Ⅰ型单纯疱疹病毒感染的细胞培养系统的建立

目的:建立稳定的HSV-1感染的细胞培养系统,为HSV-1感染性皮肤病的基础及临床研究提供稳定的平台。方法:以猴肾细胞(Vero)为繁殖细胞,选择人鼻咽癌上皮细胞(Hep-2)为靶细胞,观察HSV-1在Hep-2株中的致细胞病变效应(CPE)。结果:HSV-1在Vero细胞中能稳定、大量地繁殖;HSV-1感染Hep-2细胞后可出现明显的细胞病变;结论:以Vero为繁殖细胞,Hep-2为靶细胞的稳定的细胞培养系统可作为HSV-1感染性皮肤病的基础及临床研究的平台。     

Ⅰ型单纯疱疹病毒 gD基因片段的高效表达及抗原性分析

目的 获得高表达的Ⅰ型单纯疱疹病毒(HSV)被膜糖蛋白gD（简称gD1）基因的工程菌。方法 通过计算机分析,筛选出疱疹病毒gD1中优势抗原决定簇的基因片段。将克隆的基因片段插入表达载体pTrxA内,转化大肠杆菌Rosetta,以异丙基-β-D-硫代半乳糖苷诱导表达。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表达产物。　结果　PCR扩增出约930bp的gD1编码基因目的片段,与预期片段大小相符,经测序鉴定无基因突变;所构建pTrxA-gD1重组表达质粒阳性克隆经PCR与双酶切鉴定,与预期结果一致;含有pTrxA-gD1重组质粒的大肠杆菌Rosetta诱导后得到了高效达,SDS-PAGE显示表达产物约Mr48000（Dalton）。免疫印迹结果表明表达产物具有较好的抗原性。结论　成功构建了pTrxA-gD1表达质粒,实现了成熟gD1蛋白在大肠杆菌中的高效表达,表达产物具有好的抗原性。     

Modulation of Adhesion Molecule Expression on Rat Cortical Astrocytes During Maturation

Abstract: During development of the vertebrate CNS the functional properties of astrocytes change significantly. Many of these functional changes result from modifications in the expression of cell surface adhesion molecules on astrocytes that mediate the interactions of astrocytes with other astrocytes, neurons, and growing axons. In this study we have compared the cell surface expression of HNK-1, NCAM, and laminin on rat cortical type-I-like astrocytes during maturation in vitro and in vivo. Both the proportion of immunoreactive cells and the relative levels of expression of these antigens on different aged astrocyte populations were assayed by flow cytometry. At birth, most cortical type-I astrocytes express high levels of HNK-1 and NCAM, while ∼50% of the cells express laminin. During maturation in vitro, the proportion of cortical astrocytes that expressed these surface molecules decreased over a period of 28 days, even though cell size and glial fibrillary acidic protein content increased. During maturation in vivo, a qualitatively and temporally similar decrease in antigen expression on astrocytes was observed. This reduction in the expression of specific cell surface molecules on maturing astrocytes results from maturation of a single population of astrocytes and not differential proliferation of a nonexpressing subpopulation of astrocytes, as shown by cell cycle analysis of both immunoreactive and nonimmunoreactive cell populations. These data indicate that during maturation of rat cortical type-I-like astrocytes, the expression of cell surface adhesion molecules is regulated. Furthermore, this regulation appears to be cell autonomous and not dependent on environmental factors. Such regulation of adhesion molecule expression may have profound consequences for the functional properties of astrocytes during CNS maturation.     

Expression of HSV-1 glycoproteins in tunicamycin-treated monkey kidney cells

The role of glycosylation in transport and expression of HSV-1 glycoproteins on the surface of HSV-1-infected African green monkey kidney cells was investigated by using tunicamycin (TM). A concentration of 0.05 microgram/ml of TM inhibited the replication of HSV-1 by greater than 99%. Immunoblot analysis of TM-treated and virus-infected cells indicated that 0.05 microgram/ml of TM blocked the addition of N-linked oligosaccharides into glycoproteins B, C and D. An immunofluorescence assay of TM-treated (0.05 and 0.1 microgram/ml) and virus-infected cells demonstrated the presence of nonglycosylated gC, gD and a reduced amount of gB on the surface of infected cells. The results suggest that the addition of N-linked oligosaccharides on the studied HSV-1 glycoproteins was not necessary for their transport and expression on the virus-infected cell surface.     

