灭活SARS病毒的免疫原性的实验研究

研究灭活SARS病毒的免疫原性。SARS病毒F6 9株经甲醛灭活后 ,加入氢氧化铝佐剂 ,以 3种剂量接种BALB/c小鼠 ,定时采血 ,测定特异性抗体的滴度及其中和活性 ,同时用化学发光酶联免疫法测定抗血清与SARS病毒结构蛋白的反应特异性及其相对强度。结果发现 ,小鼠接种疫苗 4d后 ,血清中可检测到IgM抗体 ,直至 2 6d后逐渐下降 ;IgG抗体在初次免疫 8d后出现 ,4 7d时达最高峰 ,6 3d后进入稳定期 ;不同剂量组的抗体滴度具有明显剂效关系 ,低剂量组和中剂量组滴度峰值为 1∶192 0 0 ,高剂量组滴度峰值为 1∶384 0 0。中和实验结果表明 ,小鼠所产生的抗体具有中和病毒活性 ,在 6 3d时 ,低剂量组和中剂量组血清的中和效价为 1∶12 80 ,高剂量组血清的中和效价为 1∶5 12 0。抗体分类结果表明 ,小鼠抗血清中含有针对多种SARS病毒结构蛋白的特异性抗体 ,其中 ,针对N蛋白、S4蛋白和S2蛋白的抗体水平相对较高 ,而抗M抗体、抗 3CL抗体的水平相对较低。上述结果说明 ,SARS病毒F6 9株经甲醛灭活后 ,各主要结构蛋白仍保持较强免疫原性 ;免疫小鼠后 ,可以诱导产生高滴度的特异性混合抗体 ,在体外可以保护敏感细胞不受SARS病毒攻击     

桑寄生水煎液对小鼠胚胎肢芽细胞生长发育的影响

目的探讨桑寄生水提液对小鼠胚胎肢芽细胞生长发育的影响。方法取小鼠E13后肢芽制成细胞悬液,接种到细胞培养板中,分别加入0.2,0.1,0.05 g/ml桑寄生水提液和蒸馏水,设置为高,中,低,和空白对照组。培养72h后采用MTT法、甲苯胺蓝染色法、流式细胞术分别检测肢芽细胞的增殖、分化和凋亡情况。结果 (1)高剂量桑寄生水煎液抑制肢芽细胞的增殖,与空白对照组比较,差异具有显著性(P0.05);而低和中剂量促进肢芽细胞的增殖,其中低剂量与空白对照组比较差异具有显著性(P0.05)。(2)各剂量组桑寄生水煎液均诱导了肢芽细胞的分化,中和低剂量组与空白对照组比较均有显著性差异(P0.05)。(3)各剂量组均诱导了肢芽细胞坏死,高剂量组与空白对照组比较具有显著性差异(P0.05);高和低剂量诱导了肢芽细胞的凋亡,其中高剂量与空白对照组比较差异具有显著性(P0.05)。结论桑寄生水煎液高剂量抑制肢芽细胞的增殖,诱导细胞凋亡和坏死;低和中剂量促进肢芽细胞的增殖和分化。     

银杏叶提取物对AD模型小鼠海马谷氨酸转运体GLAST表达的影响

目的探讨银杏叶提取物对AD模型小鼠海马谷氨酸转运体GLAST表达的影响。方法采用双侧海马CA1区注射Aβ_(1-42)法测定正常对照组、AD模型组、AD+银杏叶提取物低剂量组(30 mg·kg~(-1)·day~(-1))、中剂量组(60 mg·kg~(-1)·d~(-1))、高剂量组(120 mg·kg~(-1)·d~(-1))动物海马中GLAST的变化。结果经银杏叶提取物处理后,模型组动物存活率增加;小鼠海马组织中Aβ_(1-42)阳性沉积明显减少,且高剂量组减少最为明显;AD模型组GLAST阳性表达明显减少(P0.05);银杏叶提取物处理组小鼠海马区GLAST阳性表达出现增加,银杏叶提取物中剂量组GLAST阳性表达增加最为显著(P0.05)。结论银杏叶提取物具有缓解Aβ诱导神经毒性的作用,对AD模型小鼠海马中谷氨酸转运体GLAST具有调节和保护作用。     

