miRNA-126对肺癌A549细胞的增殖、迁移、侵袭及EGFR/AKT/mTOR信号通路的影响

 目的: 探讨微小RNA(microRNA,miRNA)-126对肺癌A549细胞功能的影响及相关的作用机制。方法: 利用脂质体试剂Lipofectamine 2000将miRNA-126转染入肺癌A549细胞中,采用real-time PCR法检测转染后各组细胞中miRNA-126的表达;MTT法检测细胞活力;台盼蓝拒染实验检测细胞存活数目;细胞集落培养实验观察转染后细胞集落形成;划痕愈合实验检测细胞的迁移能力;Transwell小室实验检测细胞侵袭能力;Western blot法检测EGFR/AKT/mTOR通路中各蛋白水平的变化。结果: Real-time PCR结果显示,转染miRNA-126组细胞中的miRNA-126表达水平显著高于阴性对照组和空白对照组(P<0.01),而阴性对照组与空白对照组无显著变化;A549细胞转染miRNA-126模拟物后,细胞增殖和集落形成能力均呈明显下降(P<0.01);肺癌细胞的迁移和侵袭能力也受到明显抑制(P<0.01);Western blot结果显示,转染miRNA-126组细胞中EGFR/AKT/mTOR通路相关蛋白p-EGFR、p-AKT和p-mTOR的水平均有明显降低(P<0.01)。结论: miRNA-126可以显著降低肺癌A549细胞中p-EGFR、p-AKT和p-mTOR的蛋白水平,进而可能抑制其细胞增殖和迁移侵袭能力。     

Sensing of Mycobacterium tuberculosis and consequences to both host and bacillus

Mycobacterium tuberculosis (Mtb), the primary causative agent of human tuberculosis, has killed more people than any other bacterial pathogen in human history and remains one of the most important transmissible diseases worldwide. Because of the long-standing interaction of Mtb with humans, it is no surprise that human mucosal and innate immune cells have evolved multiple mechanisms to detect Mtb during initial contact. To that end, the cell surface of human cells is decorated with numerous pattern recognition receptors for a variety of mycobacterial ligands. Furthermore, once Mtb is ingested into professional phagocytes, other host molecules are engaged to report on the presence of an intracellular pathogen. In this review, we discuss the role of specific mycobacterial products in modulating the host's ability to detect Mtb. In addition, we describe the specific host receptors that mediate the detection of mycobacterial infection and the role of individual receptors in mycobacterial pathogenesis in humans and model organisms.     

Tissue factor expression by myeloid cells contributes to protective immune response against Mycobacterium tuberculosis infection

Tissue factor (TF) is a transmembrane glycoprotein that plays an essential role in hemostasis by activating coagulation. TF is also expressed by monocytes/macrophages as part of the innate immune response to infections. In the current study, we determined the role of TF expressed by myeloid cells during Mycobacterium tuberculosis (M. tb) infection by using mice lacking the TF gene in myeloid cells (TF^Δ) and human monocyte derived macrophages (MDMs). We found that during M. tb infection, a deficiency of TF in myeloid cells was associated with reduced inducible nitric oxide synthase (iNOS) expression, enhanced arginase 1 (Arg1) expression, enhanced IL‐10 production and reduced apoptosis in infected macrophages, which augmented M. tb growth. Our results demonstrate that a deficiency of TF in myeloid cells promotes M2‐like phenotype in M .tb infected macrophages. A deficiency in TF expression by myeloid cells was also associated with reduced fibrin deposition and increased matrix metalloproteases (MMP)‐2 and MMP‐9 mediated inflammation in M. tb infected lungs. Our studies demonstrate that TF expressed by myeloid cells has newly recognized abilities to polarize macrophages and to regulate M. tb growth.     

