壳聚糖微球分散体系的抑菌性研究

以乳化交联法制备的壳聚糖微米微球(CMs)为试验材料,研究其对金黄色葡萄球菌和大肠杆菌的抑菌行为,并对其浓度因素、粒径因素和抑菌机理做了探讨。结果表明CMs的抑菌作用随其浓度的增加和粒径的减小而增强。在浓度<0.7 mg/mL时,CMs对细菌的生长具有先抑制后促进的作用;当浓度≥0.7 mg/mL时对细菌有杀灭作用。CMs对两种细菌的最小抑菌浓度和最小杀菌浓度分别为0.8、1.4 mg/mL和1.0、1.8 mg/mL,表明壳聚糖微球对金黄色葡萄球菌和大肠杆菌均具有明显的抑制作用,且金黄色葡萄球菌对微球抑制作用的反应比大肠杆菌更敏感。扫描电子显微镜观察发现,细菌能迅速贴附在CMs表面,并且贴壁后的细菌发生了明显的形态变化,有的甚至完全被破坏。     

剪切力对脑微血管内皮细胞骨架蛋白的影响

利用自行研制的细胞流动小室对大鼠脑微血管内皮细胞在剪切力作用下细胞骨架蛋白的结构改变进行了初步研究,结果提示脑微血管内皮细胞在剪切力作用下,细胞形态学发生明显改变,细胞间隙增大、皱缩、脱落,细胞骨架蛋白的结构也有类似的变化,骨架蛋白沿流动方向重新排列,微丝中Ｆ－Ａｃｔｉｎ的数量增加、变粗。这些改变的直接后果是内皮细胞通透性的增加。该工作为进一步开展剪切力对微血管内皮细胞功能、代谢等方面的影响提供了实验数据     

连翘苷和黄芩苷对表皮葡萄球菌生物膜抑制作用的研究

目的通过中药有效成分连翘苷和黄芩苷分别对表皮葡萄球菌生物膜抑制作用的研究,为表皮葡萄球菌生物膜引起的相关感染提供新的治疗途径。方法体外构建表皮葡萄球菌生物膜,XTT减低法评价连翘苷、黄芩苷对表皮葡萄球菌初始黏附及生物膜内细菌代谢的影响,显微镜下观察用药后表皮葡萄球菌生物膜形态和结构改变。结果连翘苷和黄芩苷对表皮葡萄球菌生物膜的早期黏附均无抑制作用;连翘苷对表皮葡萄球菌生物膜菌的SMIC50为31.25μg/ml,而黄芩苷对表皮葡萄球菌生物膜菌的代谢无影响;在显微镜下观察,连翘苷使部分表皮葡萄球菌被膜的形态发生改变,而黄芩苷对其形态影响不显著。结论连翘苷对表皮葡萄球菌生物膜的初始黏附阶段无抑制作用,对生物膜菌的代谢和生物膜形态均有显著影响;黄芩苷对表皮葡萄球菌生物膜无显著作用。     

金黄色葡萄球菌sdhCAB操纵子影响持留菌形成机制的研究

持留菌是细菌群体中的一小部分细菌,可耐受致死浓度抗生素的处理,是引起慢性感染的重要原因。金黄色葡萄球菌(Staphylococcus aureus,S. aureus)作为常见致病菌,有重要临床意义。分别敲除sdhA和sdhB后,金黄色葡萄球菌持留菌形成水平下降,但sdhCAB操纵子对持留菌形成的作用及机制尚不明确。本研究敲除sdh操纵子,通过酸压力、氧化压力、热压力及抗生素压力实验检测敲除株的持留菌水平,转录组测序检测敲除株的代谢通路变化,高通量微生物细胞表型检测评估敲除株的代谢水平变化。结果显示,敲除sdhCAB或sdhAB后,金黄色葡萄球菌对酸压力、氧化压力的耐受能力均下降;而在抗生素压力、热压力条件下,分别仅sdhCAB敲除株、sdhAB敲除株耐受能力下降。转录组测序发现,sdhCAB敲除后三羧酸循环、甲烷代谢通路及聚合酶Ⅳ等基因表达上调,耐药相关基因、氨基酸代谢基因、糖类代谢基因、卟啉代谢基因及一些转运体基因等表达下调,提示这些通路的基因参与sdhCAB影响持留菌形成的过程。此外,高通量微生物细胞表型检测发现,敲除sdhCAB可降低金黄色葡萄球菌对琥珀酸、柠檬酸、糖原、L-天冬氨酸等64种碳源的代谢。结果提示,sdhCAB操纵子对金黄色葡萄球菌持留菌形成水平有重要影响。本研究初步阐明了sdhCAB操纵子影响持留菌形成的可能机制,为研究和治疗金黄色葡萄球菌慢性感染提供了新的思路。     