Ubc13 对胶原诱导性关节炎小鼠体内Treg / Th17 平衡的影响研究

目的:研究重组泛素结合酶2N(Ubc13)蛋白对胶原诱导性关节炎(CIA)模型小鼠体内调节性T细胞(Treg和辅助性T细胞17(Th17)平衡的影响。方法:小鼠随机分为空白对照组(空白组),CIA模型对照组(模型组),CIA+5μg Ubc13组(低剂量组),CIA+25μg Ubc13组(中剂量组),CIA+50μg Ubc13组(高剂量组)。模型组与Ubc13各剂量组小鼠第0天尾根部皮下注射胶原,第21天加强免疫,建立CIA模型。第21天开始Ubc13各剂量组分别每天皮下注射Ubc13重组蛋白,模型组注射等体积生理盐水。二次免疫56 d后,剖解小鼠,取小鼠脾淋巴细胞,流式细胞术检测Treg细胞和Th17细胞数目,实时定量PCR(qRT-PCR)检测小鼠脾脏维甲酸相关受体c(RORc)、白介素23(IL-23)、叉头转录因子p3(Foxp3)和白介素17(IL-17) mRNA水平,HE染色鉴定小鼠关节病理特征。结果:Ubc13能显著提高小鼠Treg细胞在Th细胞中的比率(P0. 05),并改善关节炎症。与模型组相比,Ubc13各剂量组脾脏中IL-23、IL-17和RORγt的表达量显著降低(P0. 05),HE染色显示Ubc13各组小鼠关节损伤均显著减轻。结论:皮下注射Ubc13可通过增加CIA模型小鼠体内Treg的数量,降低Th17细胞相关基因的表达,改善CIA小鼠关节炎。     

中国株HIV-1 gp120核酸疫苗的实验免疫研究

目的　探讨表达中国株HIV 1gp12 0基因的核酸疫苗在小鼠体内的免疫反应。方法　将表达HIV 1gp12 0的核酸疫苗质粒pVAXP经肌肉注射免疫Balb c小鼠 ,检测免疫小鼠脾CD4 +、CD8+T细胞亚群的数量 ,脾特异性CTL杀伤活性和血清抗体滴度。结果　重组质粒pVAXP免疫组小鼠脾CD4 +、CD8+T细胞亚群的数值均比对照组高 (P <0 .0 5 ) ;免疫组脾特异性CTL杀伤活性与对照组相比差异极显著 (P <0 .0 1) ;血清抗体滴度显著高于对照组 (P <0 .0 5 )。结论　表达HIV 1gp12 0基因的核酸疫苗质粒pVAXP能诱导小鼠产生特异性细胞和体液免疫。     

重组人α2b干扰素胶囊抗小鼠阴道HSV-2感染的研究

目的 研究新剂型重组人干扰素(interferon,IFN)α2b胶囊抗小鼠疱疹病毒性阴道炎的效果.方法 人工建造阴道感染HSV-2的小鼠模型,以不同剂量的IFNα2b对小鼠阴道局部用药,以阿昔洛韦(acyclovir,ACV)为阳性对照.观察药物干扰对模型小鼠发病率、死亡率及病程进展的影响.结果 试验药物能减少小鼠的病死数、发病数,减慢小鼠的病程进展,其中16000IU/天高剂量组效果最好,类似于ACV组的用药效果,4000IU/天的低剂量用药组效果最弱,8000IU/天中剂量组效果居中.结论 受试药物在小鼠体内具有良好的抗HSV-2感染所致阴道炎的作用.     

