TLR4介导汉滩病毒引起血管内皮细胞分泌IL-6和TNF-α

目的检测汉滩病毒感染血管内皮细胞后IL-6,IL-8和TNF-α的分泌变化及其与TLR4的关系。方法用5LgTCID50/mL的HTNV76-1180.2mL感染EVC-304细胞(TLR4+)和EVC-304TS4(TLR4-)分别为实验组,以病毒未感染为阴性对照组,以LPS(2μg/mL)刺激作为阳性对照。48h后取细胞培养上清,用人IL-6,IL-8和TNF-α定量EIA试剂盒分别检测IL-6,IL-8和TNF-α在两个细胞系感染前后的分泌水平。结果IL-8在两个细胞系中感染前后的变化不明显,IL-6和TNF-α在EVC-304细胞系中,HTNV感染后升高,而在TLR4表达阴性的EVC-304细胞中,感染前后变化不明显。结论在TLR4表达阳性的EVC-304细胞中IL-6和TNF-α分泌增加,血管内皮细胞EVC-304在HTNV感染后的IL-6和TNF-α分泌可能是TLR4介导的。     

汉坦病毒体外感染乳大鼠大脑皮层星形胶质细胞

目的 证实乳大鼠大脑皮层星形胶质细胞是汉坦病毒属(HV)汉滩病毒(HTNV)和汉城病毒(SEOV)感染的靶细胞,观察病毒感染星形胶质细胞后的动态变化.方法 建立乳大鼠大脑皮层星形胶质细胞的体外培养体系,用HTNV 76-118和SEOV L99分别感染乳大鼠大脑皮层星形胶质细胞,免疫荧光染色、Western印迹和RT-PCR检测病毒感染细胞后病毒NP和S基因片段的变化.结果 成功建立了乳大鼠大脑皮层星形胶质细胞的体外培养体系,并通过免疫荧光染色、Western印迹和RT-PCR证实HTNV 76-118和SEOV L99可以感染离体培养的乳大鼠大脑皮层星形胶质细胞,随感染时间推移,病毒感染细胞数和病毒量显著增加.结论 乳大鼠大脑皮层星形胶质细胞可作为HTNV和SEOV感染的靶细胞.这为研究肾综合征出血热发病机制提供了实验平台.     

曼氏血吸虫:用尾蚴糖萼制剂免疫可增加成虫负荷

曾有人发现含有糖萼(GCX)和其它尾蚴成份的尾蚴液在体外能抑制人外周血淋巴细胞对血吸虫抗原和促细胞分裂剂的增生应答。但对GCX作免疫原的作用尚未研究。作者等用十二烷基磺酸钠(SDS)和盐酸胍(GuHCl)作溶剂制备GCX,2种GCX制剂富含碳水化合物,其中去氧半乳糖的含量分别为40％及80％。又用PBS、Maalox(氢     

汉坦病毒激发人胃上皮细胞病变和凋亡

目的证实人胃粘膜上皮细胞是否为汉坦病毒属(HV)汉滩病毒型(HTN)和汉城病毒型(SEO)病毒的靶细胞,病毒对其是否有致细胞病变效应(CPE)和促凋亡作用。方法建立人胃上皮细胞(HGEC)离体培养;用HVN8、76118、Z37、Seoul8039株感染HGEC,观察细胞CPE,用直接免疫荧光法(IFA)检测病毒感染细胞和感染灶;用AnnexinV FITCkit观察HV的致HGEC凋亡和坏死作用。结果HTNV和SEOV可以感染离体培养的HGEC,形成感染灶并出现CPE;与模拟感染细胞相比,N8、76118、Z37、Seoul8039株感染的HGEC凋亡和坏死细胞比例明显增高,HTNV较SEOV促凋亡和坏死作用更强。结论HGEC可作为HTNV和SEOV感染的靶细胞,致CPE并促进细胞凋亡和坏死,在急性肾综合征出血热病人胃粘膜损害机制中起重要作用。     

