolic and signaling pathways, and in understanding their mecha- nism of action in both model plants and crops, such as Arabidopsis and rice. We also discuss emerging insights into the regulations of these pathways, their interactions with other hormonal pathways and multiple environmental signals, and the putative nature of sterols as signaling molecules.     

基于PiggyBac转座系统构建稳定表达UL19基因的小鼠乳腺癌细胞系4T1

为研究溶瘤Ⅱ型单纯疱疹病毒(oncolytic herpes simplex virus type 2, oHSV2)主要衣壳蛋白VP5蛋白(由UL19基因编码)调控免疫细胞抗肿瘤的机制,本研究通过PiggyBac转座系统构建了稳定表达VP5蛋白、近红外荧光蛋白(near-infrared fluorescent protein, iRFP)和绿色荧光蛋白(green fluorescent protein, GFP)的小鼠乳腺癌细胞4T1-iRFP-VP5-GFP细胞系。通过流式细胞术及免疫印迹法筛选GFP及VP5高表达的单克隆细胞系,检测所构建细胞系中UL19基因表达稳定性。经SYBR Green I实时荧光定量PCR、免疫印迹法检测,连续传代至15代的4T1-iRFP-VP5-GFP细胞中的UL19基因拷贝数及VP5蛋白表达量均显著高于瞬转UL19基因的4T1细胞,证明UL19基因稳定插入至4T1细胞基因组中。实时无标记动态细胞分析技术(real time cellanalysis, RTCA)监测4T1-iRFP-VP5-GFP细胞显示具有与其亲代细胞4T1相似的生长活性。进一步研究证实,NK92细胞对4T1-iRFP-VP5-GFP细胞的杀伤能力显著高于4T1细胞。本研究为阐述VP5蛋白在4T1细胞中通过HLA-E分子调控包括T细胞、NK细胞在内的免疫细胞发挥抗肿瘤功能奠定了基础。     

MEK1-ERKs级联激活方式是调控单纯疱疹病毒Ⅱ型复制的重要机制

本研究通过阐明MEK1和MEK2亚型在单纯疱疹病毒Ⅱ型(herpes simplex virus type 2,HSV2)复制中介导的Raf/MEK/ERK(简称ERK)通路活化中的作用,以期进一步阐明该通路调控病毒复制的机制.研究中应用了MEK抑制剂U0126、针对MEK1和MEK2的特异性小干扰RNA(small ...     

The anti-tumor effect of human monocyte-derived dendritic cells loaded with HSV-TK/GCV induced dying cells

Herpes simplex virus thymidine kinase (HSV-TK) gene and dendritic cells (DC) have been used as the pioneering in cancer therapy. HSV-TK gene can induce apoptosis and necrosis in tumor cells in the presence of the non-toxic prodrug ganciclovir (GCV). We investigated the anti-tumor effect of DC vaccination by introducing dying cells from HSV-TK gene treatment as an adjuvant. HepG₂-TK cell line was established by transfecting human hepatoma cell line HepG₂ (HLA-A₂ positive) with HSV-TK gene. Dying tumor cells were generated by culturing HepG₂-TK cells with GCV. After engulfed dying cells efficiently, immature DCs (imDC) derived from human monocytes were fully matured and elicited marked proliferation and cytotoxicity against HLA matched HepG₂ cells in autologous peripheral blood mononuclear cells (PBMC). It also implied that HepG₂ specific CTLs played an important role in the cytotoxicity which was primarily depended on Th1 responses. Given the feasibility of inducing dying cells by HSV-TK/GCV in vivo, our results suggest an effective method in clinical human hepatocellular carcinoma (HCC) treatment by an in vitro model of applying HSV-TK gene modified human tumor cells integrated with DC vaccination.     

Alkaline phosphodiesterase I and alkaline phosphatase I in plasma membranes of herpes simplex virus type 1 transformed hamster cells

Plasma membrane extracts from Herpes simplex virus type 1 transformed hamster embryo fibroblasts were chromatographed on Lens culinaris lectin coupled to Sepharose (LcH-Sepharose) and analysed by dodecyl sulphate polyacrylamide gel electrophoresis. Coomassie blue-staining revealed two major protein bands with apparent molecular weights of 125 000 and of about 75 000–90 000. In plasma membranes isolated from these tumor cells prior labeled with [³H]fucose or [³H]glucosamine these bands contained the highest amounts of incorporated radioactivity. Separation by LeH-Sepharose-affinity chromatography as well as metabolic labeling clearly demonstrates their glycoprotein character. The 125 000 protein coincides with alkaline phosphodiesterase I activity with a K_m of 6 · 10^?4 M for TMP p-nitrophenyl ester and is competitively inhibited by UDP-N-acetylglucosamine. This enzymatic activity is also present in normal hamster embryo fibroblasts. Gel electrophoresis of the Lens culinaris lectin-binding glycoproteins from plasma membranes of normal hamster embryo fibroblasts additionally revealed a strong alkaline phosphatase activity represented by an apparent molecular weight of 150 000, while HSV₁ hamster tumor cells contain only a very weak activity of this enzyme activity. HSV-lytically infected cells, however, have unchanged levels of alkaline phosphatase activity, whereas alkaline phosphodiesterase activity increases slightly.     

