苍龙丸治疗小鼠结核病模型的疗效观察

我国的结核病疫情仍相当严重,2000年第四次全国结核病流行病学抽样调查的结果表明,我国有5.5亿人感染了结核菌,有活动性肺结核病人451万,每年死亡人数约13万,结核病死亡率在传染病中居第一位。随着社会人口流动增加,HIV感染者增多,新型及多重耐药菌株的出现等因素,我国结核病的发病率呈明显的上升趋势[1]。     

不同剂量结核病DNA疫苗及细胞因子联合免疫的研究

目的：评价结核分枝杆菌MPT64（简称M64）和ESAT6（简称E6）DNA疫苗不同剂量联合免疫、与细胞因子联合免疫的保护效力，以及Ag85A（简称85A）和Ag5B（简称85B）DNA疫苗的保护效力。方法：将C57BL／6小鼠随机分为14组免疫：①生理盐水；②载体pVAX1 100μg；③卡介苗；④M64100μg＋E6100μg；⑤M6450μg＋E650μg；⑥M6475μg＋E625μg；⑦M6425μg＋E675μg；⑧M6425μg＋E625μg；⑨M64100μg＋IFN-γ 100μg；⑩E6100μg＋IFN-γ100μg；（11）M64100μg＋IL-12 100μg；（12）E6100μg＋IL-12 100μg；（13）85A100μg；逼（14）5B100μg。用ELISA法检测血清抗体，结核分枝杆菌通过尾静脉攻击小鼠，动态观察小鼠体重变化；攻击4周后，取肺、肝和脾观察病理改变、称重量、做菌落计数。结果：不同剂量的M64和E6DNA联合免疫、85A和85BDNA分别免疫均能诱导小鼠产生特异性抗体，但M64或E6与IFN-γ或IL-12质粒DNA共同免疫后特异性抗体水平与对照组无显著性差异；各组血清抗体亚型IgG2a均无显著升高，IgG2b均显著高于IgGI。第2、3A、8、9、11、12组在感染后4周体重明显增加，而第1、14、5、6组体重下降；第3、11、9、7组的肺菌落指数较少。第10、13组肺肉眼未见明显病变；第2～4、7、9～13组肺组织主要为不同程度的增殖性反应，第1、6、8、14组肺组织为增殖性反应与渗出性反应并存，第5组为渗出性反应。结论：DNA免疫的量需100μg才能诱导产生强的保护效力；M64和E6DNA各100μg混合免疫、85ADNA疫苗的保护效力与卡介苗相当；b164和E6DNA与IFN-γ或IL-12质粒DNA共同免疫后，明显增强其保护效力。     

刀豆蛋白A对结核病小鼠的保护作用及其机理研究

目的：观察刀豆蛋白A（ConA)对结核病小鼠的保护作用及对TH1／TH2平衡的调节。方法：以ConA 100(E2组）或500μg(E1组）腹腔内注射预处理结核病小鼠,观察生存时间、肺组织病变及菌落计数;检测脾细胞培养上清液中的IFN－γ、IL－4。结果：ConA可延长结核病小鼠的存活期。第2周,感染对照组（C组）肺组织大面积受累,而E2组病变较轻,E1组仅局部有炎性细胞浸润;第4周,C组出现严重的炎症变化,E2组病变范围小,E1组病变局限化,出现淋巴细胞结节。E1、E2组的菌落计数于第2周显著低于C组;第4周,E1组显著低于E2及C组。第2周,E1、E2组IFN－γ的水平显著高于C组及正常对照组（P＜0．01）;第4周,E1组IFN－γ水平显著高于C组及E2组（P＜0．01）,而IL－4变化不明显（P＞0．05）。结论：ConA预处理可使TH细胞以TH1应答为主,增强小鼠对结核杆菌的抵抗力。     

