Esat6-卡介苗重组疫苗的构建免疫原性及抗结核作用的初步研究

目的构建结核分枝杆菌esat6-卡介苗重组疫苗,研究其免疫原性及抗结核作用,以期获得结核病预防性或治疗性疫苗。方法通过基因工程重组技术将结核分枝杆菌保护性抗原esat6的编码基因与穿梭质粒载体pYUB295重组,采用电穿孔技术导入卡介苗中,应用聚合酶链反应(PCR)扩增、聚丙烯酰胺凝胺电泳(PAGE)鉴定重组卡介苗。通过酶联免疫吸附试验(ELISA)法检测其血清中特异性抗体,用MTS法分析其脾淋巴细胞增殖指数。通过观察esat6-卡介苗重组疫苗对结核分枝杆菌感染的预防试验中实验动物半数死亡时间、一定时间内的死亡率、大体病变、T细胞及B细胞免疫功能等指标评价esat6-卡介苗重组疫苗对结核分枝杆菌感染的预防效果。结果PCR扩增、限制性内切酶酶切、DNA测序鉴定、PAGE电泳表明成功地构建了esat6基因pYUB295重组质粒,ESAT6蛋白在卡介苗中分泌表达。重组卡介苗免疫原性试验表明:esat6-卡介苗重组疫苗组有ESAT6特异性抗体产生,45d时达到最高水平。脾淋巴细胞增殖试验表明各组刺激指数均达2.0以上,esat6重组卡介苗免疫组小鼠脾淋巴细胞的刺激指数稍高于对照组。预防试验表明:esat6-卡介苗重组疫苗组、卡介苗组与生理盐水对照组比都能延长结核分枝杆菌感染小鼠的半数死亡时间,降低2个月内的死亡率。其中esat6-卡介苗重组疫苗组效果显著,与卡介苗组比有显著差异。结核分支杆菌攻击后2个月,处死小鼠时,小鼠脏器大体病变及脏器培养结果表明:esat6-卡介苗重组疫苗免疫组小鼠比其他各组小鼠结核菌的负荷轻。抗体检测结果及淋巴细胞增殖实验各组结果差异无统计学意义。结论正确构建了esat6-卡介苗重组疫苗,esat6-卡介苗重组疫苗能提高卡介苗对结核分枝杆菌感染的预防作用。     

治疗性重组质粒对结核分枝杆菌抑菌作用的分析

应用动物试验模型主要脏器的结核分枝杆菌生长情况,观察结核分枝杆菌MPT64和ESAT6 DNA疫苗预防结核病的效力.试验将BALB/C小鼠随机分五组,其中A组生理盐水、B组载体质粒、C组卡介苗、D组重组质粒MPT64 DNA、E组:ESAT6 DNA.免疫9周后,用H37RV结核分枝杆菌强毒株攻击小鼠,分别在攻击一个月后和二个月后观察结果.     

IFN-r增加小鼠抗结核分支杆菌感染的抵抗力研究

目的 观察IFN-Γ增加小鼠抗结核分支杆菌感染的作用。方法以BALB/C小鼠接种结核分支杆菌为实验动物模型,用IFN-Γ治疗感染小鼠,予14d,28d时,采血测定小鼠IFN-Γ,IL-12,IL-18,IL-4值水平;同时计算小鼠肺、肝、脾菌落数及观察小鼠生存期。结果 治疗组小鼠脏器菌落数低于模型组(P<0.05),同时生存期延长;第14d第28d时IL-12,IL-18,IFN-Γ水平均高于模型组(P<0.05,P<0.01),IL-4水平无差异性。结论IFN-Γ有抗结核分支杆菌的作用。     

结核病病毒载体疫苗研究进展

结核病(TB)由结核分枝杆菌感染引起,其发病率和死亡率在传染性疾病中均居于前列,而疫苗是有效控制TB的重要因素。鉴于卡介苗(BCG)对成年人TB预防效果有限,新型TB疫苗(尤其是病毒载体疫苗)意义重大。该文拟对TB病毒载体疫苗的类型、效力及其研究进展做一综述。     

