靶向沉默Mcl-1基因对感染不同毒力结核杆菌小鼠腹腔巨噬细胞凋亡的影响

 目的: 通过RNA干扰技术特异性沉默 Mcl-1 基因,探讨下调 Mcl-1 基因对感染不同毒力结核杆菌小鼠腹腔巨噬细胞凋亡的影响。方法: 分别用制备好的新疆地区流行的优势强毒结核分枝杆菌临床分离株(简称强毒株)、结核分枝杆菌国际标准强毒株H37Rv(简称H37Rv)、结核分枝杆菌国际标准无毒株H37Ra(简称H37Ra)和卡介苗(BCG)菌悬液感染BALB/c小鼠,再以筛选并构建好的Mcl-1-shRNA作用于感染的小鼠模型,同时设立相应对照组,于作用后1 d、3 d、5 d和7 d提取小鼠腹腔巨噬细胞,应用实时荧光定量PCR和Western blot检测各组小鼠腹腔巨噬细胞中Mcl-1 mRNA和蛋白的表达;应用流式细胞术检测各组巨噬细胞的凋亡水平。结果: 小鼠被不同毒力的结核杆菌感染后其腹腔巨噬细胞中Mcl-1 mRNA和蛋白的表达水平均有不同程度的升高,其中以感染了强毒株和H37Rv的腹腔巨噬细胞升高最为明显(P<0.05);应用RNA干扰技术沉默 Mcl-1 基因后,Mcl-1 mRNA和蛋白的表达水平明显低于对照组(P<0.05);流式细胞术分析显示,下调 Mcl-1 基因的表达可诱导小鼠腹腔巨噬细胞凋亡。结论: 应用Mcl-1-shRNA可有效沉默 Mcl-1 在感染了不同毒力结核杆菌小鼠腹腔巨噬细胞中的表达,并能上调巨噬细胞的凋亡水平。     

下调Bcl-2家族蛋白促进MTB感染的小鼠巨噬细胞系凋亡

目的探讨Mcl-1shRNA靶向下调Mcl-1的表达后,Bcl-2家族蛋白对不同毒力结核杆菌感染的小鼠Raw264.7巨噬细胞凋亡的调控作用。方法用XJ-MTB、H37Rv、H37Ra和卡介苗(BCG)感染小鼠Raw264.7巨噬细胞,并用Mcl-1 shRNA作用于感染的巨噬细胞,流式细胞术检测细胞凋亡率;Western blot检测Bcl-2家族蛋白Mcl-1和Bax的表达;实时荧光定量PCR检测Mcl-1、Bcl-2、Bax、caspase-3与细胞色素C mRNA的表达。结果 1)不同毒力的MTB感染可诱导小鼠Raw264.7巨噬细胞的凋亡,靶向下调Mcl-1的表达可显著提高XJ-MTB和H37Rv感染的宿主巨噬细胞的凋亡率。2)XJ-MTB和H37Rv感染可显著上调Bcl-2家族抗凋亡成员Mcl-1与Bcl-2的表达,应用Mcl-1 shRNA沉默Mcl-1基因后Mcl-1与Bcl-2水平显著下降,同时增高Bax的表达。3)不同毒力的MTB感染小鼠Raw264.7巨噬细胞后caspase-3和细胞色素C表达升高,靶向下调Mcl-1可以进一步上调感染组caspase-3和细胞色素C的表达。结论 MTB感染后小鼠Raw264.7巨噬细胞中Bcl-2家族蛋白与基因的表达水平与菌株毒力相关,应用Mcl-1shRNA下调Mcl-1的表达可以促进宿主巨噬细胞的凋亡。     