结核分枝杆菌Ag85A质粒DNA疫苗治疗小鼠耐药结核病的效果研究

目的 研究结核分枝杆菌Ag85A质粒DNA疫苗治疗小鼠耐药结核病的效果.方法 用结核分枝杆菌高耐利福平低耐异烟肼临床分离株HB361尾静脉注射17～19 g的6～8周龄雌性BALB/c小鼠后,将小鼠随机均匀地分为6组,感染后第3天开始,分别用生理盐水(A组)、pVAX1空载体(B组)、利福平(C组)、微卡菌苗(D组)、Ag85A质粒DNA疫苗(E组)、利福平和Ag85A质粒DNA疫苗(F组)治疗60d,每组10只小鼠.治疗结束后3周,分别取肺和脾观察病理改变,称取重量做菌落计数.结果 治疗结束后3周,与对照组比较,D组、E组和F组肺脏病变有不同程度减轻,病变局限,病变范围分别为50%、20%、20%,2/3区域可见正常的肺泡结构,肺泡轮廓相对清晰,细胞分布均匀.与A组相比,D组、E组和F组肺脏菌落数分别减少了52%、68%、78%;脾脏菌落数依次减少了48%、65%、79%.结论 与对照组相比,Ag85A质粒DNA疫苗单独应用或与利福平联合应用治疗小鼠耐药结核病均显示疗效.     

结核病免疫应答中自噬现象的分子机制和意义

越来越多的证据表明,自噬是结核免疫反应的重要组成部分.自噬可以杀灭结核分枝杆菌、调节促炎细胞因子的分泌、增加抗原递呈功能.自噬与其他抗菌途径如维生素D3、炎性体、泛素系统存在协同作用.另一方面,结核分枝杆菌可以调控巨噬细胞的自噬.目前,自噬已成为临床重要的诊疗靶点.其能诱导自噬的药物,可以作为佐剂治疗耐药性结核;能有效诱导自噬的疫苗,可能提供更好的免疫保护作用.     

柠檬酸钠对肾缺血再灌注损伤模型小鼠肾损伤指标及炎症细胞因子的影响

目的:揭示柠檬酸钠对肾缺血再灌注损伤模型小鼠肾损伤指标及炎症细胞因子的影响.方法:将40只C57BL/6小鼠随机分为假手术组(n=10)、模型组(n=10)、低剂量组(n=10)和高剂量组(n=10).建立缺血再灌注损伤模型前7 d,低剂量组和高剂量组分别以200 mg/kg和600 mg/kg的柠檬酸钠对小鼠进行灌胃...     

精氨酸单糖苷( AF) 对免疫抑制小鼠免疫功能的影响

目的:研究精氨酸单糖苷(Arginyl-fructose,AF)对环磷酰胺(CTX)诱导的免疫抑制小鼠免疫功能的影响。方法: ICR 小鼠100 只,随机分成5 组,即正常对照组(N),免疫抑制模型组(M),免疫抑制AF 低、中、高剂量组(M-L、M-M、M-H)。试验组连续给药28 d。免疫抑制组,每周前3 天,腹腔注射80 mg/ kg 环磷酰胺,形成免疫抑制。末次给药12 h 解剖后,测定免疫器官指数,AF 对脾淋巴细胞转化及增殖、巨噬细胞吞噬功能、特异性IgG 抗体、血清中TNF-β、IL-2 含量的影响。并通过实时荧光定量PCR 检测TNF-β、IL-2 基因表达的差异。结果:AF 能够显著提高小鼠的胸腺指数和脾脏指数,促进脾淋巴细胞的自然转化与增殖,增强巨噬细胞吞噬功能,显著提高小鼠血清中特异性IgG 抗体含量,可显著提高小鼠血清中TNF-β、IL-2 含量,实时荧光定量PCR 检测结果显示,TNF-β、IL-2 mRNA 能够良好表达。结论:AF 能拮抗CTX 免疫功能低下小鼠的免疫抑制作用。     