干扰素-γ对小鼠结核病的实验研究

目的：研究干扰素γ（IFN—γ）对结核分支杆菌感染小鼠的疗效与机制。方法：将BALB／c小鼠制成结核分枝杆菌感染模型，随机分为4组，分别以IFN—γ组、左氧氟沙星组（LVFX）、联合组及磷酸盐缓冲液组（PBs，对照组）治疗。检测重要脏器菌落数，观察小鼠肺组织病理改变和平均生存时间。结果：各治疗组肝、脾、肺组织菌落数均显著低于对照组，肺部损伤亦较轻，尤其联合组效果更为明显。各治疗组小鼠平均生存期均显著高于对照组（P〈0．01）。结论：外源性IFN-γ可减少肝、脾、肺组织中结核分枝杆菌菌落数，减轻肺部病变，延长动物生存时间，促进Th，细胞的免疫应答，在抗结核分枝杆菌感染中起着重要作用。     

治疗用皮卡乙型肝炎疫苗（CHO细胞）及其组分的小鼠急性毒性实验研究

目的比较治疗用皮卡乙型肝炎疫苗（CliO细胞）及疫苗组分（重组乙型肝炎疫苗（CliO细胞）、皮卡佐剂）对小鼠的急性毒性反应。方法ICR小鼠按体重随机分为溶媒对照组,皮卡佐剂高剂量组（原液）和低剂量组（2倍稀释液）,重组乙型肝炎疫苗（CHO细胞）组（原液）,治疗用皮卡乙型肝炎疫苗（CliO细胞）高剂量组（原液）和低剂量组（2倍稀释液）,每组小鼠后肢im0．2mL受试物,给药后连续14天观察其状态及毒性反应。结果各组小鼠全部存活,摄食和体重指标正常,注射部位肌肉、外观体征、行为活动、呼吸、排泄、各组织脏器肉眼观察及组织病理学检查均未见异常。结论在本实验条件下,治疗用皮卡乙型肝炎疫苗（CliO细胞）及疫苗组份对小鼠无明显毒性反应。     

非结核分支杆菌16例药敏特点分析

目的:分析我院2008-2010年非结核分支杆菌(NTM)的耐药情况.方法:对2008年3月～2010年3月无锡市传染病医院结核病实验室所做结核分支杆菌药敏试验的279例菌株用TCH和PNB进行菌型初步鉴定,采用绝对浓度法对12种抗结核药物:异烟肼、利福平、链霉素、乙胺丁醇、吡嗪酰胺、奥氟星、对氨基水杨酸、卷曲霉素、力克肺疾、利福喷丁、丁胺卡那和丙硫异烟胺进行药敏试验,并对分离出来的16株NTM的药敏结果进行分析.结果:279例菌株中NTM分离率为5.73%,有3例全耐药;16株NTM对吡嗪酰胺呈现全耐药;除对丁胺卡那的低浓度与高浓度耐药率差异有统计学意义外,对其他10种药物低、高浓度的耐药率差异无统计学意义.说明很多非结核分支杆菌对抗结核药物呈天然的抗药性.结论:我市非结核分支杆菌处于较低的流行水平,针对非结核分支杆菌的高耐药性.需要制定新的、更有效的治疗手段,对NTM感染和NTM病应予高度重视.     

重组人白介素-2吸入给药对小鼠克雷伯肺炎感染的影响

目的研究重组人白介素-2吸入给药对小鼠克雷伯肺炎感染的保护作用。方法小鼠以滴鼻感染法进行感染。感染过程中,以鼻腔插管注入法和皮下注射给予小鼠重组人白介素-2。同时另设正常组、阴性对照组、模型组和阳性对照组(庆大霉素组)。观察各组动物肺病理变化,比较平均死亡时间和一周内死亡率,测定支气管肺泡洗涤液中总蛋白、白蛋白含量,碱性磷酸酶、乳酸脱氢酶活力。结果重组人白介素-2给药组肺病理变化与模型组和阴性对照组相比显著减轻,支气管肺泡洗涤液中总蛋白、白蛋白含量和碱性磷酸酶活力降低。结论吸入重组人白介素-2缓解感染后肺部病理变化, 为研发重组人白介素-2吸入剂提供了依据。     