细胞因子变化在疱疹病毒性脑炎中机制及作用

目的定量检测细胞因子IL2、IL10、TNFα在单纯疱疹病毒性脑炎(HSE)中的表达和变化,以探讨细胞因子变化在HSE中的机制及作用。方法实验动物分正常对照组、病毒感染组和无环鸟苷(ACV)治疗组3组。使用100LD50HSV1病毒悬液20μl接种入3周龄Balb/c小鼠颅内制造HSE模型,ACV治疗组在接     

水母雪莲乙醇提取物对Lewis肺癌荷瘤小鼠抗肿瘤免疫的影响

目的：初步探讨藏药水母雪莲(SMM)的体内外抗肿瘤免疫功能。方法：C57BL/6野生小鼠接种3LL细胞建立Lewis肺癌荷瘤小鼠模型,随机分为生理盐水组、SMM1低剂量组、SMM2中剂量组、SMM3高剂量组和环磷酰胺(CTX)组,体内连续治疗21 d。检测Lewis肺癌荷瘤小鼠实体瘤体积、质量和体质量;计算胸腺、脾脏指数;流式细胞仪检测肿瘤浸润组织淋巴细胞(TIL)的CD4⁺T、CD8⁺T百分比和CD4⁺T/CD8⁺T比例。体内MTT法检测80、100μg/ml SMM乙醇提取物对Lewis肺癌荷瘤小鼠脾CTL杀伤活性的影响;El ISA法检测CTL分泌IFN-γ的水平。结果：与生理盐水组比较,SMM乙醇提取物可显著抑制肿瘤生长并维持荷瘤小鼠体质量增长;胸腺和脾脏指数均显著升高(P<0.05);TIL中CD4⁺T和CD4⁺T/CD8⁺T水平显著升高(P<0.05),SMM1和SMM3组CD8⁺T显著降...     

茯苓多糖抑制甲醛诱导小鼠血清 DNA 加合物的实验研究

目的:观察茯苓多糖灌胃对甲醛诱导小鼠体内DNA加合物含量的影响,探讨茯苓多糖的抗遗传毒性效应。方法:40只成年昆明种雄性小鼠随机分成阳性对照组(甲醛染毒)、茯苓多糖低剂量组、中剂量组和高剂量组,每组10只。阳性对照组灌注0. 4 ml浓度为0. 5 g/L的甲醛溶液,茯苓多糖组小鼠除了灌胃0. 2 ml浓度为1. 0 g/L的甲醛溶液外,还灌胃0. 2 ml不同浓度的茯苓多糖溶液(低剂量组2. 0 g/L,中剂量组4. 0 g/L和高剂量组8. 0 g/L)。1周后摘眼球取血,用ELISA法测定各组小鼠血清DNA加合物含量。结果:4组间DNA加合物浓度差异有统计学意义(P0. 01),茯苓多糖中剂量组和高剂量组DNA加合物浓度均低于阳性对照组(P0. 01)。茯苓多糖剂量越高,DNA加合物浓度越低,呈现明显的剂量-效应关系(rs=-0. 869,P0. 01)。结论:茯苓多糖小鼠灌胃可抑制DNA加合物的形成,且存在剂量-效应关系,作用随茯苓多糖剂量的提高而增强。     

携带EBV-LMP2基因的重组MVA痘苗病毒初步免疫效果

目的 观察携带EBV lmp2基因的重组痘苗病毒疫苗在小鼠体内诱导的LMP2特异性细胞免疫应答效果.方法 使用表达EBV lmp2基因的重组痘苗病毒MVA-Lac-LMP2,采用低（2＆#215;105 pfu/只）、中（2 ＆#215; 106pfu/只）、高（2 ＆#215; 107pfu/只）三个剂量组,分别于0、2周免疫BALB/c小鼠,末次免疫1周后应用IFN-γ ELISPOT方法检测小鼠脾淋巴细胞中EBV LMP2特异性CTL应答水平.结果 MVA-Lac-LMP2能诱导小鼠产生EBV LMP2特异性的CTL应答,其中,中、高剂量组小鼠CTL水平明显高于低剂量组（P≤0.01）,中、高剂量组小鼠诱导的CTL应答水平差异没有统计学意义.结论 MVA-Lac-LMP2能够在小鼠体内诱导EBV-LMP2特异性的细胞免疫,应答水平的高低与免疫剂量有关.     