免疫低下小鼠肺部肺炎链球菌感染模型的制备

目的建立免疫低下肺部肺炎链球菌感染小鼠模型。方法将配比好的FLV病毒悬浊液腹腔注射模型组小鼠,21 d后,采用滴鼻法对小鼠进行Ⅲ型肺炎链球菌接种;正常对照组给予与药液同体积的生理盐水。结果与正常对照组比较模型组小鼠毛发光泽度欠佳,竖毛现象严重,活动度逐渐减少,饮食明显减少,体质量减轻;模型组小鼠各脏器指数比正常对照组均升高,差异有显著统计学意义(P0.05),免疫低下肺部肺炎链球菌感染模型小鼠血清中IL-6、PCT表达水平均比正常对照组增高(P0.05)。结论采用FLV病毒悬浊液结合滴鼻法滴注肺炎链球菌建立的免疫低下肺部肺炎链球菌感染小鼠模型,为研究免疫低下肺部肺炎链球菌感染药物干预提供了较理想的实验模型。     

结核分枝杆菌Rv2450蛋白诱导小鼠免疫应答的实验研究

目的:以原核表达的结核分枝杆菌Rv2450蛋白在小鼠体内诱导体液和细胞免疫应答。方法:采用皮下包埋的方法,以预先转移到硝酸纤维素膜上的原核表达的Rv2450蛋白免疫小鼠(10只)3次,每次间隔2周。用间接ELISA法检测免疫小鼠血清特异性抗体的滴度。末次免疫完成后4周,处死3只免疫小鼠并分离脾淋巴细胞,体外经PPD(2μg/孔)刺激后,用MTT比色法检测免疫小鼠脾淋巴细胞的增殖指数。用ELISA法检测脾淋巴细胞悬液中IFN-γ、IL-10及IL-12的水平。结果:Rv2450蛋白免疫小鼠血清特异性抗体的滴度为1∶3200,淋巴细胞增殖指数为3.76±0.19。免疫小鼠脾淋巴细胞培养液中IFN-γ、IL-10及IL-12的含量,分别为(1740±19)ng/L、(678±15)ng/L、(469±13)ng/L,均高于各组的生理盐水对照组(P<0.05)。结论:Rv2450有可能作为新型结核疫苗的候选组分。     

Differential cytokine gene expression and secretion after phagocytosis by a human monocytic cell line of Toxoplasma gondii compared with Mycobacterium tuberculosis.

Toxoplasma gondii infection may be clinically silent in immunocompetent individuals but may cause fatal disease in immunocompromised patients such as those with HIV infection. Proinflammatory cytokines are known to be important in murine resistance to T. gondii but there are no data from human models of infection. We have investigated whether phagocytosis of T. gondii, of Mycobacterium tuberculosis (a pathogen which elicits a granulomatous host immune response) and of inert latex particles by THP-1 cells, a human monocytic line, caused gene expression and secretion of tumour necrosis factor (TNF), IL-6 and IL-8. These cytokines are important in recruitment and activation of T lymphocytes, and both TNF and IL-6 may have direct antitoxoplasmacidal and antimycobacterial activity. Phagocytosis of T. gondii by THP-1 cells resulted in minimal gene expression and secretion of TNF, IL-6 and IL-8 similar to that following phagocytosis of inert latex particles. In contrast, phagocytosis of M. tuberculosis resulted in increased gene expression of TNF and IL-8 as well as increased secretion of all three cytokines, particularly IL-8. These observations may partially explain the frequency of non-inflammatory host responses to T. gondii in immunocompetent individuals.     

Mycobacterium bovis with different genotypes and from different hosts induce dissimilar immunopathological lesions in a mouse model of tuberculosis