姜黄素下调PI3K/AKT/mTOR通路抑制人肝癌细胞系增殖

目的观察姜黄素对体外培养人肝癌细胞系HL-7702增殖的影响,并探讨其作用机制。方法体外培养HL-7702细胞,不同浓度的姜黄素(0、10、20、40和80μmol/L)处理48 h。PI3K/AKT抑制剂LY294002联合姜黄素处理细胞。分别用MTT法检测细胞增殖,用Western blot法检测细胞内PI3K、AKT、m TOR、Bax、Bcl-2及活化的caspase-3的蛋白含量。结果姜黄素可以浓度依赖性的抑制HL-7702细胞增殖,增加细胞中PI3K、AKT和m TOR的磷酸化水平(P0.05)。姜黄素可诱导HL-7702细胞凋亡,增加Bax和活化的caspase-3的含量(P0.05),减少Bcl-2的含量(P0.05)。LY294002与姜黄素联用后,姜黄素对细胞增殖及凋亡无明显作用。结论姜黄素可抑制人肝癌细胞系HL-7702增殖,诱导细胞凋亡,作用机制可能与其下调PIK/AKT/m TOR信号通路的活性有关。     

Prostaglandin transporter,SLCO2A1, mediates the invasion and apoptosis of lung cancer cells via PI3K/AKT/mTOR pathway

Treatment of lung cancer involves regulation of various key factors in many signaling pathways. The prostaglandin transporter, solute carrier organic anion transporter family member 2A1 (SLCO2A1), is a promising regulatory factor of cancer cells. By analyzing the invasion and apoptosis status of lung cancer cells, and detecting the expression changes of key factors in PI3K/AKT/mTOR pathway after overexpression and knockdown of SLCO2A1 in vitro, this study intended to investigate the function of SLCO2A1 in mediating lung cancer cells. Results showed overexpression of SLCO2A1 could induce the invasion of lung cancer cells, and its knockdown inhibited the invasion and induced the apoptosis of cells. mTOR, AKT and S6 in PI3K/AKT/mTOR pathway were not affected by SLCO2A1. But the expression levels of p-mTOR, p-AKT and p-S6 were up-regulated or down-regulated with the overexpression or knockdown of SLCO2A1. Thus SLCO2A1 was inferred to mediate the invasion and apoptosis of lung cancer cells via PI3K/AKT/mTOR pathway. These results implied SLCO2A1 could be a regulatory factor of the invasion and apoptosis of lung cancer cells and serve as a promising target for lung cancer therapy.     

二甲基三十六烷基铵及卡介苗多糖核酸佐剂在结核亚单位疫苗加强BCG免疫中的效应研究

目的 探讨二甲基三十六烷基铵(dimo-thylidioctyl ammonium bromide,DDA)和卡介苗多糖核酸(BCC-PSN)佐剂在结核融合蛋白疫苗加强卡介苗(BCG)免疫中的不同免疫辅助效应.方法 选择DDA、BCG-PSN作为融合蛋白AMM(Ag85B-MPT64190-198-Mtb8.4)的佐剂,在BCG初免小鼠后,AMM疫苗加强免疫两次,其中一组联合使用两种佐剂(DDA/BCG-PSN),另一组单独以DDA作为佐剂,同时设立BCG或磷酸缓冲液(PBS)免疫组为对照.应用ELISA及ELISPOT检测免疫小鼠的体液与细胞免疫反应,最后一次加强免疫后第12周,以H37Rv尾静脉攻毒并检测小鼠肺脾组织细菌载量和病理改变,评价不同佐剂疫苗的保护效果.结果 在BCG初免基础上,联合佐剂组(AMM/DDA/BCG-PSN)和单独佐剂组(AMM/DDA)加强免疫两次后,脾脏淋巴细胞经抗原Ag85B和PPD(purified protein derivative)刺激后,皆可产生分泌较BCG组高的IFN-γ.毒力株攻击后菌落形成单位(colony-forming unit,CFU)计数显示,联合佐剂组(AMM/DDA/BCG-PSN)脾脏荷菌量少于PBS组和BCG组(P<0.05);而单独佐剂组(AMM/DDA)肺部荷菌量少于PBS组和BCG组(P<0.05).组织病理分析结果 表明AMM/DDA/BCG-PSN组肺组织病理损伤较轻,而AMM/DDA组病理损伤个体差异较大.结论 DDA是较为理想的结核亚单位疫苗佐剂,能诱导较强的细胞免疫和免疫保护作用;BCG-PSN可能具有免疫调节作用,可以减轻免疫病理损伤.     