不同毒力结核分枝杆菌感染小鼠对肺泡巨噬细胞的凋亡率及其时相性变化的影响

目的:探讨不同毒力结核分枝杆菌感染小鼠肺泡巨噬细胞的凋亡率及其时相性变化。方法:不同毒力结核分枝杆菌悬液分别经小鼠尾静脉注射、复制及鉴定各组小鼠感染模型,各组小鼠感染模型复制成功后第1、3、5、7、9、11、13、15天,进行肺泡灌洗,收集小鼠肺泡灌洗液,获取感染小鼠肺泡巨噬细胞,以激光共聚焦显微镜技术检测及鉴定结核分枝杆菌感染小鼠肺泡巨噬细胞,流式细胞术检测上述各时间点、各组感染小鼠肺泡巨噬细胞的凋亡率,比较各组感染小鼠肺泡巨噬细胞凋亡率的时相性变化。结果:激光共聚焦显微镜检测结果显示,结核分枝杆菌国际标准强毒株H37Rv株和卡介苗菌株(BCG)均被小鼠肺泡巨噬细胞大量吞噬。流式细胞技术检测结果显示:结核分枝杆菌国际标准强毒株H37Rv株感染小鼠模型组和卡介苗菌株(BCG)感染小鼠模型组,在小鼠感染模型复制成功后1～9天,小鼠肺泡巨噬细胞的凋亡率逐渐升高,9天时两组小鼠肺泡巨噬细胞的凋亡率均达最高,随着时间的延长,两组小鼠肺泡巨噬细胞的凋亡率呈现逐渐降低趋势。结核分枝杆菌国际标准强毒株H37Rv株感染小鼠组,感染小鼠的肺泡巨噬细胞的凋亡率明显高于卡介苗菌株(BCG)感染小鼠组,差异有统计学意义(P<0.05)。结论:感染小鼠的肺泡巨噬细胞的凋亡率与结核分枝杆菌的毒力强弱呈正相关。     

结核分枝杆菌诱导的THP-1细胞转化及其相关信号传导通路的研究

目的 了解结核分枝杆菌(Mycobacterium tuberculosis)体外诱导人单核细胞株THP-1向类上皮细胞(EC)转化的效应及其相关信号传导通路和调控作用.方法 建立人结核分枝杆菌H37Rv株、牛分枝杆菌bovis株、草分枝杆菌phlei株THP-1细胞感染模型.采用间接免疫荧光法检测感染前后THP-1细胞表面单核细胞或巨噬细胞分化抗原CD115和EC分化抗原CD82的表达情况.采用Sandwich ELISA Kits检测感染前后THP-1细胞p38MAPK(p38丝裂原活化蛋白激酶)、Akt1(丝/苏蛋白激酶)和STAT3(信号转导因子和转录活化因子)磷酸化水平.采用特异性阻断剂,了解各信号通路阻断前后CD115和CD82表达水平变化.结果 3株分枝杆菌感染的THP-1细胞均出现CD115表达减弱和CD82明显表达的现象.H37Rv株或bovis株感染的THP-1细胞可出现一过性p38MAPK磷酸化水平上调,但PI3K(磷脂酰肌醇3-激酶)/Akt和STAT3磷酸化水平均无明显变化.p38MAPK、PI3K/Akt或JAK/STAT通路被阻断后,上述3株分枝杆菌感染的THP-1细胞仍表达CD115.JAK/STAT通路被阻断时,各分枝杆菌感染的THP-1细胞仍表达CD82.但p38MAPK、PI3K/Akt通路被阻断时,H37Rv株和bovis株感染的THP-1细胞CD82表达消失.结论 结核分枝杆菌H37Rv株和bovis株感染后可诱导THP-1细胞向EC转化,p38MAPK、PI3K/Akt参与和调控了该转化过程,其中以p38MAPK通路较为重要.     