With the hypothesis that genetic variability of Mycobacterium bovis could influence virulence and immunopathology, five M. bovis strains were selected from an epidemiological study in Argentina on the basis of their prevalence in cattle and occurrence in other species. We then determined the virulence and the immunopathology evoked by these strains in a well‐characterized mouse model of progressive pulmonary tuberculosis. The reference strain AN5 was used as a control. BALB/c mice infected with this M. bovis reference strain showed 50% survival after 4 months of infection, with moderate bacillary counts in the lung. Two weeks after inoculation, it induced a strong inflammatory response with numerous granulomas and progressive pneumonia. In contrast, strain 04‐303, isolated from a wild boar, was the most lethal and its most striking feature was sudden pneumonia with extensive necrosis. Strain 04‐302, also isolated from wild boar but with a different spoligotype, induced similar pathology but to a lesser extent. In contrast, strains 534, V2 (both from cattle) and 02‐2B (from human) were less virulent, permitting higher survival after 4 months of infection and limited tissue damage. Strain AN5 and the cattle and human isolates induced rapid, high and stable expression of interferon (IFN)‐γ and inducible nitric oxide synthase (iNOS). In contrast, the more virulent strains induced lower expression of IFN‐γ, tumour necrosis factor‐α and iNOS. Interestingly, these more virulent strains induced very low expression of murine beta defensin 4 (mBD‐4); whereas, the control strain AN5 induced progressive expression of this anti‐microbial peptide, peaking at day 120. The less virulent strains induced high mBD‐4 expression during early infection. Thus, as reported with clinical isolates of M. tuberculosis, M. bovis also showed variable virulence. This variability can be attributed to the induction of a different pattern of immune response.     

八种中药多糖对小鼠巨噬细胞释放细胞因子的影响

常规提取黄芪、枸杞、当归、香菇、地黄、蒲公英、板蓝根、柴胡等8味中药的多糖组份,体外与小鼠腹腔巨噬细胞共同孵育,ELISA法检测培养液中TNF-α、IL-1β、IL-6、IL-8等细胞因子的含量,并与巨噬细胞对照组比较,借以观察黄芪等中药的多糖对巨噬细胞释放细胞因子的影响,探索巨噬细胞甘露糖受体在中药多糖免疫干预效应中的作用。结果表明,黄芪、枸杞、当归、香菇和蒲公英等5味中药多糖组份一定浓度组TNF-α、IL-1β、IL-6、IL-8中一个或多个细胞因子水平明显升高(P<0.05或P<0.01),且具有浓度差异性(P<0.05或P<0.01)。提示黄芪等5味中药多糖组分刺激巨噬细胞释放细胞因子是他们干预机体免疫功能的部分机制。     

The 19-kD antigen and protective immunity in a murine model of tuberculosis

The 19-kD antigen is a cell wall-associated lipoprotein present in Mycobacterium tuberculosis and in bacille Calmette-Guérin (BCG) vaccine strains. Expression of the 19-kD antigen as a recombinant protein in two saprophytic mycobacteria-M. vaccae and M. smegmatis-resulted in abrogation of their ability to confer protection against M. tuberculosis in a murine challenge model, and in their ability to prime a DTH response to cross-reactive mycobacterial antigens. Induction of an immune response to the 19-kD antigen by an alternative approach of DNA vaccination had no effect on subsequent M. tuberculosis challenge. These results are consistent with a model in which the presence of the 19-kD protein has a detrimental effect on the efficacy of vaccination with live mycobacteria. Targeted inactivation of genes encoding selected antigens represents a potential route towards development of improved vaccine candidates.     

以结核分枝杆菌H_(37)Rv莽草酸脱氢酶为靶点的高通量筛选模型的建立及应用

目的建立以结核分枝杆菌莽草酸脱氢酶为靶点的新型抗结核药物高通量筛选模型;用此模型筛选莽草酸脱氢酶抑制剂;进一步评价化合物对莽草酸脱氢酶活性的影响。方法表达并纯化结核分枝杆菌H37Rv莽草酸脱氢酶;利用还原型辅酶II(NADPH)在溶液中的光吸收,测定酶的活性,构建了该酶抑制剂的高通量筛选模型;用Z′因子法评价该模型的可靠性,并对5万余个化合物进行筛选;测定了各抑制剂的IC50并对抑制剂6186050的酶抑制动力学进行了研究;用菌液稀释法评价了抑制剂对某些临床分离菌株包括耐药菌株的影响。结果得到了重组莽草酸脱氢酶;测得比活力为20987U/mg,所建的莽草酸脱氢酶高通量筛选模型Z′因子为0.76,符合高通量筛选的要求;对5万余个化合物进行筛选得到9个抑制率较高的化合物;抑制剂6186050为竞争性可逆抑制剂;抑制剂6230384和6186050对海分枝杆菌的最低抑菌浓度(MIC)都是32μg/ml。结论建立了稳定性好、灵敏度较高的结核分枝杆菌莽草酸脱氢酶抑制剂高通量药物筛选模型,应用该模型筛选得到的抑制剂可能具有抑菌活性。     