Combined treatment of therapeutic laser and herbal application improves the strength of repairing ligament

The present study investigated the effects of combined therapeutic laser and herbal medication protocols on injured medial collateral ligaments (MCLs) of rat knees. Fully 36 rats were evenly divided into 9 groups. Right MCLs of groups 1 to 6 and 8 were transected, while that of groups 7 and 9 remained intact. After surgery, group 1 was treated with 1 session of high-dosed laser; group 2 with 9 sessions of low-dosed laser; group 3 with an herbal plaster; groups 4 and 5 received combined treatments of groups 1 and ss and 2, and 3 respectively; groups 6 and 7 received only bandaging; groups 8 and 9 received placebo laser and no treatment, respectively. All MCLs were subjected to biomechanical testing at 3 weeks postsurgery. Results revealed significant differences among groups in ultimate tensile strength (UTS) and stiffness (p < 0.01). Combination of multiple low-dosed laser treatment with herbal treatment (group 5) resulted in higher UTS than either no treatment (groups 6 and 8), single high-dosed laser treatment (group 1), multiple low-dosed laser treatment (group 2), or herbal treatment (group 2) alone. We concluded that combined applications of laser and herb can enhance further biomechanical properties of repairing rat MCLs than separate applications at 3 weeks postinjury.     

Combined Treatment of Therapeutic Laser and Herbal Application Improves the Strength of Repairing Ligament

The present study investigated the effects of combined therapeutic laser and herbal medication protocols on injured medial collateral ligaments (MCLs) of rat knees. Fully 36 rats were evenly divided into 9 groups. Right MCLs of groups 1 to 6 and 8 were transected, while that of groups 7 and 9 remained intact. After surgery, group 1 was treated with 1 session of high-dosed laser; group 2 with 9 sessions of low-dosed laser; group 3 with an herbal plaster; groups 4 and 5 received combined treatments of groups 1 and ß and 2, and 3 respectively; groups 6 and 7 received only bandaging; groups 8 and 9 received placebo laser and no treatment, respectively. All MCLs were subjected to biomechanical testing at 3 weeks postsurgery. Results revealed significant differences among groups in ultimate tensile strength (UTS) and stiffness (p < 0.01). Combination of multiple low-dosed laser treatment with herbal treatment (group 5) resulted in higher UTS than either no treatment (groups 6 and 8), single high-dosed laser treatment (group 1), multiple low-dosed laser treatment (group 2), or herbal treatment (group 2) alone. We concluded that combined applications of laser and herb can enhance further biomechanical properties of repairing rat MCLs than separate applications at 3 weeks postinjury.     

The role of antigen presentation in the ontogeny of immune responses to herpes simplex virus.

The ability of epidermal antigen presenting cells (APC) to induce immune responses to herpes simplex virus (HSV) has been studied in mice of differing ages. Using an in vivo model of HSV infection we have demonstrated that neonatal epidermal cells (EC) induce specific suppression of DH to HSV in normal syngeneic adult mice. The suppression is transferable and mediated by T cells of the Lyt1+, Lyt2-, L3T4+ phenotype. The ability of EC to induce suppression persists up to 4 weeks of age, whereas EC from mice 6 weeks of age or older induce positive DH responses to the virus. This correlates with the susceptibility of mice of the different ages to HSV infection and their ability to mount DH responses to the virus.     

Treatment of HSV-1 infection with immunoglobulin or acyclovir: comparison of their effects on viral spread, latency, and reactivation.