巨噬细胞的自噬及其在抗结核分枝杆菌感染中的作用

白噬是广泛存在于真核细胞内的一种溶酶体依赖性的自降解途径,可通过降解长寿蛋白和受损细胞器维持细胞内的平衡。近年来的研究发现,巨噬细胞的自噬还是固有免疫和适应性免疫的重要组成部分,可参与胞内感染病原体的清除。目前已发现有多种途径参与自噬的诱导和调节。在感染的巨噬细胞内,诱导自噬的发生能促进吞噬体和溶酶体的融合,抑制胞内结核分枝杆菌（Mtb）的存活。但同时Mtb也可通过某些机制抑制巨噬细胞自噬的发生以逃避巨噬细胞的杀伤,进而长期持留于巨噬细胞内。深入了解巨噬细胞自噬与胞内Mtb相互作用的机制,有助于人类更好的预防和控制结核病。     

Intragranulomatous necrosis in pulmonary granulomas is not related to resistance against Mycobacterium tuberculosis infection in experimental murine models induced by aerosol

Intragranulomatous necrosis is a primary feature in the natural history of human tuberculosis (TB). Unfortunately, this phenomenon is not usually seen in the experimental TB murine model. Artificial induction of this necrosis in pulmonary granulomas (INPG) may be achieved through aerosol inoculation of lipopolysaccharide (LPS) 3 weeks after Mycobacterium tuberculosis infection. At week 9 post-infection, the centre of primary granulomas became larger, showing eosinophilic necrosis. Interestingly, INPG induction was related to mice strains C57BL/6 and 129/Sv, but not to BALB/c and DBA/2. Furthermore, the same pattern was obtained with the induction of infection using a clinical M. tuberculosis strain (UTE 0335R) that naturally induces INPG. In all the mice strains tested, the study of pulmonary mRNA expression revealed a tendency to increase or to maintain the expression of RANTES, interferon-gamma, tumour necrosis factor and iNOS, in both LPS- and UTE 0335R-induced INPG, thus suggesting that this response must be necessary but not sufficient for inducing INPG. Our work supports that INPG induction is a local phenomenon unrelated to the resistant (C57BL/6 and BALB/c) or susceptible (129/Sv and DBA/2) background of mice strains against M. tuberculosis infection.     

The rs5743708 gene polymorphism in the TLR2 gene contributes to the risk of tuberculosis disease

Background: It has been reported that one single nucleotide polymorphisms (SNPs) rs5743708 in TLR2 gene might be associated with the susceptibility to tuberculosis disease. Owing to mixed and inconclusive results, we conducted a meta-analysis to systematically summarize and clarify the association between the rs5743708 gene polymorphism in the TLR2 gene and the risk of tuberculosis disease. Methods: A systematic search of studies on the association of the rs5743708 gene polymorphism in the TLR2 gene with susceptibility to tuberculosis disease was conducted in PubMed. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were used to pool the effect size. Results: A total of nineteen case-control studies from 13 articles on rs2910164 and 3 studies on the rs5743708 gene polymorphism in the TLR2 gene and the risk of tuberculosis disease were included. A significant relationship between the rs5743708 gene polymorphism in the TLR2 gene and tuberculosis disease was discovered in an allelic genetic model (OR: 2.801, 95% CI: 2.130-3.683, P=0.000), a homozygote model (OR: 5.795, 95% CI: 1.982-16.941, P=0.001), a heterozygote model (OR: 2.628, 95% CI: 1.888-3.569, P=0.000), a dominant genetic model (OR: 2.786, 95% CI: 2.003-3.877, P=0.000) and a recessive genetic model OR: (5.568, 95% CI: 1.907-16.255, P=0.002). In sub-group analysis base on ethnicity, significance was observed between the Caucasian group and the Asian group. Conclusions: The rs5743708 gene polymorphism in the TLR2 gene contributes to the risk of tuberculosis disease. Individuals with the rs5743708 gene polymorphism in the TLR2 gene are under a higher risk for tuberculosis disease.     

