蜡螟抗白假丝酵母菌自噬机制的初步研究

目的探究白假丝酵母菌感染蜡螟时蜡螟的自噬相关通路蛋白的表达情况。方法用一定量的白假丝酵母菌的活化孢子感染蜡螟,经过12h,解剖蜡螟收集蜡螟细胞并裂解细胞,用真菌活性检测试剂盒检测孢子活性;解剖蜡螟取肠道组织并用PI染死细胞。取不同感染时间段的淋巴细胞,用裂解液裂解,离心取上清,用Western blot法检测上清液中的Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平。结果活化的孢子注射至蜡螟体内后,其活性受到抑制;蜡螟的肠道细胞被定位在上面的菌丝损伤并且孢子活性受到抑制。随着蜡螟感染白假丝酵母菌时间的递增,其自身的Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平在不断增高且在感染24h最高。结论白假丝酵母菌感染蜡螟后,蜡螟的淋巴细胞和肠道细胞通过升高Dectin-1、ROS、LC3Ⅰ/Ⅱ的表达水平发挥杀伤孢子的作用。     

阴道白假丝酵母菌水解酶的研究进展

外阴阴道白假丝酵母菌病是常见的妇产科感染性疾病,对其致病微生物白假丝酵母菌的研究方向已从大范围的筛查病因过渡到小范围的深入研究,本文系统综述了近年来对白假丝酵母菌水解酶的研究发现和前沿进展。     

白假丝酵母菌耐药与抗氰呼吸的相关性研究

目的探讨白假丝酵母菌的耐药情况及其与抗氰呼吸的相关性。方法用真菌药敏测定试剂盒测定从临床分离出来的37株白假丝酵母菌的耐药性,并从中选出5株耐药菌和5株敏感菌进行抗氰呼吸的研究。结果白假丝酵母菌对益康唑的耐药率最高,达54.1%,耐药白假丝酵母菌的抗氰呼吸速率均值为(17.56±6.75)nmol/(min.A620),敏感白假丝酵母菌的抗氰呼吸速率均值为(7.99±5.80)nmol/(min.A620),耐药白假丝酵母菌的抗氰呼吸速率明显升高,且耐药菌株抗氰呼吸速率占总呼吸的比例明显高于敏感菌株(P0.05),差异具有显著性。结论兰州市区白假丝酵母菌对益康唑耐药性较高,且白假丝酵母菌的耐药与抗氰呼吸途径相关。     

呼吸道感染中白假丝酵母菌的致病性与细胞外水解酶关系

将84株呼吸道分离的白假丝酵母菌分为致病组和非致病组,采用卵黄培养基法检测细胞外磷脂酶的活力,用牛血清白蛋白琼脂培养基法检法分泌型天冬氨酸蛋白酶的活力,并用RT-PCR的方法检测这2种酶相关基因PLB1和SAP2的表达情况,分析其组间差别,结果表明致病组菌株的分泌型天冬氨酸蛋白酶的活力要高于非致病组(P=0.034<0.05),细胞外磷脂酶活力二组未见差别,致病组的PLB1和SAP2基因的表达均高于非致病组(P<0.05),PLB1和SAP2的表达存在着明显的正相关(r=0.776,P<0.01),提示分泌型天冬氨酸蛋白酶是呼吸道白假丝酵母菌重要的毒力因子,PLB1和SAP2的表达上调可能影响菌株的毒力,这2个基因的表达量有正相关的关系。     

克柔假丝酵母菌通过Dectin-1/Syk信号通路活化巨噬细胞自噬

目的探究Dectin-1/Syk信号通路在克柔假丝酵母菌激活RAW264.7细胞自噬中的作用。方法以特异性抗体封闭RAW264.7细胞表面TLR-2、TLR-4及Dectin-1受体,免疫蛋白印记检测克柔假丝酵母菌刺激后LC3II的表达量;通过白皮杉醇及Raf-1抑制剂分别阻断RAW264.7细胞Syk及Raf-1磷酸化,观察对克柔假丝酵母菌激活细胞自噬的影响;采用SiMi Transfection Reagents转染Atg5siRNA,检测不同时间段RAW264.7细胞对克柔假丝酵母菌的杀菌率。结果封闭细胞膜Dectin-1、阻断Syk磷酸化显著抑制克柔假丝酵母菌诱导RAW264.7细胞LC3II的表达,而封闭细胞膜TLR-2或TLR-4,以及阻断Raf-1磷酸化对于克柔假丝酵母菌刺激下LC3II的表达无显著影响。敲低Atg5后RAW264.7细胞在感染6h后对克柔假丝酵母菌的杀菌率显著降低。结论 Dectin-1/Syk信号通路介导了克柔假丝酵母菌激活RAW264.7细胞自噬,并且自噬功能参与了该细胞对克柔假丝酵母菌的杀灭作用。     