汉滩病毒结构蛋白与该病毒感染诱导表达的多种热休克蛋白的研究

目的研究汉滩病毒(HTNV)感染乳鼠诱导其脑组织表达热休克蛋白(HSPs)及其与病毒结构蛋白的相互关系。方法选出生2～3d的昆明乳鼠实验性感染HTNV,取感染后8d的乳鼠脑组织制成组织匀浆液,用双特异性抗体夹心酶联免疫吸附法(ELISA)及免疫共沉淀方法分析病毒核衣壳蛋白(HTNVNP)和囊膜糖蛋白G2(HTNVG2)与94000葡萄糖调节蛋白(GRP94)、HSP70、HSP27三种HSPs的关系。结果HTNV感染乳鼠诱导其脑组织表达GRP94、HSP70;HTNVNP同时与GRP94、HSP70、HSP27相互作用,呈复合物形式存在;HTNVG2也与HTNVNP和HSP27存在相互作用,形成HTNVG2NPHSP27非共价复合物。结论HTNV感染可诱导表达多种HSPs,这些HSPs同时与病毒结构蛋白发生相互作用形成非共价复合物,表明在病毒感染和病毒结构蛋白的合成、转运等过程中有多种HSPs伴侣分子的参与,其相互作用机制有待进一步研究。     

应用RNA聚合酶Ⅰ反向遗传操作技术拯救汉滩病毒84FLi株微基因组

目的 建立基于汉滩病毒(HTNV)84FLi株L片段的RNA聚合酶Ⅰ微基因组拯救体系.方法 将含有氯霉素乙酰转移酶(CAT)编码区的cDNA插入含有HTNV 84FLi株L片段5'端和3'端非编码区的质粒内的两个非编码区之间,将此eDNA嵌合体(polⅠ表达盒)克隆人人polⅠ肩动子和终止子之间,分别获得正义和反义方向的RNA polⅠ转录报告质粒.用报告质粒转染293T细胞或等量293T和HTNV感染的Vero混合培养细胞,在HTNV的L蛋白和核衣壳蛋白表达质粒共转染后48 h收获细胞,检测CAT活性.用上清液感染293T细胞,了解CAT活性的传代能力.结果 构建了正义和反义方向的HTNV 84FLi株L片段微基因组RNA聚合酶Ⅰ报告质粒pLvRNA-CAT和pLcRNA-CAT,用此质粒转染细胞后能检测到CAT的表达,且CAT活性能在拯救病毒微基因组中传代.结论 应用RNA聚合酶Ⅰ反向遗传操作技术成功拯救了HTNV 84FLi株微基冈组.     

机械力对血管内皮细胞一氧化氮合成的调控机制及致动脉粥样硬化作用

<正>血流机械力变化导致的动脉粥样硬化主要在于它破坏了血管内皮细胞和平滑肌细胞的一氧化氮(NO)生成的系统平衡〔1〕。本文重点讨论血流机械力〔剪切力和环壁张力对内皮细胞NO的生成和NO合酶(NOS)〕表达的调控机制,并强调其介导血管内皮细胞的感染、氧自由基的产生及最终引发血管壁重建(主要是平滑肌细胞参与)的机制。1剪切力对血管内皮细胞NO生成及NOS表达的影响1.1剪切力影响血管内皮细胞生成NO单向的剪切力通过     