乌骨藤提取物通过调控PI3K/AKT/mTOR信号通路抑制黑色素瘤细胞的活力并诱导细胞凋亡

目的:探究乌骨藤提取物(Marsdenia tenacissima extract,MTE)对黑色素瘤细胞活力及凋亡的作用及机制。方法:用不同浓度(0、50、100和200 mg/L) MTE处理小鼠皮肤黑色素瘤B16-F10细胞24 h或不同浓度MTE处理不同时间(0、24、48、72和96 h),MTT法检测细胞活力。流式细胞术检测细胞凋亡情况,Western blot法检测细胞增殖、凋亡和PI3K/AKT/mTOR通路相关蛋白的表达,同时检测通路激动剂胰岛素样生长因子1(IGF-1)处理细胞后p-PI3K及细胞增殖和凋亡相关蛋白的表达。结果:根据预实验结果,选择MTE作用时间为72 h。MTE浓度为100 mg/L和200 mg/L时能明显抑制B16-F10细胞活力及增殖相关蛋白Ki67和PCNA的表达,并且还可诱导B16-F10细胞凋亡,提高凋亡相关蛋白cleaved caspase-3和cleaved caspase-9的蛋白水平;同时,MTE还可显著降低p-PI3K、p-AKT和mTOR的蛋白水平;此外,激动剂IGF-1可明显减弱MTE抑制细胞活力及诱导细胞凋亡的作用。结论:MTE可通过下调PI3K/AKT/mTOR通路活性抑制黑色素瘤细胞活力,诱导细胞凋亡。     

结核杆菌抗原激活MEK-ERK信号通路延迟中性粒细胞自发性凋亡的作用

探讨结核杆菌抗原(Mtb-Ag)对中性粒细胞自发性凋亡的影响。取健康成人外周血分离中性粒细胞,加Mtb-Ag培养24 h,或用Mek抑制剂PD98059预处理30 min,Annexin V染色流式细胞术观察细胞凋亡;并用Western blot印迹法检测Mek活化情况。与中性粒细胞体外培养24 h后自发凋亡(55%±6%)相比,加入Mtb-Ag(1.125 mg/ml)后,中性粒细胞凋亡率(31%±3%)明显降低;并且Mtb-Ag可诱导Mek的激活;而PD98059(50μmol/L)预处理可阻断Mtb-Ag的抑制凋亡作用。Mtb-Ag对中性粒细胞自发凋亡有抑制作用,这一作用涉及Mek-Erk途径。     

刺激人γδT细胞增殖的结核杆菌多肽抗原的生物学特性分析

目的 :分析可刺激人γδT细胞增殖的结核杆菌多肽抗原(Mtb Ag)的某些生物学特性。方法 :以结核杆菌分泌蛋白与经透析或链霉蛋白酶处理的Mtb Ag体外分别刺激人外周血单个核细胞 (PBMC)增殖 ,培养 10d时 ,采用流式细胞仪分析细胞的表型。结果 :透析后的Mtb Ag刺激γδT细胞增殖的活性未见明显降低 ;而经链霉蛋白酶处理的Mtb Ag促进γδT细胞增殖的活性则显著下降。另外 ,与结核杆菌分泌蛋白相比较 ,Mtb Ag刺激γδT细胞增殖的活性显著增强。 结论 :Mtb Ag中可刺激人γδT细胞增殖的成分是一种相对分子质量 (Mr)大于 10 0 0 0、非分泌性、对蛋白水解酶敏感的多肽抗原     

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.     

人γδT细胞TCR分子与结核杆菌多肽抗原特异性结合的证据

目的探讨γδT细胞对结核杆菌抗原(Mtb—Ag)识别的分子机制。方法采集人外周血(PB)，分别经抗TCRγδ-PE和FITC标记的Mtb—Ag及其纯化的不同组分，结核杆菌高分子量蛋白组分(Mtb—HW-Ag)、低分子量多肽组分(Mtb—LW-Ag)或Mtb-Ag峰3单一主肽抗原(Mtb-Ag-Pk3)进行双标记染色，或使用过量Mtb—AS先与细胞预处理，然后再做上述染色，用流式细胞仪检测γδT细胞上结合的抗TCRγδ-PE荧光强度变化。将人外周血PBMC经Mtb—Ag刺激24h后，使用抗TCRγδ-PE和Mtb—Ag-FITC染色，用激光共聚焦显微镜观察γδT细胞表面TCRγδ分子的聚集。结果经流式细胞仪分析，Mtb—Ag及其纯化的Mtb—LW-Ag、Mtb-Ag-Pk3均可与γδT细胞发生特异性直接结合，而且Mtb—Ag可有效阻断抗TCRγδ单抗与γδT细胞的结合。而Mtb—HW-Ag与γδT细胞并无特异性结合效应。经激光共聚焦显微镜观察，Mtb—Ag可显著激活γδT细胞而诱导TCRγδ分子发生功能性聚集化。结论γδT细胞通过其表面的TCRγδ分子可直接结合Mtb—Ag，而Mtb-Ag-Pk3多肽可能是Mtb—Ag发挥其结合效应的有效成分。Mtb—Ag可激活γδT细胞并诱导TCRγδ分子聚集，而可能参与功能性脂筏(raft)的形成。     

