栀子提取物ZG对单纯疱疹病毒1型吸附量的影响

目的 观察栀子提取物ZG对单纯疱疹病毒1型（HSV-1）吸附过程中病毒吸附量的影响，以探讨其抗病毒作用机理。方法 采用异硫氰酸荧光素（fluorescein isothiocyanate，FTrc）作为荧光探针标记病毒，以肝素钠为对照，借助流式细胞仪，通过收集多种不同的加药方式后细胞表面的荧光强度，来考察病毒吸附过程中病毒吸附量的变化及栀子提取物ZG对此变化的影响。结果 单纯疱疹病毒1型在Hep-2细胞表面吸附存在饱和性、特异性；肝素钠3种加药方式均能明显降低病毒吸附量，先加药后吸附及吸附和加药同时进行两种方式，优于先吸附后加药的方式，其吸附抑制率分别为84．76％和82．02％；栀子提取物ZG3种加药方式均能明显降低病毒吸附量，其中以吸附和加药同时进行组最为显著，吸附抑制率为68．46％。结论 栀子提取物ZG可能是通过作用于病毒吸附的多个环节来达到抑制HSV-1的吸附作用。     

栀子提取物ZG对单纯疱疹病毒1型吸附后宿主细胞膜电位的影响

经我试验组反复验证栀子提取物ZG在体内外均有明显的抗病毒感染作用，其作用机理之一可能是通过抑制病毒吸附来发挥抗病毒作用。通常情况下，稳定的膜电位是维持细胞正常功能所必需的；而在病毒感染过程中，细胞膜跨膜电位差（△φ）则作为促使病毒内化的主要能态来源。国外有研究证实在HSV-1感染8h后HeLa细胞出现明显的去极化，而国内未见病毒吸附过程中膜电位变化的报道。     

加味佛手散对大鼠红细胞膜微粘度及超微结构的影响

以被动吸烟雄鼠及孕鼠为模型，探讨加味佛手散对红细胞膜流动性及红细胞超微结构的作用。用荧光探剂ＡＮＳ和ＤＰＨ分别标记红细胞膜脂的不同区段，观察加味佛手散对红细胞膜流动性的影响，结果显示，除ＤＰＨ标记的孕鼠红细胞膜流动性外，其它各组模型动物膜脂流动性明显低于相应对照组及用药组（Ｐ均＜０．０１）；扫描电镜及透射电镜下异形红细胞数，模型组明显多于对照组及用药组（Ｐ均＜０．００１）。用药组以上各项指标明显改善，而与对照组无明显差异（Ｐ均＞０．０５）。研究结果提示，加味佛手散有保护红细胞正常膜脂流动性及超微结构的作用，从而有利于改善红细胞的变形能力，增加微循环血流灌注     

自由基对红细胞膜分子流动性的影响

为探明自由基引起红细胞流变性改变的作用机理，我们对体外自由基作用三十分钟前后红细胞膜剪切弹性模量、细胞表面指数、膜脂质分子和蛋白分子流动性进行了比较分析。发现自由基作用后，红细胞膜剪切弹性模量、膜脂质分子和蛋白分子的相对旋转时间分别增加了４９．８％、４９．５％和１９１．４％，而红细胞表面指数无变化；此外，随着自由基浓度的降低以上变化也随之减轻；在自由基损伤后的第三小时，上述改变部分回复。本研究说明，自由基引起脂质和蛋白分子流动性降低共同导致红细胞膜剪切弹性模量的增加，并且一定浓度自由基对红细胞流变性的损伤是部分可逆的。     

日本鬼鲉毒腺提取物对大鼠红细胞损伤及治疗的实验研究

目的 探讨毒鲉科棘毒鱼日本鬼鲉毒腺提取物对大鼠红细胞的结构和细胞膜流动性的影响，及其与氧化损伤的关系和有效的治疗方法。方法 以两种不同浓度的粗提液作用于大鼠红细胞，光、电镜下观察红细胞形态变化及荧光偏振实验检测红细胞的流动性的变化。结果 两种浓度毒素提取液均可使大鼠红细胞发生棘突样变、明显肿胀及异形性改变，出现溶血现象，并随作用时间、剂量的增加而显著；毒液组红细胞膜流动性显著降低；维生素C组可部分阻断毒素的作用。结论 蜇伤局部容易感染和难愈合的原因可能与红细胞变形及其流动性下降密切相关。抗氧化剂维生素C可以减轻毒液的作用，毒鲉科棘毒鱼蜇伤的机制可能与部分氧化损伤机制有关。     