抗单纯疱疹病毒单克隆抗体的研究——Ⅱ.单克隆抗体介导细胞毒效应的研究

建立了单克隆抗体(McAb)介导细胞毒作用(ADCC)~(51)Cr释放试验的测定力法。确定了最适工作条件。ADCC测定结果表明,5株抗HSV McAb介导ADCC的活性不同:McAb 1A12、2A8和1G8无ADCC活性;而1D10和2C5两株McAb作1:10稀释时,~(51)Cr释放率分别为27.09％和25.07％,稀释至1:100或1:1000时仍有ADCC活性。结果提示,不同的McAb抗原决定族诱导产生的抗体,在介导ADCC免疫保护作用上有差异,并为McAh治疗临床单纯疱疹病毒感染的可能性提供了实验资料。     

子宫颈糜烂病毒病因的探讨

491份宫颈拭子病毒分离结果表明:糜烂宫颈单纯疱疹病毒(HSV)分离阳性率(30.8％)是正常宫颈(2.6％)的11.8倍,用人干扰素治疗一个疗程后,病毒分离率下降至疗前的1/4.36例糜烂宫颈活体组织DNA分子杂交表明,乳头瘤病毒16型(HPV-16)阳性者占52.8和,HPV-18占17.9％,HPV-6B占28.1％,HPV-11占7.7％,251例宫颈糜烂患者经人(?)D型基因工程干扰素双盲对比治疗后,总有效率达93.8％,显效率达60％,分析临床疗效与HSV分离率的变化表明,临床有效病例中有35％(49/140)在治疗后病毒阴转,有57％在疗前疗后均未分离出HSV,有5％在疗前疗后保持阳性不变,有2.9％疗前阴性,疗后阳性,上述结果表明,HSV和HPV与慢性宫颈炎有一定关系。     

溶瘤I型单纯疱疹病毒的研究进展

溶瘤病毒(Oncolytic virus,OV)是可以靶向感染并杀伤肿瘤细胞的一类病毒,其中溶瘤I型单纯疱疹病毒(Oncolytic herpes simplex virus type 1,OHSV-1)是目前研究最多的溶瘤病毒之一,可通过多种策略进行构建,已有多种OHSV-1进入临床试验,大量结果显示其具有较好的安全性和有效性。本文主要介绍OHSV-1的分子生物学特性与优势、主要的开发及靶向性策略、各类OHSV-1的研究进展以及目前存在的问题等。     

HSV-1溶瘤病毒的研究进展

1型单纯疱疹病毒（Herpes simplex virustype1,HSV-1）感染效率高且易于通过基因工程改造,己广泛应用于肿瘤治疗研究和临床实验。溶瘤HSV-1可通过基因工程改造或从HSV-1自发突变株中筛选获得。研究证实溶瘤HSV-1能够有效抑杀肿瘤细胞,可通过多种机制靶向肿瘤细胞,溶瘤HSV-1与放化疗联合使用治疗肿瘤的研究也取得了理想的结果。目前,已有多个溶瘤HSV-1进入临床试验。     

单纯疱疹病毒致病模型的研究

对单纯疱疹病毒（HSV）感染小白鼠致病特点进行了观察。小白鼠感染HSV第4天后开始发病,感染后2h血液内可分离出病毒,第48小时病毒血症水平和病毒检出率较高。不同组织病毒分布不同,脑、神经节在感染后第72小时病毒滴度较高,心、肝组织在第5天达到高峰。结果说明所建立的HSV致病模型可用于评价抗HSV药物。     

Isatin-β-thiosemicarbazones as potent herpes simplex virus inhibitors

A series of isatin-β-thiosemicarbazones have been designed and evaluated for antiviral activity against herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) in a plaque reduction assay. Their cytotoxicity was examined using human rhabdomyosarcoma cells (RD cells). Several derivatives of isatin-β-thiosemicarbazone exhibited significant and selective antiviral activity with low cytotoxicity. It was found that the thiourea group at thiosemicarbazone and the NH functionality at isatin were essential for their antiherpetic activity. The synthesis and structure-activity relationship studies are presented.