结核感染与结核菌素皮试结果的相关性研究

目的:研究结核菌素(Purified Protein Derivation,PPD)皮试在诊断潜伏结核感染和结核病中的意义.方法:2008年1月～2009年12月怀疑结核感染的2 714例小儿PPD皮试,结果登记并随访分析,PPD阳性率在不同年龄组间、不同性别、有无卡介苗接种的卡疤组间进行χ2检验.结果:28例皮试硬结反应出现在4～7 d;潜伏结核的感染率为22.2%(603/2714),结核病为5.8%(158/2714),卡介苗病为0.3%(8/2714);PPD +～+++的阳性率在各年龄组间、有无卡疤组间比较差异有统计学意义,在性别间比较差异无统计学意义.结论:因PPD皮试安全且不良反应小推荐为常规的结核病筛查,结果观察时间建议为3～5 d;皮试的颜色与肿胀边界在判断结核感染上更有意义.     

盐酸米托蒽醌与卡介苗膀胱灌注预防肿瘤复发的疗效

目的对比盐酸米托蒽醌与卡介苗膀胱灌注预防膀胱癌术后复发的效果。方法将100例膀胱癌经尿道电切除术后患者随机分成2组,一组患者膀胱灌注盐酸米托蒽醌,另一组膀胱灌注卡介苗,各组均灌注3～42个月,观察两组复发情况及不良反应。结果盐酸米托蒽醌组复发率为12%,不良反应率为8%,卡介苗组复发率为20%,不良反应率为64%;两组肿瘤复发率差异无统计学意义(P>0.05),但卡介苗不良反应发生率高于盐酸米托蒽醌(P<0.01)。结论盐酸米托蒽醌及卡介苗均能有效预防膀胱癌术后复发,但盐酸米托蒽醌膀胱灌注操作简单,不良反应小,是一种安全有效的膀胱局部化疗药物。     

Biotechnology in the development of new vaccines and diagnostic reagents against tuberculosis

Tuberculosis (TB) is a disease of global concern. About one third of the world population is infected with Mycobacterium tuberculosis. Every year, approximately 8 million people get the disease and 2 million die of TB. The currently available vaccine against TB is the attenuated strain of Mycobacterium bovis, Bacillus Calmette Guerin (BCG), which has failed to provide consistent protection in different parts of the world. The commonly used diagnostic reagent for TB is the purified protein derivative (PPD) of M. tuberculosis, which is nonspecific because of the presence of antigens crossreactive with BCG and environmental mycobacteria. Thus there is a need to identify M. tuberculosis antigens as candidates for new protective vaccines and specific diagnostic reagents against TB. By using the techniques of recombinant DNA, synthetic peptides, antigen-specific antibodies and T cells etc., several major antigens of M. tuberculosis have been identified, e.g. heat shock protein (hsp)60, hsp70, Ag85, ESAT-6 and CFP10 etc. These antigens have shown promise as new candidate vaccines and/or diagnostic reagents against TB. In addition, recent comparisons of the genome sequence of M. tuberculosis with BCG and other mycobacteria have unraveled M. tuberculosis specific regions and genes. Expression and immunological evaluation of these regions and genes can potentially identify most of the antigens of M. tuberculosis important for developing new vaccines and specific diagnostic reagents against TB. Moreover, advances in identification of proper adjuvant and delivery systems can potentially overcome the problem of poor immunogenicity/short-lived immunity associated with protein and peptide based vaccines. In conclusion, the advances in biotechnology are contributing significantly in the process of developing new protective vaccines and diagnostic reagents against TB.     

Nanoparticle-based adjuvant for enhanced protective efficacy of DNA vaccine Ag85A-ESAT-6-IL-21 against Mycobacterium tuberculosis infection

The goal of this study was to evaluate the protective efficacy of a cationic nanoparticle-based DNA vaccine expressing antigen 85A (Ag85A) and 6-kDa early secretory antigen target (ESAT-6) of Mycobacterium tuberculosis as well as cytokine interleukin-21 (IL-21) against M. tuberculosis infection. The results of this indicated that the anti–M. tuberculosis immune responses were induced in mice that had received the different DNA vaccines. More importantly, compared with using DNA vaccine Ag85A-ESAT-6-IL-21 alone, the nanoparticle-based DNA vaccine Ag85A-ESAT-6-IL-21 showed a statistically significant increase in the protective efficacy against M. tuberculosis infection in the immunized mice. We concluded that the nanoparticle-based DNA vaccine induced a strong immune response and markedly inhibited the growth of the M. tuberculosis in the mice. These findings highlighted the potential utility of Fe₃O₄-Glu-polyethyleneimine nanoparticles encapsulated with the DNA vaccine as a prophylactic vaccine in the M. tuberculosis–infected mouse model.From the Clinical EditorThis study emphasizes the potential utility of Fe₃O₄-Glu-polyethyleneimine nanoparticles encapsulated with DNA vaccine against TB as a prophylactic vaccine. The authors demonstrated a strong immune response and marked growth inhibition of mycobacterium tuberculosis in the mice.     