We compared immunoglobulin (IgG) and acyclovir (ACV) therapies on the establishment, maintenance, and reactivation from latency of HSV-1(McKrae) in a mouse ocular infection model. Mice were given one intraperitoneal (IP) dose of human IgG 24 h after infection (Day 1 p. i.) or ACV in the drinking water from Days 1 to 7 p.i. Both treatments allowed similar percentages of mice to survive the infection and decreased ocular virus shedding as compared with untreated controls. At most time points, there were no differences between IgG- and ACV-treated animals with respect to tissue virus titers or in the rates of virus reactivation during explant cocultivation. However, after ultraviolet exposure, HSV reactivated in 30% of ACV-treated mice compared with 90% of IgG-treated mice (P = 0.02). Also by quantitative PCR, we found more latent HSV-1 DNA copies in IgG-treated mice compared with those given ACV (P = 0.02). IgG treatment protects mice from HSV-1 infection essentially as well as ACV does. Nonetheless, it permits higher levels of latent infection and subsequent in vivo reactivation. These studies have implications for the mechanism by which IgG functions to attenuate HSV infections and for its potential value as a therapeutic agent in humans.     

Tolerance and suppression of immunity to herpes simplex virus: different presentations of antigens induce different types of suppressor cells.

In this report, we examine tolerance (hyporesponsiveness) and suppression of delayed hypersensitivity (DH) to herpes simplex virus (HSV) in mice, using two different forms of tolerogen: HSV particles and HSV-infected spleen cells. The intravenous injection of mice with either HSV particles or spleen cells 7 days before subcutaneous immunization with virus induced a profound state of unresponsiveness. This unresponsive state was mediated, at least in part, by suppressor T cells (Ts), which were demonstrated by passive transfer to naive recipients. However, different types of Ts were induced depending on the form of the tolerogen. The injection of HSV particles induced Ts which suppressed the induction but not the expression of DH. On the other hand, the injection of HSV spleen cells induced two types of Ts: one which inhibited the induction of the DH response and one which inhibited the expression of DH to HSV. Both tolerance and Ts are virus specific (i.e., the DH response to an unrelated virus was not inhibited) but not type specific for HSV type 1 and HSV type 2. Since both virus particles and virus-infected cells may be present in the blood during HSV infection, the induction of this type of immune regulation may influence the outcome of both acute and latent HSV infections.     

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.     

Survey of acyclovir-resistant herpes simplex virus in the Netherlands: prevalence and characterization.

BACKGROUND: Widespread and frequent use of acyclovir (ACV) for treatment, suppressive therapy and prophylaxis of herpes simplex virus (HSV) infections and its over the counter availability may be associated with emergence of HSV resistance. OBJECTIVES: To determine the prevalence of ACV-resistant HSV isolates in different patient groups between 1999 and 2002 in the Netherlands. STUDY DESIGN: A total of 542 isolates, 410 HSV-1 and 132 HSV-2, from 496 patients were screened for reduced susceptibility to ACV. A newly developed ELVIRA HSV screening assay was used that allowed a high throughput screening. The genotypic analysis of the HSV thymidine kinase gene was performed to identify resistance-associated mutations. RESULTS: Thirteen isolates, 8 HSV-1 and 5 HSV-2, from 10 patients (2%) were found resistant to ACV. A single ACV-resistant strain was identified among isolates from 368 immunocompetent patients (0.27%; 95% confidence interval [CI], 0.007%-1.5%), whereas in nine isolates from 128 immunocompromised patients resistant HSV was identified (7%; 95% CI, 3.26%-12.93%). The highest frequency of ACV-resistant HSV was associated with bone marrow transplantation: four patients out of 28 (14.3%) shed resistant virus. In addition, resistant virus was obtained from two HIV-positive patients, one patient with a hematological malignancy and two patients on immunosuppressive drugs. Further testing showed that none of the isolates was resistant to foscarnet. Several new mutations were identified in the thymidine kinase gene of these resistant isolates, and their effect on ACV-resistance is discussed. CONCLUSIONS: Our study shows that the prevalence of ACV resistance is low in immunocompetent patients (0.27%), whereas ACV-resistant HSV infections occur relatively frequently in immunocompromised patients (7%; P < 0.0001). This emphasizes the need for drug susceptibility monitoring of HSV infections in immunocompromised patients with persisting infections despite antiviral therapy.     