不同毒力结核分枝杆菌感染对巨噬细胞铁蛋白及铁转运蛋白表达的影响

目的探讨结核分枝杆菌国际标准强毒株H37Rv株(简称H37Rv株)和卡介苗菌株(简称BCG菌株)分别感染巨噬细胞后,各感染组巨噬细胞内铁蛋白(Fn)和铁转运蛋白(FPN)表达量及其时相性变化。方法分别用结核分枝杆菌H37Rv株和BCG菌株感染巨噬细胞RAW264.7细胞株,于感染后1、6、12、18、24 h,应用ELISA检测各组感染巨噬细胞培养上清液中Fn和FPN的含量;应用Western blot技术检测上述时间点各组感染巨噬细胞内Fn的表达量。结果不同菌株感染巨噬细胞后,巨噬细胞内Fn随处理时间延长表达逐渐增强;感染1 h,正常对照组Fn的表达高于H37Rv组和BCG组;感染6 h,感染组表达高于正常对照组,感染12 h,H37Rv组高于正常对照组,差异具有统计学意义(P<0.05);感染18 h和24 h,H37Rv组>BCG组>正常对照组,差异具有统计学意义(P<0.05)。FPN的表达量随时间变化逐渐降低。与正常对照组相比,H37Rv组、BCG组FPN的表达均呈现较低水平,为H37Rv组相似文献   

抑制Mcl-1表达调控结核感染的小鼠腹腔巨噬细胞凋亡的机制初探

目的:应用Mcl-1-shRNA质粒抑制不同毒力结核分枝杆菌(MTB)菌株感染的小鼠腹腔巨噬细胞中Mcl-1的表达,通过观察Bcl-2和Bax表达的变化探讨其调控机制。方法:制备不同毒力MTB菌株悬液,分别感染BALB/c小鼠,再用Mcl-1-shRNA质粒处理感染小鼠模型,并同时设立对应的对照组,于处理后1 d、3 d、5 d和7d处死小鼠并收集腹腔巨噬细胞。应用流式细胞术检测不同处理时间、不同毒力菌株感染时小鼠腹腔巨噬细胞的凋亡率,real-time PCR和Western blot检测Bcl-2和Bax的表达。结果:Mcl-1-shRNA质粒处理后,不同毒力MTB菌株感染的小鼠巨噬细胞凋亡率均比对照组有不同程度的增高,其中以BCG和H37Ra组最明显(P 0. 05); Bcl-2的mRNA和蛋白水平显著减少,而Bax的mRNA和蛋白的表达均显著增加,以BCG感染组较为显著,且二者mRNA的比值与菌株毒力呈负相关(P 0. 05)。结论:抑制Mcl-1的表达可显著促进不同毒力MTB菌株感染的小鼠腹腔巨噬细胞凋亡,其调控机制可能与Bcl-2和Bax蛋白的表达及MTB菌株毒力密切相关。     

脂多糖诱导大鼠膀胱上皮细胞MAPK/ERK与Stat3的表达

目的证实体外培养的大鼠膀胱上皮细胞是否可以通过MAPK及STAT信号途径调节脂多糖诱导的炎症反应。方法分离幼龄SD大鼠膀胱粘膜,培养上皮细胞,分为四组:对照组、脂多糖组、PD98059+脂多糖组及AG490+脂多糖组,采用Real time-PCR及Western blot技术检测ERK及Stat3表达量的变化。结果脂多糖可显著激活膀胱上皮细胞中ERK及Stat3的表达,PD98059及AG490能够部分减低脂多糖刺激后两种基因的相对表达量。结论 MAPK/ERK及STAT信号通路可能参与脂多糖诱导的大鼠膀胱上皮细胞的炎症反应。     