ARL4C stabilized by AKT/mTOR pathway promotes the invasion of PTEN-deficient primary human glioblastoma

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) deficiency in primary human glioblastoma (GBM) is associated with increased invasiveness and poor prognosis with unknown mechanisms. Therefore, how loss of PTEN promotes GBM progression remains to be elucidated. Herein, we identified that ADP-ribosylation factor like-4C (ARL4C) was highly expressed in PTEN-deficient human GBM cells and tissues. Mechanistically, loss of PTEN stabilized ARL4C protein due to AKT/mTOR pathway-mediated inhibition of ARL4C ubiquitination. Functionally, ARL4C enhanced the progression of GBM cells in vitro and in vivo. Moreover, microarray profiling and GST pull-down assay identified that ARL4C accelerated tumor progression via RAC1-mediated filopodium formation. Importantly, targeting PTEN potently inhibited GBM tumor progression in vitro and in vivo, whereas overexpression of ARL4C reversed the tumor progression impaired by PTEN overexpression. Clinically, analyses with patients' specimens validated a negative correlation between PTEN and ARL4C expression. Elevated ARL4C expression but PTEN deficiency in tumor was associated with poorer disease-free survival and overall survival of GBM patients. Taken together, ARL4C is critical for PTEN-deficient GBM progression and acts as a novel prognostic biomarker and a potential therapeutic candidate. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

结核杆菌Ag85A/GM-CSF嵌合表达质粒的构建及表达

目的：探讨对结核病DNA疫苗进行基因修饰的新方法。方法：以结核杆菌H37Rv株基因组DNA为模板，通过PCR扩增获得结核杆菌Ag85A抗原蛋白基因的成熟肽编码区，以经PHA诱导后的小鼠脾组织总RNA为模板，通过RT－PCR获得小鼠粒细胞－巨噬细胞集落刺激因子的cDNA，将二者定向克隆入质粒pBK-CMV中构建含嵌合目的基因的重组质粒，转染培养的COS7细胞后检测嵌合目的基因的表达。结果：成功构建了小鼠粒细胞－巨噬细胞集落刺激因子cDNA与结核杆菌Ag85A抗原基因的真核嵌合表达质粒，重组质粒能在COS7细胞中进行稳定表达。结论：本研究首次将细胞因子基因与结核杆菌免疫保护性抗原基因嵌合构建嵌合DNA疫苗，为进一步研究其在动物体内的免疫原性和免疫保护性奠定了基础。     

TLR2 engagement on CD4+ T cells enhances effector functions and protective responses to Mycobacterium tuberculosis

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4⁺ T cells and upregulate TCR‐triggered IFN‐γ secretion and cell proliferation in vitro. Here we examined the role of CD4⁺ T‐cell‐expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag‐specific T‐cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4⁺ T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1‐like response was observed in the context of both polyclonal and Ag‐specific TCR stimulation. To evaluate the role of T‐cell TLR2 in priming of CD4⁺ T cells in vivo, naive MTB Ag85B‐specific TCR transgenic CD4⁺ T cells (P25 TCR‐Tg) were adoptively transferred into Tlr2^?/? recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam₃Cys‐SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN‐γ‐secreting P25 TCR‐Tg T cells 1 week after immunization. P25 TCR‐Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4⁺ T cells increases MTB Ag‐specific responses and may contribute to protection against MTB infection.     

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.