结核病疫苗研发面临的挑战

结核病（tuberculosis ,TB）是由结核杆菌（Mycobacterium tuberculosis ,M tb）引起的传染病,每年有近1000万例 TB 新发病例,150万人死于 TB 。卡介苗是唯一能够为婴幼儿提供保护作用的疫苗,但它却不能预防成年人 TB 以及控制 TB 流行。随着耐多药 TB 的播散和 M tb／HIV 双重感染人数的不断上升,在世界范围内控制 TB 的形势变得愈发严峻。新疫苗的研发是 TB 防治的重要内容之一,目前全世界已有至少15种 TB 候选疫苗进入临床试验。此外,适宜的动物模型也将对新型疫苗研发以及宿主抗 TB 免疫机制研究起重要作用。     

Tuberculosis vaccines: current status and future prospects

There is an urgent need to develop more effective tuberculosis vaccines as chemotherapy and Bacille Calmette-Guérin (BCG) have failed to control the current epidemic. BCG does have some protective effect in childhood, so using a second vaccine to boost BCG would be the most ethical and logistically feasible strategy. The cost of tuberculosis efficacy trials will be high and return on investment into the development of a tuberculosis vaccine will be low. Incentives such as orphan drug status could encourage industrial interest. As more vaccines enter into early clinical trials, there is an urgent need for the identification of correlates of protection to aid decisions about which vaccines should go forward into efficacy testing. Research efforts that focus on reducing the cost and risk of conducting clinical trials will be of direct benefit to tuberculosis vaccine development.     

肿瘤坏死因子和一氧化氮在N-甲基-N-(3，4-亚甲二氧基苯甲酰)甲基-乙酰胺抗小鼠免疫性肝损伤中的作用

研究了N-甲基-N-（3，4-亚甲二氧基苯甲酰）甲基-乙酰胺（SY-640）的保肝作用及其机理. 给小鼠iv活卡介苗(BCG)12 d 后再iv脂多糖 (LPS)诱导小鼠血浆一氧化氮 (NO), 肿瘤坏死因子(TNF), 谷丙转氨酶(GPT), 谷草转氨酶 (GOT)剧烈升高及严重的肝损伤. 以SY-640给小鼠ig(每日一次，连续10 d)，显著降低BCG＋LPS 诱导的肝损伤小鼠血浆GPT，GOT和TNF水平的升高, 血浆NO水平的升高更加显著, 肝损伤减轻. 以单甲基精氨酸抑制NO的生成, SY-640的上述作用被抵消. SY-640对正常小鼠血浆NO，GPT，GOT水平无影响. 可见，SY-640 的保肝作用与其升高血浆NO, 降低血浆TNF水平有关.     

Enhancement of immunogenicity of murine lymphocytic leukemia cells by transfection with BCG heat shock protein 70 gene

The effects of BCG heat shock protein 70 (BCG HSP70) gene transfection on tumorigenicity and immunogenicity of murine lymphocytic leukemia cell line (L1210) were studied. After HSP70 gene transfection, the tumor cells became strongly immunogenic and lost their tumorigenicity in syngeneic mice. It mainly exhibited that tumor growth was slow or without the formation of tumor, mean survival time of mice was significantly prolonged and a marked stimulating effect on L1210 specific Th1 cells detected by IFN-γ ELISPOT assay. Tumor-bearing mice treated with the L1210-HSP70 cells showed thorough coagulation necrosis and abundant CD8 + T lymphocyte infiltration. Meanwhile, as the tumor vaccine, the HSP70-transfected tumor cells could induce a protective immune response in vivo. It showed that the tumor growth was significantly inhibited, tumor diameter was markedly reduced and the survival time of tumor-bearing mice was further prolonged. Immunization with it also resulted in regression of the established L1210 tumor and prolonged survival time of mice. These results suggest that gene transfection of BCG HSP70 could effectively improve the immunogenicity of tumor cells and it may be used as a suitable candidate gene-modified cell vaccine for cancer immunotherapy.     