活菌H37Ra与灭活菌H37Rv感染小鼠腹腔巨噬细胞的实验研究

目的:探讨小鼠腹腔接种结核分枝杆菌活菌H37Ra、灭活菌H37Rv后,诱导巨噬细胞免疫物质的表达差异,探讨活菌H37Ra及灭活菌H37Rv作为新的结核候选疫苗的可行性。方法:分别培养BALB/c小鼠腹腔巨噬细胞,用活菌H37Ra,灭活菌H37Rv感染10小时后用抗酸染色方法,观察各组巨噬细胞吞噬情况并计算吞噬率。将活菌H37Ra、灭活菌H37Rv、生理盐水分别接种BALB/c小鼠腹腔,免疫30天后取小鼠腹腔巨噬细胞,RT-PCR法检测小鼠腹腔巨噬细胞IL-12P40、TNFα-、IFNγ-的基因转录水平;ELISA法检测细胞因子IL-12P40、TNFα-、IFNγ-的表达;Griess法、化学法检测小鼠腹腔巨噬细胞分泌NO、H2O2水平;流式细胞仪检测IFNγ-诱导的CD40L的变化情况。结果:巨噬细胞对活菌H37Ra和灭活菌H37Rv的吞噬率分别为(55.71±8.42)%、(14.82±2.12)%。与灭活菌相比,活菌H37Ra有更强的被吞噬能力(P<0.01)。活菌H37Ra免疫小鼠腹腔巨噬细胞后能显著诱导巨噬细胞的IL-12P40、TNF及IFNγ-基因的转录;细胞因子IL-12P40、TNFα-、IFNγ-、NO及H2O2高水平分泌;同时活菌H37Ra刺激IFNγ-诱导的巨噬细胞表面CD40L的高表达。结论:活的结核分枝杆菌H37Ra能显著诱导小鼠腹腔巨噬细胞产生更多的保护性免疫物质,有利于免疫应答,有可能作为新的结核候选疫苗,而灭活菌H37Rv不能显著激活小鼠腹腔巨噬细胞分泌保护性免疫物质,不宜作为新的结核候选疫苗。     

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。     

MAPK信号通路参与调控大鼠腹腔巨噬细胞表达CRH

为了解终末巨噬细胞表达促肾上腺皮质激素释放激素(CRH)的胞内信号转导机制,明确丝裂原活化蛋白激酶(MAPK)信号通路与脂多糖(LPS)诱导的大鼠腹腔表达CRH的关系。我们利用体外培养的大鼠腹腔巨噬细胞,采用RT-PCR及ELISA方法观察MAPK信号通路中关键激酶特异性阻断剂(PD98059、SB203580、SP600125)对LPS诱导的CRH表达的影响。结果显示,LPS促进了大鼠腹腔巨噬细胞表达CRH,MAPK三条主要信号通路中关键激酶特异性阻断剂均剂量依赖性地抑制了LPS诱导的CRH表达。因此,我们认为LPS可以促进终末巨噬细胞表达CRH,而MAPK信号通路参与了该过程。     

Growth of Virulent and Avirulent Mycobacterium tuberculosis Strains in Human Macrophages

Mycobacterium tuberculosis H37Rv causes progressive disease in animals, whereas the H37Ra strain does not. The relevance of this difference in virulence to human infection is uncertain because these strains have been shown to have similar growth rates in human macrophages. To evaluate the intracellular growth of M. tuberculosis strains in macrophages under conditions similar to those encountered in vivo, we infected human monocyte-derived macrophages with H37Ra, H37Rv, or one of four isolates from tuberculosis patients at a low bacillus-to-macrophage ratio. H37Rv and the patient isolates grew significantly faster than H37Ra, based on the numbers of CFU and acid-fast bacilli. These findings did not result from extracellular mycobacterial growth, differential macrophage viability, or bacillary clumping. In contrast to other published results, these findings indicate that the virulence characteristics of M. tuberculosis strains in animal models are relevant to human tuberculosis infection.     