中重度烧伤早期红细胞膜流动性的实验研究和临床观察

采用ＤＰＨ荧光探针标记、荧光测定和光镜观察烧伤大鼠中重度烫伤早期红细胞膜流动性、血浆过氧化脂质含量、红细胞异形率的动态变化，以及不同的处理方法对其影响。同时对同期１０例中重度烧伤病进行观察，发现中重度烧伤早期红细胞膜流动性明显下降，血浆脂质过氧化物增多，红细胞异形率增高，这些改变与烧伤面积成正相关，提示烧伤后氧自由基是损伤红细胞膜流动性的因素之一。应用抗自由基药物有保护红细胞膜结构和功能的作用，有     

结核分枝杆菌诱导宿主巨噬细胞凋亡机制初探

目的探讨结核杆菌感染中巨噬细胞凋亡的信号传导通路及分子机理。方法用透射电镜、荧光染色对凋亡细胞的形态特征进行分析；琼脂糖凝胶电泳检测巨噬细胞DNA片段化水平；流式细胞术测定不同时期小鼠巨噬细胞凋亡率、膜表面Toll样受体2(TLR2)和胞内Bcl-2蛋白的水平。结果结核杆菌强毒力株H37Rv感染组的巨噬细胞凋亡率在1～7d内逐渐升高，至7d时最高可达32．2％，感染的9～11d以后，凋亡率逐渐降低；H37Rv感染组Bcl-2水平于感染9～11d后逐渐升高，与卡介苗(BCG)组比较差异有统计学意义；H37Rv感染组和BCG组的巨噬细胞膜表面的ⅡB．2水平均高于正常对照组；各组组内不同时期的TLB2水平无明显变化。结论结核杆菌H37Rv株在感染的早期对宿主巨噬细胞有较强的致凋亡作用，而Bcl-2表达的增加能显著抑制这种凋亡。结核杆菌在体内感染过程中，TLR2蛋白可能与巨噬细胞凋亡及Bcl-2表达无关。     

黄柏及其主要成分对细胞膜流动性的影响(英)

目的：观察具有免疫抑制作用的药物黄柏及其3种主要成分：小檗碱、药根碱与巴马汀对正常小鼠脾细胞膜流动性的影响。 方法： 以1，6－二苯基，1，3，5－己三烯（DPH）作为荧光探针，利用荧光偏振法测定脾细胞膜脂质区的流动性。 结果： 在无ConA刺激时，黄柏的水提物可提高小鼠脾细胞膜的流动性，6.25％的浓度与对照组相比有极显著性差异；小檗碱与药根碱可降低脾细胞膜的流动性，与对照组相比均有显著性差异；巴马汀可提高脾细胞膜的流动性，与对照组相比有极显著性差异。经ConA刺激后，黄柏的水提物、小檗碱、药根碱及巴马汀均可降低脾细胞膜流动性，与对照组相比有显著性差异。 结论： 上述结果提示这些药物的免疫抑制作用有可能是通过降低淋巴细胞膜的流动性来实现的。     