黄芪A_6组分与无环鸟苷联合对Ⅰ型单纯疱疹病毒感染小鼠的治疗作用

黄芪Ａ＿６组分与无环鸟苷联合对Ⅰ型单纯疱疹病毒感染小鼠的治疗作用左丽,董熙昌,孙晓娟（贵阳医学院微牛物学教研室,贵阳５５０００４）关键词黄芪,无环鸟苷,药物协同作用,Ⅰ型中纯疱疹病毒,小鼠单纯疱疹病毒（ＨＳＶ）是人类最常见的病原之一,可引起多种疱疹性...     

Process Formation in Astrocytes: Modulation of Cytoskeletal Proteins

Studies on primary astrocytes cultured in vitro have shown that process formation involves changes in cytoskeletal proteins and release of tension on the substratum. Actin filament reorganization has previously been found to be the major cytoskeletal change occurring during process formation. These changes are relatively rapid with breakdown of the actin web and release of contacts occur within 15 min. of cyclic AMP treatment. The former is regulated by myosin light chain (MLC) and actin depolymerizing factor (ADF), with MLC involved in the initial release of contractile tension and ADF in both initial and longer term actin breakdown. Our results show that the dephosphorylation of MLC is due to the phosphorylation and inactivation of myosin light chain kinase (MLCK) in response to cyclic AMP. To further study the mechanisms underlying the process formation in astrocytes we used endothelin-1 (ET-1), a vasopeptide which has been shown to inhibit process formation in astrocytes and sodium fluoride which is a general phosphatase inhibitor. We observe an increase in phosphorylation of MLC on inhibition of process formation. To study the role of adhesion in process formation we used suspension cultures of astrocytes. Our results with the astrocytes in suspension suggest that the process formation in astrocytes is adhesion dependent and the changes in ADF and MLC occur only when there is process formation.     

UHRF1在结肠癌中的表达及其与细胞增殖的关系

目的:探讨UHRF1(Ubiquitin-like with plant homeodomain and ring覱nger domains 1)在结肠癌组织中的表达,及其与结肠癌细胞增殖的关系。方法:采用免疫组织化学方法检测62例结肠癌组织和癌旁正常对照组织的UHRF1表达,统计分析其表达和临床病理资料和预后关系。采用si RNA抑制UHRF1表达,分析其与细胞增殖的关系。结果:结肠癌组织中UHRF1高表达,癌旁正常组织低表达。62例结肠癌组织中33例UHRF1阳性,阳性率为53.2%。UHRF1表达与淋巴结转移、远处器官转移和Dukes'分期相关(P0.05)。高表达UHRF1结肠患者的生存时间明显较低表达者短(P0.05)。单因素分析显示淋巴结转移、远处器官转移、UHRF1和Dukes'分期与患者累积生存率相关(P0.05)。多因素分析显示UHRF1可作为结肠癌患者预后的独立危险因子(P0.05)。采用si RNA转染HCT116后,UHRF1表达显著降低,细胞增殖被抑制。结论:UHRF1参与结肠癌细胞增殖,与结肠癌患者预后密切相关。     

HSV-1感染对人星形胶质瘤细胞增殖、凋亡和细胞周期的影响

目的:研究单纯疱疹病毒1型(Herpes simplexvires,HSV-1)感染对人星形胶质瘤细胞U251增殖、凋亡和细胞周期的影响.方法:以感染复数(MOI)为5的HSV-1感染体外培养的U251细胞,在感染后24 h、48 h、72 h和96 h用倒置显微镜观察U251细胞的形态改变:用MTT法、流式细胞术观察HSV-1感染对U251细胞增殖、凋亡和细胞周期的影响.结果:①U251细胞在感染24h后开始出现细胞融合,48 h后开始出现典型的细胞病变效应(Cytopathic effect,CPE),72h后超过80%的细胞出现CPE,.96h后细胞大部分死亡.②MTT法显示HSV-1感染U251细胞24 h、48 h、72 h及96 h的U251细胞OD值均低于对照组(P<0.05).③HSV-1感染U251细胞12h后凋亡率与对照组无显著差异(P0.05),感染24h和36h后凋亡率比相应对照组有显著差别(p<0.05).④HSV感染12h、24h和36h后均可引起U251细胞S期细胞增多和G0/G1期细胞减少,24 h后G2/M期细胞比例开始增加.结论:HSV.1能感染体外培养的U251细胞,抑制其增殖,促进其凋亡并影响其细胞周期.     