以肽脱甲酰基酶为靶点的抗结核药物高通量筛选模型的建立和应用

目的建立以肽脱甲酰基酶(PDF)为靶点的抗结核药物高通量筛选模型,应用该模型筛选得到活性微生物发酵液粗提物样品。方法以结核分枝杆菌H37Rv基因组为模板,扩增肽脱甲酰基酶的基因片段def,构建表达载体pET-28a-def,表达并纯化结核分枝杆菌PDF酶;基于PDF水解三肽底物for-Met-Ala-Ser释放出游离NH2,而游离NH2可与荧光胺反应产生荧光的原理,利用测定所产生荧光值的方法,建立高通量药物筛选模型;使用该模型对12400个微生物发酵液粗提物样品进行筛选,同时以耻垢分枝杆菌为检定菌,平板纸片法检测样品的抗菌活性,并检测所得阳性样品的细胞毒性。结果成功构建了表达载体pET-28a-def;所建立的模型稳定可行,可用于以肽脱甲酰基酶为靶点的抗结核药物的高通量筛选;用该模型对12400个微生物发酵液粗提物样品进行筛选,最终得到8个对肽脱甲酰基酶抑制活性和抗耻垢分枝杆菌活性均较好的阳性样品,阳性率0.06%;其中5个样品的细胞毒性较小。结论建立了灵敏度好、稳定性高的结核分枝杆菌肽脱甲酰基酶抑制剂高通量药物筛选模型,应用该模型所得到的阳性样品具有进一步深入研究的意义。     

Mycobacterium tuberculosis and the intimate discourse of a chronic infection

Mycobacterium tuberculosis is an extremely successful pathogen that demonstrates the capacity to modulate its host both at the cellular and tissue levels. At the cellular level, the bacterium enters its host macrophage and arrests phagosome maturation, thus avoiding many of the microbicidal responses associated with this phagocyte. Nonetheless, the intracellular environment places certain demands on the pathogen, which, in response, senses the environmental shifts and upregulates specific metabolic programs to allow access to nutrients, minimize the consequences of stress, and sustain infection. Despite its intracellular niche, Mycobacterium tuberculosis demonstrates a marked capacity to modulate the tissues surrounding infected cells through the release of potent, bioactive cell wall constituents. These cell wall lipids are released from the host cell by an exocytic process and induce physiological changes in neighboring phagocytes, which drives formation of a granuloma. This tissue response leads to the generation and accumulation of caseous debris and the progression of the human tuberculosis granuloma. Completion of the life cycle of tuberculosis requires damaging the host to release infectious bacteria into the airways to spread the infection. This damage reflects the pathogen's ability to subvert the host's innate and acquired immune responses to its own nefarious ends.     

Comparison of the immune response against Mycobacterium tuberculosis antigens between a group of patients with active pulmonary tuberculosis and healthy household contacts.

The mycobacterial antigens and the factors related to protection for the development of active tuberculosis are not known. In a natural model of tuberculosis, we studied 10 patients with active pulmonary tuberculosis (non-protective immune response) and 38 healthy household contacts (protective immune response). We tested the lymphocyte proliferative response by T cell Western blotting to eight different antigen fractions and to two purified mycobacterial antigens of 30 and 64 kD. Patients with active tuberculosis recognized fractions with molecular weights of 80-114, 60-80, 28-41 and 14-19 kD. Household contacts recognized the same fractions except the 14-19 kD. The response to the 64-kD antigen was not significantly different between groups. In contrast, 10% of the patients with active tuberculosis and 73% of the household contacts responded to the 30-kD antigen. The humoral response against the 30-kD antigen by ELISA showed a significantly higher production of antibodies in tuberculosis patients compared with household contacts. We conclude that patients with active pulmonary tuberculosis develop an immune response characterized by poor proliferative response to the 30-kD antigen with a strong humoral response, whereas the opposite occurs in healthy subjects infected by Mycobacterium tuberculosis.