宫颈癌与多种病原微生物感染、细胞因子及硒元素含量相关性研究

目的 探讨多种病毒及支原体性病原微生物感染、细胞因子和微量元素硒（Se)含量与宫颈癌发病的相关性。方法 PCR法检测宫颈癌组织中人乳头瘤病毒、疱疹病毒、人巨细胞病毒、EB病毒、沙眼衣原体和解脲支原体的感染（75份非宫颈癌患者正常宫颈组织作对照）；ELISA法检测血清中细胞因子IL－2R、TNF的含量；荧光光度技术检测血清中硒（Se) 元素含量。结果 在195份宫颈癌患者的癌组织标本中，病原微生物感染阳性者为166份（85．1％），其中16，18和35型人乳头瘤病毒感染60份（30．8％）；6和11型人乳头瘤病毒感染5份（2．6％）；2型单纯疱疹病毒感染60份（30．8％）；1型单纯疱疹病毒感染4份（2．1％）；EB病毒感染3份（1．5％）；人巨细胞病毒感染6份（3．1％）；沙眼衣原体感染11份（5．6％）和解脲支原体感染17份（8．7％）。对照组75份正常宫颈组织中病原微生物感染5份（6．7％）；EB病毒感染1份（1．3％）；沙眼衣原体感染3份（4．0％）和解脲支原体感染3份（4．0％）。62份宫颈癌和36份正常人血清中，白细胞介素－2受体（x^-=356.44U/ml)和肿瘤坏死因子（x^-=373.48pg/ml)浓度均比对照组明显升高（P＜0．001）。硒含量（x^-=0.058mg/ml)比对照组水平低（P＜0．05）。结论 山西宫颈癌的发病与多种病原微生物感染有关，尤其16，18和35型人乳头瘤病毒和2型单纯疱疹病毒感染率较高，同时伴有白细胞介素－2受体和肿瘤坏死因子水平的升高与硒元素的低下。     

红细胞膜脂肪酸组成对膜脂区流动性的影响

采用高效液相色谱法（ＨＰＬＣ）和荧光偏振技术同时测定了４２例正常成人红细胞膜脂肪酸成分与膜流动性，并进一步探讨了膜脂肪酸组成对膜流动性的影响。结果表明：（１）正常人红细胞膜脂肪酸主要由廿二碳六烯酸（Ｃ２２：６ｎ—３）、花生四烯酸（Ｃ２０：４ｎ—６）、亚油酸（Ｃ１８：２ｎ—６）、油酸（Ｃ１８：１ｎ—９）、软脂酸（Ｃ１６：０）和硬脂酸（Ｃ１８：０）等六种脂肪酸组成，其中花生四烯酸含量最高（２９％）。其次为软脂酸（１６．９％），而不饱和酸占７１．６％。（２）红细胞膜微粘度与软脂酸和硬脂酸呈明显正相关；与廿二碳六烯酸和花生四烯酸呈明显负相关。提示红细胞膜脂肪酸构成对膜流动性有重要影响。     

Effect of Gardenia extract ZG on the adsorption quantity of herpes simplex virus type 1 （HSV-1）

To observe the effect of Gardenia extract ZG on the adsorption quantity of herpes simplex virus type 1 (HSV-1) so as to explore the mechanism of its antiviral activity, fluorescein isothiocyanate (FITC) was used as the fluorescent probe to label viruses and heparin sodium was used as control. Meanwhile , the effect of Gardenia extract ZG on the adsorption quantity on the surface of Hep-2 cells was determined by flow cytometry. It was demonstrated that adsorption of HSV-1 on the surface of Hep-2 cells exhibited the character of saturation and specificity and heparin sodium could prevent attachment of viruses on these cells. These results are in accord with those reported previously. It was also proved that the manner of drug-use prior to adsorption or simultaneous use of drug and adsorption was better than adsorption prior to drug-use, and the inhibition rates of the former and latter manner were 84. 76% and 82.92% respectively. Three manners of drug-use with Gardenia extract ZG were all effective to reduce the adsorption quantity of viruses, especially the manner of simultaneous use of drug and adsorption with an adsorption inhibition rate of 68.46% . From the above observation, it is apparent that the mechanism of anti-viral activity of Gardenia extract ZG may be via several steps involved in the HSV-1 adsorption.     

Differences in the morphology of herpes simplex virus infected cells

The two types of herpes simplex virus (HSV-1, HSV-2) induced significantly different alterations in the morphology and permeability of infected cells. HEp-2 cells infected with HSV-1 (strain THEA) were characterized by the formation of polynuclear syncytia. In contrast, after infection with HSV-2 (strain D316, DD), the cells were rounded up. The HSV-1 strains KOS and LS5039 and the HSV-2 strain 196 induced both types of cytopathic effect. As shown by comparative scanning and transmission electron microscopy newly synthesized virus particles of the various strains of HSV-1 were generally found to be restricted to smooth areas of the cell surface. In these areas the number of microvilli was reduced in comparison to uninfected cells. However, the progeny viruses of the strains of HSV-2 were mainly connected with protrusions of the cell membrane (microvilli and filopodia). The morphological changes in cells infected with either type of HSV were associated with different functional alterations of the cell membrane. The membranes of HEp-w cells became more stable after infection with HSV-1. This is characterized by a reduced permeability for 51Cr as well as by a decreased sensitivity to the detergent Triton-X-100. HSV-2 induced opposite effects on the stability of the membrane in infected cells. In contrast to these findings with HEp-2 cells, opposite results were obtained with primary chick embryo fibroblasts: Infection with HSV-1 rendered the cell membrane more permeable for 51Cr and a reduction of the 51Cr-release was achieved by infection with HSV-2. The results show that HSV-cell interactions depend on the type of the virus as well as on the type of the infected cell.     