氟康唑体外诱导白假丝酵母菌耐药基因表达的研究

目的 对白假丝酵母菌耐药机制进行研究.方法 将临床分离对氟康唑敏感的白假丝酵母菌种,经体外诱导产生耐药.半定量PCR检测敏感株、耐药株、回复敏感株多药耐药基因CDR1、CDR2、MDR1和转录调控因子TAC1编码基因表达水平的变化,并对TAC1编码基因进行测序.结果 与敏感株和回复敏感株比较,耐药株CDR1、CDR2相对表达量增高,发现1株TAC1 N977D氨基酸置换.结论 氟康唑体外诱导白假丝酵母菌产生耐药的机制与CDR1、CDR2表达相关.TAC1基因突变在诱导耐药中的机制有待进一步研究.     

白假丝酵母菌生物膜的研究进展

随着医疗水平的不断发展,越来越多的医疗操作、医疗设备和药物可能导致人体正常的微生物平衡被打破,使得机会致病菌白假丝酵母菌的感染呈现逐年上升的趋势。白假丝酵母菌在宿主或医疗器械表面形成生物膜的能力是一个十分关键的毒力因素。生物膜可以帮助白假丝酵母菌成功逃避宿主免疫并产生较强的耐药性,从而导致难治性真菌感染。本文从白假丝酵母菌生物膜的形成过程、生物膜相关的主要基因和影响生物膜毒力的因素3个方面介绍近年来的研究进展,为进一步研究白假丝酵母菌生物膜的形成机制提供参考。     

白假丝酵母激活的巨噬细胞自噬促进MHCⅡ抗原提呈

目的 探究白假丝酵母（Candida albicans）激活的Raw264.7细胞自噬在主要组织相容性复合体Ⅱ类分子（MHCⅡ）抗原提呈以及协同刺激分子表达中的作用。方法 C. albicans刺激Dectin-1单克隆抗体封闭或白皮杉醇阻断的Raw264.7细胞,Western blot法检测LC3Ⅱ表达量,RT-PCR检测CD80与CD86的表达。免疫荧光实验观察有无3-MA预处理的GFP-LC3-Raw264.7细胞与C. albicans共孵育后MHCⅡ与LC3在胞浆的分布,ELISA法检测有无封闭Dectin-1或3-MA预处理的Raw264.7细胞与C. albicans共孵育不同时间段后IL-6的分泌。结果 阻断Dectin-1或Sky后C. albicans诱导的LC3Ⅱ表达降低。LC3、MHCⅡ与胞内C. albicans存在显著的共定位关系,阻断自噬后C. albicans与MHCⅡ的共定位明显减弱。C. albicans引发Raw264.7细胞表达CD80与CD86 mRNA,封闭Dectin-1或阻断自噬后二者转录水平降低。C. albicans通过Dectin-1引发Raw264.7细胞分泌IL-6,阻断自噬对IL-6分泌无显著影响。结论 C. albicans通过Dectin-1/Sky通路激活巨噬细胞自噬,自噬体的构建促进MHCⅡ招募至胞内C. albicans,并促进协同刺激分子的表达。     

白假丝酵母菌体外生物膜药敏性不同检测方法的比较

目的比较不同的方法对白假丝酵母菌体外生物膜药敏性检测的差异。方法分别采用菌落计数法(CFU)、AlamarBlue试剂法、XTT减低法、MTT法对白假丝酵母菌体外48h成熟生物膜的药敏性进行检测,并将AlamarBlue试剂法、XTT减低法、MTT法与CFU法进行比较,观察其相关性及差异性。结果 AlamarBlue试剂法、XTT减低法、MTT法与CFU法都有较高的相关性,相关系数分别为r=0.969、r=0.971、r=0.982(P0.01);与CFU法的差异分别为(0.093±0.127)、(0.054±0.113)、(0.013±0.066),其差异无统计学意义(P0.05)。结论 AlamarBlue试剂法、XTT减低法、MTT法均可替代CFU法对白假丝酵母菌生物膜进行药敏检测。MTT法与CFU法的相关性最大,差异性最小;AlamarBlue试剂作为一种新的试剂,操作简单,对细胞及人类无毒害,更加适合高通量检测。     