利用RNAi技术分析VP16对HSV-1增殖过程的影响

目的:探讨在RNAi技术条件下,HSV-1感染Vero细胞过程中,病毒间层蛋白VP16的生物学功能。方法:用T7RNA聚合酶体外合成针对VP16 mRNA的3种T7siRNAs。用Lipofectamine 2000脂质体转染Vero细胞后接种HSV-1,感染后12、24、36、48、60和72h,分别收取实验组和对照组标本,Karber法计算病毒滴度值,绘制子代病毒一步生长曲线。结果:病毒子代一步生长曲线显示,转染siRNAs的实验组Vero细胞接种HSV-1后24h(24h pi),病毒滴度明显低于对照组;12h pi也观察到病毒滴度低于对照组的现象;siRNAs具有协同作用,三种siRNAs同时作用时干扰效果最为明显;实验组病毒滴度高峰出现时间后移12h左右。结论:HSV-1间层蛋白VP16对病毒复制、成熟起重要的生物学作用。     

扇贝糖胺聚糖对感染HSV-I小鼠免疫功能的影响研究

目的：研究扇贝裙边糖胺聚糖（glycosaminoglycan from Scallop Skirt,SS—GAG）对感染单纯疱疹病毒I型（herpes simplex virustype,HSV-I）小鼠免疫功能的影响。方法：通过在无菌条件下给予小鼠注射扇贝糖胺聚糖SS．GAG,连续11天,并在给药第三天给小鼠腹腔注射HSV—I病毒悬液建立小鼠感染模型,用MTT等方法观察SS—GAG对HSV—I感染小鼠腹腔巨噬细胞吞噬活性的影响、对脾脏指数、胸腺指数的影响以及对脾淋巴细胞转化能力等免疫指标的影响。结果：与病毒对照组相比,扇贝糖胺聚糖SS-GAG低剂量组、中剂量组、高剂量组均能显著增强HSV．I感染小鼠的腹腔巨噬细胞的吞噬活性和HSV—I感染小鼠的脾脏指数和胸腺指数（P〈0．01）,并且能促进其脾淋巴细胞转化增殖能力（P〈0．01）。结论：扇贝糖胺聚糖在体内有一定的抗I型单纯疱疹病毒作用。其抗病毒作用可能与增强机体免疫功能有关。     

Modulation of Glutamine Synthesis in Cultured Astrocytes by Nitric Oxide

1. Previous results suggest that glutamine synthesis in brain could be modulated by nitrix oxide. The aim of this work was to assess this possibility.2. As glutamine synthetase in brain is located mainly in astrocytes, we used primary cultures of astrocytes to assess the effects of increasing or decreasing nitrix oxide levels on glutamine synthesis in intact astrocytes.3. Nitric oxide levels were decreased by adding nitroarginine, an inhibitor of nitric oxide synthase. To increase nitric oxide we used S-nitroso-N-acetylpenicillamine, a nitric oxide generating agent.4. It is shown that S-nitroso-N-acetylpenicillamine decreases glutamine synthesis in intact astrocytes by 40–50%. Nitroarginine increases glutamine synthesis slightly in intact astrocytes.5. These results indicate that brain glutamine synthesis may be modulated in vivo by nitric oxide.     

HSV-1UL-12基因的克隆、表达及产物复性

目的:克隆及表达单纯疱疹病毒Ⅰ型(HSV-1)UL12基因,对其表达产物HSV-1碱性核酸酶(AN)进行复性,为建立抗HSV-1药物的分子筛选模型奠定了基础,对于寻找抗病毒药物具有重要意义.方法:从HSV-1感染的Vero细胞中提取HSV-1基因组DNA,通过PCR克隆出UL12基因并对其测序;然后将HSV-1 UL12基因先克隆至pMD19-T载体,再亚克隆到pET32a表达载体上,并将重组载体转化到E.coli Rosetta菌中进行表达;最后用梯度盐酸胍对表达出的包涵体进行复性.结果:成功构建了pET32a-UL12重组载体,经序列比对,与GenBank上公布的HSV-1国际标准毒株17株的UL12基因序列的同源性达到了99.2%.在E.coli Rosetta菌中以包涵体的形式表达,经复性得到有活性的HSV-1碱性核酸酶(AN).AN同时具有核酸外切酶和核酸内切酶的活性,与核酸作用60min时酶活性最高.结论:重组载体pET32a-UL12经表达、复性可获得有活性的HSV-1 AN.     