Mycobacterium tuberculosis blocks crosslinking of annexin-1 and apoptotic envelope formation on infected macrophages to maintain virulence

Macrophages infected with attenuated Mycobacterium tuberculosis strain H37Ra become apoptotic, which limits bacterial replication and facilitates antigen presentation. Here we demonstrate that cells infected with H37Ra became apoptotic after the formation of an apoptotic envelope on their surface was complete. This process required exposure of phosphatidylserine on the cell surface, followed by deposition of the phospholipid-binding protein annexin-1 and then transglutaminase-mediated crosslinking of annexin-1 through its amino-terminal domain. In macrophages infected with the virulent strain H37Rv, in contrast, the amino-terminal domain of annexin-1 was removed by proteolysis, thus preventing completion of the apoptotic envelope, which resulted in macrophage death by necrosis. Virulent M. tuberculosis therefore avoids the host defense system by blocking formation of the apoptotic envelope, which leads to macrophage necrosis and dissemination of infection in the lung.     

Effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta and RANTES mRNA expression in guinea pig cells exposed to attenuated and virulent mycobacteria

The effect of Mycobacterium bovis BCG vaccination on interleukin-1 beta (IL-1 beta) or regulated-upon-activation, normally T-cell-expressed and -secreted chemokine (RANTES) mRNA expression in guinea pig spleen cells stimulated with concanavalin A, lipopolysaccharide (LPS), phorbol myristate acetate (PMA) plus ionomycin, or purified protein derivative (PPD) was studied in vitro. Similarly, peritoneal exudate cell-derived macrophages from na?ve and BCG-vaccinated guinea pigs were infected with M. bovis BCG, Mycobacterium avium, the attenuated Mycobacterium tuberculosis H37Ra strain, or virulent strains H37Rv and Erdman of M. tuberculosis. Total RNA was subjected to Northern blot analysis using probes generated from guinea pig IL-1 beta or RANTES cDNA. Although IL-1 beta and RANTES mRNA could be detected in the spleen cells from na?ve animals stimulated with LPS or PMA plus ionomycin, the levels were significantly enhanced after BCG vaccination. mRNA expression was also elevated in macrophages infected with live mycobacteria after BCG vaccination. However, macrophages infected with the virulent H37Rv strain of M. tuberculosis showed 75 to 90% reductions in IL-1 beta expression and 25 to 60% reductions in RANTES mRNA expression compared with macrophages infected with the attenuated H37Ra strain. The IL-1 beta mRNA levels peaked as soon as 1 h after PPD stimulation and 4 h after M. tuberculosis H37Rv infection of macrophages. In contrast, RANTES mRNA expression was delayed until 48 h after infection. These results indicate that molecular mediators produced in response to various stimuli associated with protective immunity against mycobacteria are upregulated after BCG vaccination; however, a significantly weaker response was observed with virulent M. tuberculosis. These initial studies indicate that BCG vaccination has a positive effect on IL-1 beta and RANTES mRNA expression by host cells in a highly relevant animal tuberculosis model.     

Mycobacterium bovis BCG vaccination augments interleukin-8 mRNA expression and protein production in guinea pig alveolar macrophages infected with Mycobacterium tuberculosis

Alveolar macrophages are likely the first cell type to encounter Mycobacterium tuberculosis in a pulmonary infection, resulting in the production of chemokines. In order to evaluate this response, alveolar macrophages harvested from nonvaccinated and Mycobacterium bovis BCG-vaccinated guinea pigs were infected in vitro with live M. tuberculosis H37Ra or H37Rv (multiplicity of infection, 1:1) or cultured with lipopolysaccharide (10 micro g/ml) for 3, 12, and 24 h. Interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) mRNA expression was determined by real-time PCR. Culture supernatants were assayed for guinea pig IL-8 protein by using a human IL-8 enzyme-linked immunosorbent assay kit. Alveolar macrophages harvested from BCG-vaccinated guinea pigs produced significantly more mRNA and protein for IL-8 than alveolar macrophages harvested from nonvaccinated guinea pigs at 12 and 24 h poststimulation or postinfection. Infection with attenuated M. tuberculosis (H37Ra) stimulated alveolar macrophages isolated from BCG-vaccinated guinea pigs to produce significantly more IL-8 mRNA than did alveolar macrophages infected with a virulent strain (H37Rv) at 12 and 24 h postinfection. Significant MCP-1 mRNA production was also detected in stimulated or infected alveolar macrophages; however, prior vaccination did not significantly affect levels of MCP-1 mRNA. Alveolar macrophages isolated from BCG-vaccinated guinea pigs produced significantly more IL-8 mRNA and protein when stimulated for 24 h with heat-killed H37Ra, heat-killed H37Rv, and H37Rv cell wall, but not mannose-capped lipoarabinomannan (ManLAM), than did cells stimulated with media alone. These observations indicate that prior vaccination may alter very early events in the M. tuberculosis-infected lung.     