白假丝酵母菌滞留菌形成相关机制的研究进展

白假丝酵母菌属于临床侵袭性条件致病菌,可引起从浅表黏膜到危及生命的全身感染性疾病,其形成的生物膜是为生物膜内细胞提供结构支架和保护模式的结构化菌体群落,其产生的滞留菌是一种随机且对抗真菌药物高度耐受的细胞亚群。由于生物膜与滞留菌两种因素的存在,致使临床治疗侵袭性真菌感染时面临极大挑战,因此,研究白假丝酵母菌滞留菌形成机制对目前临床治疗侵袭性真菌感染具有重要意义。本文对白假丝酵母滞留菌形成的主要调控因素(生物膜的形成、氧化应激反应、蛋白酶系统、TOR-RAS-CAMP-PKA信号转导途径和菌种自身因素),以及滞留菌与临床疾病的相关性进行综述。     

Resveratrol prevents RANKL-induced osteoclast differentiation of murine osteoclast progenitor RAW 264.7 cells through inhibition of ROS production

The bone protective effects of resveratrol have been demonstrated in several osteoporosis models while the underlying mechanism is largely unclear. In the present study, we evaluated the effects of resveratrol on differentiation and apoptosis of murine osteoclast progenitor RAW 264.7 cells. We found that resveratrol at non-toxic concentrations dose-dependently inhibited RANKL-induced osteoclast differentiation and induced apoptosis. Resveratrol has been shown to be an activator of Sirt1, a NAD⁺ dependent protein deacetylase, and has been demonstrated to mimic estrogen. However, we found that although Sirt1 protein was abundantly expressed in RAW264.7 cells, the specific Sirt1 inhibitor EX-527 could not attenuate the inhibition of osteoclastogenesis mediated by resveratrol. Also, the effects of resveratrol could not be attenuated by ICI-182780, a high affinity estrogen receptor antagonist. The central role of reactive oxygen species (ROS) in RANKL-induced osteoclast differentiation has recently been clarified. We found that resveratrol suppressed RANKL-induced ROS generation in a concentration dependent manner. We postulate that the direct inhibitory effects of resveratrol on osteoclastogenesis are mediated via inhibition of ROS generation.     

New insights into an old story: Pollen ROS also play a role in hay fever

Reactive oxygen species (ROS) can exhibit negative and benign traits. In plants, ROS levels increase markedly during periods of environmental stress, and defense against pathogen attack. ROS form naturally as a by-product of normal oxygen metabolism, and evenly play an essential role in cell growth. The short ROS lifespan makes them ideal molecules to act in cell signaling, a role they share in both plants and animals. A particular plant organism, the pollen grain, may closely interact with human mucosa and an allergic inflammatory response often results. Pollen grain ROS represent a first, crucial signal which primes and magnifies a cascade of events in the allergic response.     

Down-regulation of neprilysin (EC3.4.24.11) expression in vascular endothelial cells by laminar shear stress involves NADPH oxidase-dependent ROS production