3种痰热清胶囊小鼠体内抗流感病毒作用的比较

目的：研究3种不同处方的痰热清胶囊抗流感病毒鼠肺适应株的作用，并比较三者抗病毒作用的强弱。方法：以病毒滴鼻感染小鼠，观察痰热清胶囊对感染病毒小鼠的保护作用，以及对小鼠肺指数及肺病变的影响。结果：3种处方的痰热清胶囊均可使小鼠死亡率明显降低，存活时间明显延长。含熊去氧胆酸及熊胆粉提取物的痰热清胶囊能明显降低流感病毒感染小鼠的肺指数．不含熊去氧胆酸及熊胆粉提取物的痰热清胶囊无此作用。与模型对照组比较，三者对肺病变分级的影响差别无显著性意义。结论：3种处方的痰热清胶囊对感染流感病毒小鼠均有一定的保护作用，其中不含熊胆粉提取物及熊去氧胆酸的痰热清胶囊保护作用较弱，含熊去氧胆酸与含熊胆粉提取物的痰热清胶囊保护作用无明显差异。     

BCG-infected adherent mononuclear cells release cytokines that regulate group 1 CD1 molecule expression

Increasing evidence is now available showing that CD1-restricted T cell responses against non-peptide mycobacterial antigens could play a role in the immune resistance against tuberculosis. BCG, widely used in anti-tubercular vaccination, shares various constituents with Mycobacterium tuberculosis, but does not provide full protection. In the present study we have investigated the pattern of group 1 CD1 molecule expression in adherent mononuclear cells (AMNC) of human peripheral blood, infected in vitro with BCG. Shortly after exposure to BCG, both BCG-positive and BCG-negative AMNC showed a moderate CD1 expression elicited by BCG-induced release of GM-CSF presumably acting through an autocrine and a paracrine mechanism. This was demonstrated using two-color flow cytometry with green fluorescent BCG and anti-CD1 PE-labeled antibodies. However, high CD1 expression induced by exogenously added GM-CSF in AMNC was reduced if target cells were cocultivated with BCG. Monoclonal antibodies against IL-10 partially restored CD1 expression, thus showing that IL-10, released from infected AMNC, is involved, at least in part, in CD1 negative modulation. Therefore, through a complex cytokine network, including not yet identified factor(s), BCG triggers but does not allow full expression of CD1 on AMNC. It cannot be excluded that this mechanism could play a role in the limited efficiency of BCG vaccination.     

复方一枝蒿颗粒对防治手足口病的体内外药效学研究

目的 通过观察复方一枝蒿颗粒在体内外对肠道病毒的抑制作用,评价复方一枝蒿颗粒防治手足口病的药效作用。方法 采用复方一枝蒿颗粒的3岁儿童临床2倍、等倍和1/2倍剂量分别对肠道病毒EV71 H株感染的Babl/c乳鼠手足口病动物模型进行预防性给药和治疗性给药,通过观察乳鼠感染的严重程度、死亡数,来计算动物死亡率、死亡保护率和生命延长率;采用细胞病变的CPE法观察复方一枝蒿颗粒对肠道病毒EV71 H株,BrCr株,柯萨奇病毒B3、B4、B5株的抑制作用;采用real time PCR法观察复方一枝蒿颗粒对肠道病毒EV71 BrCr株在RD细胞中增殖的抑制作用。结果 复方一枝蒿颗粒对肠道病毒EV71 H株感染的乳鼠模型具有明显的治疗作用,其可降低感染程度和死亡率,具有明显的死亡保护和延长生命的作用;复方一枝蒿颗粒在无明显毒性的浓度下,对肠道病毒EV71 H株、BrCr株具有明显的抑制作用,其能明显抑制EV71 BrCr株在细胞内的增殖。结论 复方一枝蒿颗粒在体内外对手足口病均具有较好的治疗作用,其作用机制可能与抑制肠道病毒在细胞内增殖有关。