Deletion of the PH-domain and Leucine-rich Repeat Protein Phosphatase 1 (Phlpp1) Increases Fibroblast Growth Factor (Fgf) 18 Expression and Promotes Chondrocyte Proliferation

Endochondral ossification orchestrates formation of the vertebrate skeleton and is often induced during disease and repair processes of the musculoskeletal system. Here we show that the protein phosphatase Phlpp1 regulates endochondral ossification. Phlpp1 null mice exhibit decreased bone mass and notable changes in the growth plate, including increased BrdU incorporation and matrix production. Phosphorylation of known Phlpp1 substrates, Akt2, PKC, and p70 S6 kinase, were enhanced in ex vivo cultured Phlpp1^−/− chondrocytes. Furthermore, Phlpp1 deficiency diminished FoxO1 levels leading to increased expression of Fgf18, Mek/Erk activity, and chondrocyte metabolic activity. Phlpp inhibitors also increased matrix content, Fgf18 production and Erk1/2 phosphorylation. Chemical inhibition of Fgfr-signaling abrogated elevated Erk1/2 phosphorylation and metabolic activity in Phlpp1-null cultures. These results demonstrate that Phlpp1 controls chondrogenesis via multiple mechanisms and that Phlpp1 inhibition could be a strategy to promote cartilage regeneration and repair.     

Radiolabelled Pyrimidine Nucleosides to Monitor the Expression of HSV-1 Thymidine Kinase in Gene Therapy

Abstract

Selective radiolabelling and imaging of transduced HSV tk expressing cells was studied using [¹²³I]IVFRU, [¹²⁵I]FIRU and [¹²⁵I]FIAU. Although all three radionucleosides accumulated in the KBALB-STK transduced murine tumour line in vitro and in vivo, [¹²⁵I]FIRU provided optimal performance in terms of selectivity for HSV tk expressing cells and % of injected dose accumulating in the tumor.     

Src Homology-2 Domain-Containing Protein Tyrosine Phosphatase (SHP) 2 and p38 Regulate the Expression of Chemokine CXCL8 in Human Astrocytes

CXCL8, one of the first chemokines found in the brain, is upregulated in the brains and cerebrospinal fluid of HIV-1 infected individuals suggesting its potential role in human immune deficiency virus (HIV)-associated neuroinflammation. Astrocytes are known to be the major contributors to the CXCL8 pool. Interleukin (IL)-1β activated astrocytes exhibit significant upregulation of CXCL8. In order to determine the signaling pathways involved in CXCL8 regulation in astrocytes, we employed pharmacological inhibitors for non-receptor Src homology-2 domain-containing protein tyrosine phosphatase (SHP) 2 and mitogen-activated protein kinases (MAPK) pathway and observed reduced expression of CXCL8 following IL-1β stimulation. Overexpression of SHP2 and p38 enzymes in astrocytes led to elevated CXCL8 expression; however, inactivating SHP2 and p38 with dominant negative mutants abrogated CXCL8 induction. Furthermore, SHP2 overexpression resulted in higher SHP2 and p38 enzyme activity whereas p38 overexpression resulted in higher p38 but not SHP2 enzyme activity. Phosphorylation of SHP2 was important for phosphorylation of p38, which in turn was critical for phosphorylation of extracellular signal regulated kinase (ERK). Thus, our findings suggest an important role for SHP2 in CXCL8 expression in astrocytes during inflammation, as SHP2, directly or indirectly, modulates p38 and ERK MAPK in the signaling cascade leading to CXCL8 production. This study provides detailed understanding of the mechanisms involved in CXCL8 production during neuroinflammation.     

Local CD4 and CD8 T-Cell Reactivity to HSV-1 Antigens Documents Broad Viral Protein Expression and Immune Competence in Latently Infected Human Trigeminal Ganglia

Herpes simplex virus type 1 (HSV-1) infection results in lifelong chronic infection of trigeminal ganglion (TG) neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1–infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.