人巨细胞病毒临床分离株感染血管内皮细胞伴肾素基因表达

目的 探讨巨细胞病毒(Cytomegalovirus,CMV)是否感染血管内皮细胞伴肾素表达.方法 (1)用107pfu(空斑形成单位)/mlCMV临床分离株BI-5和实验室型CMV AD169分别与106腹主动脉内皮细胞和人脐静脉内皮细胞共同孵育,在23小时后、第3天、第7天、第10天和第14天分别收集培养上清200μI第14天用PBS缓冲液洗细胞3次,收获细胞.每组实验均设培养液代替病毒液的无感染对照;(2)COBAS定量PCR检测培养上清中CMV DNA拷贝数;(3)PCR检测感染细胞中CMV pol基因;(4)RT-PCR、Real time RT-PCR和Western blot检测肾素在感染细胞内的表达.结果 (1)BI-5和AD169感染静脉和动脉细胞后,其形态学变化相似,无细胞裂解病理效应;(2)AD169感染细胞不同时间培养上清中CMV DNA拷贝数无明显增加,BI-5呈增殖趋势;(3)BI-5感染动脉细胞CMV DNA拷贝数和肾素表达量均大于静脉细胞.结论 临床分离株CMV以非裂解形式在血管内皮细胞持续存在并诱导肾素基因表达,血管内皮细胞分泌肾素可能是CMV感染引起心血管疾病的新机制.     

汉滩病毒感染EA.hy926细胞上调固有免疫相关分子表达的研究

目的　探讨汉滩病毒（hantaan virus, HTNV）感染EA.hy926细胞诱导抗病毒固有免疫相关分子的表达,为建立HTNV感染的细胞模型提供依据。方法　将HTNV感染EA.hy926细胞,利用间接免疫荧光和实时荧光定量PCR技术于不同感染时间段,检测EA.hy926细胞中模式识别受体、细胞因子及抗病毒相关分子的mRNA表达水平。结果　HTNV感染EA.hy926细胞后,随着感染时间的持续,核蛋白mRNA相对表达倍数显著上调,且不同感染时间段差异具有统计学意义（P＜0.05）;与未处理组相比,HTNV感染EA.hy926后,Toll样受体3、RIG-I和MDA5模式识别受体mRNA相对表达倍数均显著上调（P均＜0.05）,IL-6、IL-10、IFN-β和CCL5细胞因子mRNA相对表达倍数均显著上调（P均＜0.05）,ISG15、MxA、OAS1、IFITM1和IFITM3抗病毒相关分子mRNA相对表达倍数均显著上调（P均＜0.05）。结论　HTNV感染EA.hy926细胞后,可上调抗病毒固有免疫相关分子的表达,该细胞可以作为体外HTNV感染的细胞模型。     

流感病毒感染与血管内皮细胞功能变化的体外实验研究

目的探讨流感病毒感染人脐静脉内皮细胞后,细胞间黏附分子1和血管细胞黏附分子1表达的变化,以期从细胞和分子水平探讨流感病毒感染在动脉粥样硬化形成中的作用。方法分别运用染料结合实时荧光定量聚合酶链反应方法、流式细胞术和酶联免疫吸附实验,检测流感病毒感染的不同时段(0、24、48、72 h)人脐静脉内皮细胞分泌的细胞间黏附分子1、血管细胞黏附分子1的表达情况。结果流感病毒感染人脐静脉内皮细胞后,通过以上3种方法检测细胞间黏附分子1和血管细胞黏附分子1的变化,0 h有基础水平的表达,随感染时间延长,表达量逐渐增高,感染24 h时达高峰,48 h开始回落,72 h表达量仍维持在较高的水平,显著高于未加病毒的对照组,各时段与对照组比较,均有统计学意义(P<0.05)。结论流感病毒感染人脐静脉内皮细胞后,细胞间黏附分子1和血管细胞黏附分子1表达增加,因此推测流感病毒感染导致血管内皮细胞功能障碍,流感病毒感染可能参与动脉粥样硬化的炎症反应。     

Intracerebral chondroitinase ABC and heparan sulfate proteoglycan glypican improve outcome from chronic stroke in rats