Analysis of herpes simplex virus entering into cells of oral origin

The entry of herpes simplex virus (HSV) into an oral epithelial cell line, primary normal human oral keratinocytes (NHOK) and gingival fibroblasts (GF) was examined. Infection of these cells by HSV-1 and HSV-2 was blocked by heparin. Further examination indicated that heparin reduced viral attachment but not penetration. Moreover, neomycin inhibited HSV-1 infection more effectively than HSV-2 infection in GF, but not in NHOK. In conclusion, our results elucidated some aspects of the HSV entry process into oral cells and revealed some differences in HSV entering into NHOK and GF.     

Early interactions of human herpesvirus 6 with lymphoid cells: role of membrane protein components and glycosaminoglycans in virus binding

A microassay was developed to detect human herpesvirus 6 (HHV-6) binding to its cellular receptor using flow cytometry. Comparable results were obtained either by using HHV-6 preparations conjugated with fluorescein isothiocyanate or by indirect immunofluorescent labeling of membrane-bound virus using as primary antibody a monoclonal antibody specific for the HHV-6 gp60/110 envelope glycoprotein. Virus attachment to the plasma membrane was specific and saturable. As expected, among cell lines of various origin, maximum binding was detected on human T-lymphoid cells (HSB-2). Papain digestion of HSB-2 cells prevented HHV-6 attachment and reduced significantly virus infection, indicating the involvement of a protein-based receptor in the attachment step. After removal of the protease, virus receptors were resynthesized and their regeneration was prevented partially by cycloheximide, an inhibitor of protein synthesis. Unexpectedly, only high concentrations (mg/ml) of soluble heparan sulfate and heparin inhibited HHV-6 binding and infection. Under the same conditions, few micrograms (per ml) of heparin suppressed completely herpes simplex type 1 (HSV-1) attachment to the same cell line. Treatment of HSB-2 cells with heparitinase and heparinase, at doses that reduced significantly HSV-1 attachment, had little effect on HHV-6 binding to the cell membrane, indicating a different requirement of heparan sulfate-containing glycosaminoglycans for the two herpesviruses. These data suggest that protein components of the cellular membrane play an essential role in HHV-6 binding and infection while heparan sulfate-glycos-aminoglycans appear to be involved only partially in virus-receptor interaction.     

栀子提取物T9对单纯疱疹病毒1型感染小鼠脑内VP16mRNA的影响

目的 观察不同时间栀子提取物T9对单纯疱疹病毒1型(HSV-1)感染后的BALB/c小鼠脑内间层蛋白16(VP16)的影响,探讨栀子提取物T9抗HSV-1的作用机制.方法 采用对HSV-1易感的BALB/c小鼠,以HSV-1 VP16为研究对象,借助RT-PCR方法检测栀子提取物T9对小鼠脑内HSV VP16的影响.结果 栀子提取物T9各给药组在同一时间内VP16 mRNA相对表达量均低于病毒对照组.结论 栀子提取物T9能够使HSV感染小鼠脑内VP16 mRNA相对表达量降低,可能是它抗HSV的作用机制之一.     

Passage of herpes simplex virus type 1 on chick embryo fibroblasts confers virulence for chick embryos

The pathogenesis of chorioallantoic membrane (CAM) infection with herpes simplex virus 1 and 2 (HSV-1 and HSV-2) as well as chick embryo fibroblast (CEF) passaged HSV-1 was studied. It was found that HSV-2 is at least a million-fold more virulent than HSV-1 as measured by pfu/LD50 ratios for the embryo. Serial passage of HSV-1 in vitro on CEF cells selected for a virus (CEFP10) which, unlike its parental HSV-1 strain, is able to kill the embryo. The restriction endonuclease maps of CEFP10 and its parental strain are indistinguishable and the mechanism of the increased virulence of CEFP10 was demonstrated to be its enhanced replication in chick embryo cells both in vivo and in vitro. In contrast, despite its inferior replicative ability, HSV-2 was found to have a biologically important specific invasiveness function that is not simply related to overall viral replication. Finally, the ability to isolate HSV-1 (CEFP10) virulent for the chick embryo after passage in vitro illustrates that tissue culture passage of HSV in appropriate cells may actually increase virulence for the animal host.     