Mannosylated lipoarabinomannan balances apoptosis and inflammatory state in mycobacteria-infected and uninfected bystander macrophages

To assess the role of mannosylated lipoarabinomannan (ManLAM) in the inflammatory and apoptotic response of mycobacteria-infected and uninfected, bystander cells we applied a mouse macrophage model of infection with avirulent strains--Mycobacterium bovis BCG, Mycobacterium tuberculosis (MTB) H37Ra and compared with a virulent MTB H37Rv strain infection. ManLAM contributed to the infection of macrophages by protection from apoptosis with stabilized Bcl-2 expression and down-regulated Bax expression for infected cells (BCG) or with stabilized Bcl-2 expression for uninfected bystander target cells (H37Ra). Additionally, ManLAM up-regulated FasL expression on the infected cells. Active extracellular signal-regulated kinase (ERK1/2) in BCG and H37Rv infection provided an anti-apoptotic effect by stabilization of anti-apoptotic Bcl-2 expression in the infected cells. Inhibitors specific for c-Jun-NH2-terminal kinase or stress-activated kinase (JNK) and p38 kinase decreased apoptosis of infected cells (BCG, H37Ra) and of uninfected bystanders (H37Ra) by down-regulating Bax. ManLAM significantly down-regulated production of pro-inflammatory IL-12 and TNF-alpha and activation of JNK by both avirulent strains. We conclude that by stabilization of Bcl-2 expression, down-regulation of JNK activity and down-regulation of pro-inflammatory cytokines production ManLAM can contribute to suppression of apoptosis and inflammatory reaction of uninfected, bystander cells.     

Induction of Cell Death in Human Macrophages by a Highly Virulent Korean Isolate of Mycobacterium tuberculosis and the Virulent Strain H37Rv

Recent studies have suggested that virulent strains of Mycobacterium tuberculosis induce apoptosis in macrophages less often than do attenuated strains. K-strain, which belongs to the Beijing family, is the most frequently isolated clinical strain of M. tuberculosis in Korea. In this study, we investigated the differential induction of cell death in human monocytic THP-1 cells by K-strain and H37Rv, a virulent but laboratory-adapted strain of M. tuberculosis . Although no significant difference in growth rate was observed between the cells exposed to K-strain and those exposed to H37Rv, the levels of protective cytokines such as tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-12p40 were lower in K-strain-infected cells than in H37Rv-infected cells. Cell viability assays showed that both K-strain and H37Rv, but not heat- or streptomycin-killed bacteria, induced THP-1 cell death in a TNF-independent manner. In contrast, double staining with fluorochrome-labelled inhibitors of caspase and propidium iodide and lactate dehydrogenase release assays revealed that K-strain induced significantly higher levels of necrotic cell death, rather than apoptosis, in THP-1 cells than did H37Rv. Anti-apoptotic Bcl-2 , Mcl-1 , Bfl-1 and Bcl-xL in the cells were significantly upregulated following infection with K-strain compared with H37Rv, whereas Bax was slightly upregulated in response to infection with both H37Rv and K-strain. These results suggest that the highly virulent K-strain keeps cellular apoptosis as a host defense mechanism to a minimum and induces necrosis in macrophages.