Physical and chemical constraints imposed by the periinfarct glial scar may contribute to the limited clinical improvement often observed after ischemic brain injury. To investigate the role of some of these mediators in outcome from cerebral ischemia, we treated rats with the growth-inhibitory chondroitin sulfate proteoglycan neurocan, the growth-stimulating heparan sulfate proteoglycan glypican, or the chondroitin sulfate proteoglycan-degrading enzyme chondroitinase ABC. Neurocan, glypican, or chondroitinase ABC was infused directly into the infarct cavity for 7 d, beginning 7 d after middle cerebral artery occlusion. Glypican and chondroitinase ABC reduced glial fibrillary acidic protein immunoreactivity and increased microtubule-associated protein-2 immunoreactivity in the periinfarct region, and glypican- and chondroitinase ABC-treated rats showed behavioral improvement compared with neurocan- or saline-treated rats. Glypican and chondroitinase ABC also increased neurite extension in cortical neuron cultures. Glypican increased fibroblast growth factor-2 expression and chondroitinase ABC increased brain-derived neurotrophic factor expression in these cultures, whereas no such effects were seen following neurocan treatment. Thus, treatment with glypican or enzymatic disruption of neurocan with chondroitinase ABC improves gross anatomical, histological, and functional outcome in the chronic phase of experimental stroke in rats. Changes in growth factor expression and neuritogenesis may help to mediate these effects.     

Heparanase, tissue factor, and cancer

Heparanase is an endo-beta- D-glucuronidase that is capable of cleaving heparan sulfate side chains of heparan sulfate proteoglycans on cell surfaces and the extracellular matrix, activity that is strongly implicated in tumor metastasis and angiogenesis. Evidence was provided that heparanase overexpression in human leukemia, glioma, and breast carcinoma cells results in a marked increase in tissue factor (TF) levels. Likewise, TF was induced by exogenous addition of recombinant heparanase to tumor cells and primary endothelial cells, induction that was mediated by p38 phosphorylation and correlated with enhanced procoagulant activity. TF induction was further confirmed in heparanase-overexpressing transgenic mice and correlated with heparanase expression levels in leukemia patients. Heparanase was also found to be involved in the regulation of tissue factor pathway inhibitor (TFPI). It was shown that heparanase overexpression or exogenous addition induces two- to threefold increase of TFPI expression. Similarly, heparanase stimulated accumulation of TFPI in the cell culture medium. Extracellular accumulation exceeded, however, the observed increase in TFPI at the protein level and appeared to be independent of heparan sulfate and heparanase enzymatic activity. Instead, a physical interaction between heparanase and TFPI was demonstrated, suggesting a mechanism by which secreted heparanase interacts with TFPI on the cell surface, leading to dissociation of TFPI from the cell membrane and increased coagulation activity, thus further supporting the local prothrombotic function of heparanase. As heparins are strong inhibitors of heparanase, in view of the effect of heparanase on TF/TFPI pathway, the role of heparins' anticoagulant activity may potentially be expanded.     

Lipoprotein modulation of subendothelial heparan sulfate proteoglycans (perlecan) and atherogenicity

Heparan sulfate proteoglycans (HSPGs) are key constituents of subendothelial extracellular matrix that play an important role in the assembly and structure of the basement membrane, regulation of basement membrane permeability, growth factor activity and cellular adhesion. Vascular HSPGs decrease during inflammation, atherosclerosis and diabetes. Recent studies showed that HSPGs are negatively regulated by atherogenic molecules and positively regulated by antiatherogenic agents. Extracellular matrix HSPG, perlecan, appears to be a key target of regulation by these agents. At least two levels of regulation appear to control perlecan HSPG in matrix; a change in core protein expression or a change in heparan sulfate metabolism. Atherogenic levels of low-density lipoprotein (LDL), oxidized LDL and lysolecithin decrease not only perlecan core protein synthesis but also enhance heparan sulfate degradation by stimulating endothelial secretion of heparanase. ApoE and apoE-HDL, in contrast, increase perlecan core protein as well as sulfation of heparan sulfate. Increased perlecan in endothelial cells was associated with increased antithrombin-binding and antiproliferative heparan sulfates. Moreover, modulation of perlecan appears to have a direct effect on smooth muscle cell growth. Thus, lipoprotein modulation of vascular perlecan may play a key role in the modulation of atherogenesis.     