A radioimmunoassay for herpes simplex virus (HSV) thymidine kinase

A sensitive RIA for HSV-1 thymidine kinase (TK) has been developed. This assay is based on competition for the binding site of a rabbit antibody against purified HSV-1 TK, between a purified ³H-labeled HSV-1 TK and a sample containing an unknown amount of viral TK. The assay is capable of detecting 8 ng or more of the HSV enzyme. Purified HSV-1 TK denatured to <1% of its original kinase activity is as effective in binding to the antibody as is native HSV-1 TK. Viral TK is detectable at ranges of 150–460 ng/mg protein of cell extract from infected cells or cells transformed by HSV or HSV genetic material. HSV-2 TK appears highly cross-reactive, VZV TK is slightly less so, and the vaccinia TK shows little or no cross-reactivity. This RIA may serve as a tool for monitoring the expression of the HSV TK during an active herpes virus infection, a latent ganglionic infection, or in neoplastic cells which may have arisen by viral transformation.     

Evaluation of Antiviral Activities of Four Local Malaysian Phyllanthus Species against Herpes Simplex Viruses and Possible Antiviral Target

Nucleoside analogues such as acyclovir are effective antiviral drugs against herpes simplex virus infections since its introduction. However, with the emergence of acyclovir-resistant HSV strains particularly in immunocompromised patients, there is a need to develop an alternative antiherpetic drug and plants could be the potential lead. In this study, the antiviral activity of the aqueous extract of four Phyllanthus species were evaluated against herpes simplex virus type-1 (HSV-1) and HSV-2 in Vero cells by quantitative PCR. The protein expressions of untreated and treated infected Vero cells were studied by 2D-gel electrophoresis and Western blot. This is the first study that reported the antiviral activity of P. watsonii. P. urinaria was shown to demonstrate the strongest antiviral activity against HSV-1 and HSV-2, with SI >33.6. Time-of-addition studies suggested that the extract may act against the early infection stage and the replication stage. Protein expression studies indicated that cellular proteins that are involved in maintaining cytoskeletal structure could be potential target for development of antiviral drugs. Preliminary findings indicated that P. urinaria demonstrated potent inhibitory activity against HSV. Hence, further studies such as in vivo evaluation are required for the development of effective antiherpetic drug.     

Use of monoclonal antibodies for analysis of antibody-dependent immunity to ocular herpes simplex virus type 1 infection.

Monoclonal antibodies specific for the five major glycoproteins of herpes simplex virus type 1 (HSV-1) were tested for their capacity to mediate immunity to ocular HSV-1 infection. The specificity of the immunoglobulin made by each monoclone was determined by immunoprecipitation of [14C]glucosamine-labeled polypeptides from detergent-solubilized HSV-1-infected cells. Of the five monoclonal antibodies studied, two immunoprecipitated glycoproteins gA/B, one immunoprecipitated glycoprotein gC, one immunoprecipitated glycoprotein gD, and one immunoprecipitated glycoprotein gE. All five were effective in passively transferring immunity to mice when they were given 4 to 24 h after HSV-1 infection on an abraded cornea. Four of the monoclonal antibodies were also evaluated for their capacity to neutralize HSV-1 and to promote complement-mediated cell lysis and antibody-dependent cellular cytotoxicity. It was found that none of these in vitro assays correlated with the protective activity of the antibodies in vivo. In fact, one of the monoclonal antibodies was unreactive in all three immunological reactions, even though it was highly effective in promoting recovery from HSV-1 induced ocular disease in vivo. The results suggest that antibodies can interact in vivo with virus-specific glycoproteins gA/B, gC, gD, and gE to initiate recovery from HSV-1-induced ocular disease, and that the therapeutic effectiveness of a specific monoclonal antibody does not correlate with its immunological reactivity in vitro.