Neprilysin (NEP, neutral endopeptidase, EC3.4.24.11), a zinc metallopeptidase expressed on the surface of endothelial cells, influences vascular homeostasis primarily through regulated inactivation of natriuretic peptides and bradykinin. Earlier in vivo studies reporting on the anti-atherosclerotic effects of NEP inhibition and on the atheroprotective effects of flow-associated laminar shear stress (LSS) have lead us to hypothesize that the latter hemodynamic stimulus may serve to down-regulate NEP levels within the vascular endothelium. To address this hypothesis, we have undertaken an investigation of the effects of LSS on NEP expression in vitro in bovine aortic endothelial cells (BAECs), coupled with an examination of the signalling mechanism putatively mediating these effects. BAECs were exposed to physiological levels of LSS (10 dynes/cm², 24 h) and harvested for analysis of NEP expression using real-time PCR, Western blotting, and immunocytochemistry. Relative to unsheared controls, NEP mRNA and protein were substantially down-regulated by LSS (≥50%), events which could be prevented by treatment of BAECs with either N-acetylcysteine, superoxide dismutase, or catalase, implicating reactive oxygen species (ROS) involvement. Employing pharmacological and molecular inhibition strategies, the signal transduction pathway mediating shear-dependent NEP suppression was also examined, and roles implicated for Gβγ, Rac1, and NADPH oxidase activation in these events. Treatment of static BAECs with angiotensin-II, a potent stimulus for NADPH oxidase activation, mimicked the suppressive effects of shear on NEP expression, further supporting a role for NADPH oxidase-dependent ROS production. Interestingly, inhibition of receptor tyrosine kinase signalling had no effect. In conclusion, we confirm for the first time that NEP expression is down-regulated in vascular endothelial cells by physiological laminar shear, possibly via a mechanotransduction mechanism involving NADPH oxidase-induced ROS production.     

Adherence of Candida albicans to host cells

Abstract Research devoted to uncovering the mechanisms of adherence of Candida albicans to human tissue is reviewed. The physical aspects of adherence of the fungus to host cells and the biochemical and molecular features, as far as they are known, are discussed. Relevant pre- and post-adherence events in the pathogenesis of disease caused by this fungus are also noted. Putative adhesins and surface receptors of C. albicans for host proteins are discussed in detail.     

Apoptosis of phagocytic cells induced by Candida albicans and production of IL-10

Macrophages co-incubated with Candida albicans strain CR1 in vitro showed early signs of apoptosis, but evolved to necrosis after 2 h. In this study, we investigated whether strain CR1 caused apoptosis or necrosis of macrophages after its inoculation into mice peritoneal cavity, and whether this correlated with the secretion of IL-10. Peritoneal macrophages from mice that received an inoculum of C. albicans CR1 showed signs of apoptosis and necrosis from 30 min to 2 h afterwards, whereas heat-killed C. albicans did not cause those effects. IL-10 production was low during the first 6 h post-infection, when macrophages predominated in the peritoneal exudate, whereas its higher production after 24 h correlated with an increase of neutrophils in the exudate. Treatment of CR1 with pepstatin (an inhibitor of proteinases) prevented the process of apoptosis and significantly reduced IL-10 production, suggesting that the increased production of IL-10 was caused by processes occurring during the initial phase of infection, such as apoptosis, necrosis and uptake of death cells.     

Azole sensitivity in Candida albicans

Abstract In the present study, we assessed the influence of three culture media on the susceptibility 'in vitro' of twenty four clinical strains belonging to Candida albicans against three imidazole-derivatives and also, we investigated the situation of azole sensitivity in three of these strains.     

The enhancing action of D-galactosamine on lipopolysaccharide-induced nitric oxide production in RAW 264.7 macrophage cells

The effect of D-galactosamine (D-GalN) on nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophage cells was examined. D-GalN augmented the production of NO, but not tumor necrosis factor (TNF)-alpha in LPS-stimulated RAW 264.7 cells. Pretreatment of D-GalN augmented the NO production whereas its post-treatment did not. D-GalN augmented the NO production in RAW 264.7 cells stimulated with either TNF-alpha and interferon-gamma. The augmentation of LPS-induced NO production by D-GalN was due to enhanced expressions of an inducible type of NO synthase mRNA and proteins. Intracellular reactive oxygen species (ROS) were exclusively generated in RAW 264.7 cells stimulated with D-GalN and LPS. Scavenging of intracellular ROS abrogated the augmentation of NO production. It was therefore suggested that D-GalN might augment LPS-induced NO production through the generation of intracellular ROS.     

紫外线对白念珠菌的影响

紫外线作为重要的环境因子之一,能显著影响包括白念珠菌在内的多种生物的生长及生理过程.研究发现白念珠菌受紫外线照射后菌丝形成被抑制,孢子形成增多且没有向光性;脉冲紫外线辐射可通过多靶点程序使白念珠菌失活;rad 51缺陷株比rad 52缺陷株更易受紫外线损害,同时紫外线可导致白念珠菌杂合性丢失.研究还发现UVC治疗可明显减少烧伤后真菌微生物感染,核黄素/UVA治疗可明显抑制白念珠菌生长.因此紫外线对白念珠菌有一定的抑制作用.