Varied low density lipoprotein binding property of proteoglycans synthesized by vascular smooth muscle cells cultured on extracellular matrix

Earlier we showed that the extracellular matrix (ECM) secreted by vascular cells modulated proteoglycan synthesis by vascular smooth muscle cells in culture and altered the proteoglycan characteristics. In this study, we tested the hypothesis that these ECM-mediated alterations increased the affinity of the proteoglycans for plasma low density lipoprotein (LDL). Newly synthesized proteoglycans were isolated from smooth muscle cells cultured on the ECMs secreted by vascular endothelial cells, smooth muscle cells, or THP-1 macrophages and their binding affinity for LDL determined. Proteoglycans from all cultures contained sub-fractions that bound LDL with low and high affinity. However, compared with the cells plated on the endothelial cell ECM, the cells plated on the smooth muscle cell ECM and macrophage ECM synthesized significantly more high affinity proteoglycans. Removal of collagen, elastin, and chondroitin sulfates from the smooth muscle cell ECM and chondroitin sulfates from the macrophage ECM increased the production of high affinity proteoglycans by 15-22%. However, neutralization of fibronectin from both ECMs decreased the high affinity proteoglycans by 20%. Removal of matrix-bound growth factors had no effect on the synthesis of high affinity proteoglycans. Compared with the low affinity proteoglycans, the high affinity proteoglycans were larger, more sulfated and contained higher proportions of chondritin sulfate, dermatan sulfate, and N-sulfated heparan sulfate chains. These results suggest that the ECM-mediated alterations in vascular smooth muscle cell proteoglycans may lead to increased deposition of LDL in the arterial wall.     

Surfen, a small molecule antagonist of heparan sulfate

In a search for small molecule antagonists of heparan sulfate, we examined the activity of bis-2-methyl-4-amino-quinolyl-6-carbamide, also known as surfen. Fluorescence-based titrations indicated that surfen bound to glycosaminoglycans, and the extent of binding increased according to charge density in the order heparin > dermatan sulfate > heparan sulfate > chondroitin sulfate. All charged groups in heparin (N-sulfates, O-sulfates, and carboxyl groups) contributed to binding, consistent with the idea that surfen interacted electrostatically. Surfen neutralized the anticoagulant activity of both unfractionated and low molecular weight heparins and inhibited enzymatic sulfation and degradation reactions in vitro. Addition of surfen to cultured cells blocked FGF2-binding and signaling that depended on cell surface heparan sulfate and prevented both FGF2- and VEGF₁₆₅-mediated sprouting of endothelial cells in Matrigel. Surfen also blocked heparan sulfate-mediated cell adhesion to the Hep-II domain of fibronectin and prevented infection by HSV-1 that depended on glycoprotein D interaction with heparan sulfate. These findings demonstrate the feasibility of identifying small molecule antagonists of heparan sulfate and raise the possibility of developing pharmacological agents to treat disorders that involve glycosaminoglycan–protein interactions.     

Phosphomannopentaose Sulfate (PI‐88): Heparan Sulfate Mimetic with Clinical Potential in Multiple Vascular Pathologies

The sulfated oligosaccharide PI‐88 is a potent antiangiogenic, antitumor and anti‐metastatic agent derived from yeast. It is primarily composed of sulfated phosphomannopentaose and phosphomannotetraose oligosaccharide units and is presently under evaluation in Phase II clinical trials for anticancer efficacy. PI‐88 inhibits the heparan sulfate‐degrading enzyme heparanase, exhibits antiangiogenic activity and has anticoagulant properties mediated by heparin cofactor II. It also inhibits vascular smooth muscle cell proliferation, kinase signalling and arterial intimal thickening following balloon injury. Many heparan sulfate‐binding growth factors require heparan sulfate as a co‐receptor in order to effectively deliver growth signals to cells. Thus, the antiangiogenic and antirestenotic activity of PI‐88 may be at least partially due to this highly sulfated oligosaccharide competing with the interaction of growth factors, such as FGF‐2 and VEGF, with cell surface heparan sulfate. This heparan sulfate mimetic has, therefore, multiple functions and therapeutic potential in a variety of vascular disorders.     