HSV excretion after bone marrow transplantation: a 4-year survey

BACKGROUND: Herpes simplex virus (HSV) oral excretions are common after bone marrow transplantation (BMT). OBJECTIVE: We report a 4-year systematic survey of HSV excretions in an adult population who underwent BMT (289 transplantations). STUDY DESIGN: Patients received either intravenous ACV treatment when mucositis occurred or systematic intravenous ACV prophylaxis from initiation of the BMT conditioning until the end of aplasia. All patients were followed up for HSV excretions. RESULTS: Twenty-eight patients (9.7%) excreted HSV. The occurrence of HSV excretions was similar in both allogeneic and autologous transplant patients. The incidence was significantly lower when ACV was systematically used after transplantation (2.5%) compared to when ACV was implemented for mucositis (12%). ACV-resistant HSV was detected in three patients who received allogeneic transplantation, representing 27% of allogeneic recipients excreting HSV. CONCLUSION: HSV infection prophylaxis with high dose of intravenous ACV resulted in a decreased incidence of HSV excretion. Nevertheless, the risk of emergence of ACV resistance, especially among allogeneic transplant patients, appears to be identical whatever the route and dose of ACV prophylaxis.     

Frequency of acyclovir-resistant herpes simplex virus in clinical specimens and laboratory isolates

The proportion of acyclovir (ACV)-resistant herpes simplex virus (HSV) isolates in clinical specimens and laboratory isolates was determined. HSV isolates in clinical specimens and laboratory isolates were cultured in the absence or presence of 5 microg of ACV per ml. The frequency of ACV-resistant HSV was calculated as (average virus titer in the wells with ACV)/(average virus titer in the wells without ACV). The mutation frequency of HSV type 1 isolates in clinical samples (directly from patient lesions) was 7.5 x 10(-4) +/- 2.5 x 10(-4) (mean +/- standard error), and that of HSV type 2 isolates was 15.0 x 10(-4) +/- 4.6 x 10(-4). The mutation frequencies of isolates derived in the laboratory from these clinical samples were very similar. Similarly, the 50% inhibitory concentrations for isolates in clinical samples and laboratory isolates were identical. The frequencies of ACV-resistant HSV types 1 and 2 were in a narrow range of 7.5 x 10(-4) to 15.0 x 10(-4) among isolates in clinical specimens and did not change for laboratory-derived pools of viral isolates.     

Simplified test for detecting the resistance of herpes simplex virus to acyclovir

The detection of herpes simplex virus (HSV) antigen by means of an enzyme amplified ELISA was investigated for rapid screening of acyclovir (ACV) resistance. Vero cell monolayers were inoculated in the presence of different concentrations of ACV. When cytopathic effect was present, the culture supernatants were tested by ELISA. The absorbance values were found to correlate with the results of virus yield and plaque reduction assays. The comparison between absorbance values obtained in the presence of 10 microM ACV and in the absence of drug provided the basis for a simplified sensitivity test. The use of a single ACV concentration allowed discrimination between ACV-resistant and ACV-sensitive reference strains, the detection of ACV-resistant virus mixed in the proportion of 10% with ACV-sensitive virus, and a study of the emergence of an ACV-resistant virus population in serial samples taken from experimental rabbit keratitis. The simplified susceptibility assay is a sensitive and convenient method for rapid screening of HSV resistance to ACV.