Inhibition of heparanase-mediated degradation of extracellular matrix heparan sulfate by non-anticoagulant heparin species

Incubation of human platelets, human neutrophils, or highly metastatic mouse lymphoma cells with sulfate-labeled extracellular matrix (ECM) results in heparanase-mediated release of labeled heparan sulfate cleavage fragments (0.5 less than Kav less than 0.85 on Sepharose 6B). This degradation was inhibited by native heparin both when brought about by intact cells or their released heparanase activity. Degradation of heparan sulfate in ECM may facilitate invasion of normal and malignant cells through basement membranes. The present study tested the heparanase inhibitory effect of nonanticoagulant species of heparin that might be of potential use in preventing heparanase mediated extravasation of bloodborne cells. For this purpose, we prepared various species of low-sulfated or low-mol-wt heparins, all of which exhibited less than 7% of the anticoagulant activity of native heparin. N-sulfate groups of heparin are necessary for its heparanase inhibitory activity but can be substituted by an acetyl group provided that the O-sulfate groups are retained. O-sulfate groups could be removed provided that the N positions were resulfated. Total desulfation of heparin abolished its heparanase inhibitory activity. Heparan sulfate was a 25-fold less potent heparanase inhibitor than native heparin. Efficiency of low-mol-wt heparins to inhibit degradation of heparan sulfate in ECM decreased with their main molecular size, and a synthetic pentasaccharide, representing the binding site to antithrombin III, was devoid of inhibitory activity. Similar results were obtained with heparanase activities released from platelets, neutrophils, and lymphoma cells. We propose that heparanase inhibiting nonanticoagulant heparins may interfere with dissemination of bloodborne tumor cells and development of experimental autoimmune diseases.     

Heparin stimulates proteoglycan synthesis by vascular smooth muscle cells while suppressing cellular proliferation.

We studied the effect of heparin on proteoglycan synthesis by bovine aortic smooth muscle cells in culture. Confluent, growth-arrested cells were incubated with [35S]sulfate, [3H]glucosamine or [3]serine in the presence of 0-600 micrograms/ml heparin. Metabolically labeled proteoglycans secreted into the culture medium and associated with the cell layer were analyzed. In cultures treated with heparin there was a dose-dependent increase in [35S]sulfate incorporation into secreted proteoglycans which reached a maximum (35% above controls) at 100 micrograms/ml heparin. At higher concentrations of heparin, the stimulatory activity declined and finally disappeared. Radioactivity in cell-associated proteoglycans increased significantly (16% above controls) only in cultures treated with 100 micrograms/ml heparin. Heparin also produced similar increases in the incorporation of [3H]glucosamine and [3H]serine into secreted and cell-associated proteoglycans. While chondroitin sulfate, dermatan sulfate and heparan sulfate were elevated in the media, only chondroitin sulfate and heparan sulfate were increased in the cell layer. Heparin did not alter the degradation of proteoglycans. Heparin, while inhibiting the proliferation of subconfluent smooth muscle cells, also stimulated to a greater extent the incorporation of [35S]sulfate into proteoglycans. Other glycosaminoglycans, such as heparan sulfate, dermatan sulfate, heparin hexasaccharide and Sulodexide caused a significant but lesser stimulation of proteoglycan synthesis, while chondroitin sulfates and hyaluronic acid had no effect. Gel filtration chromatography of proteoglycans and their constituent glycosaminoglycans from heparin-treated and untreated cultures showed no differences in their molecular size. The results indicate that heparin can stimulate proteoglycan synthesis by vascular smooth muscle cells irrespective of their state of proliferation. This might have implications in vessel wall repair and arterial wall lipid deposition.