家蝇幼虫抗菌肽MDL-2对细菌细胞渗透性及代谢功能影响

研究了家蝇幼虫抗菌肽MDL-2与细菌相互作用时,抗菌肤MDL-2对细菌细胞壁的溶解作用、细胞膜渗透性和代谢的影响.抗菌肽MDL-2在抗菌过程中首先与细菌的细胞壁相互作用,使其破裂,抗菌肽对革兰氏阴性细菌大肠杆菌细胞壁的作用有浓度依赖性,而对革兰氏阳性细菌金黄色葡萄球菌MDL-2在较低的浓度时即可发生细胞壁破坏作用;抗菌...     

柠檬提取物抑菌、杀灭病毒作用机制研究

目的观察柠檬提取物对细菌生物膜的消除作用,对柠檬提取物抑制多重耐药金黄色葡萄球菌的机制进行初步研究,以及对环境中富集的病毒消除作用的研究。方法采用高分子滤膜法制备金黄色葡萄球菌生物膜后,加入柠檬提取物分别作用1、2和3h,通过细菌菌落计数判定不同生物膜的细菌存活数;采用SDS—PAGE电泳法,观察在柠檬提取物作用下菌体蛋白质的变化;EcoRⅠ酶切金黄色葡萄球菌DNA,观察酶切图谱的改变;在低渗环境下动态观察柠檬提取物在不同作用时间对细菌细胞壁的影响;通过柠檬提取物对病毒作用后RNA量的变化,说明柠檬提取物具有杀灭病毒的作用。结果柠檬提取物对生物膜中的细菌具有杀菌作用,且随作用时间的延长而逐渐增强;柠檬提取物对细菌蛋白质的组成和表达量均有一定影响,同时细菌DNA的EcoRⅠ酶切图谱发生改变;柠檬提取物作用15min时细菌呈现明显的胞壁缺损现象,即细菌个体胀大,呈现大球形;经柠檬提取物作用后,RNA病毒被杀灭。结论结果显示柠檬提取物对耐药金黄色葡萄球菌所形成的生物膜具有明显抑制作用;在柠檬提取物作用下,菌体蛋白合成量发生改变,同时对细菌的DNA和细胞壁有明显的影响。柠檬提取物处理病毒悬液后,病毒RNA量显著减少,说明病毒RNA结构被破坏。     

微酸性电解水对人员手部和即食蔬菜消毒效果的研究

本实验研究了微酸性电解水对大肠杆菌、金黄色葡萄球菌菌悬液的杀灭作用,以及微酸性电解水对人员手部冲洗及黄瓜和生菜浸泡法的消毒效果.结果表明,采用有效氯浓度为80 mg/L的微酸性电解水对含有0.3％有机干扰物的大肠杆菌菌悬液,作用1 min,杀灭对数值大于6;对含有0.3％有机干扰物BSA的金黄色葡萄球菌菌悬液,作用2 ...     

大黄素对金黄色葡萄球菌的抑菌作用机制

以金黄色葡萄球菌为供试菌,通过测定大黄素对其细胞膜的通透性、可溶性蛋白质和呼吸代谢的影响,来阐述大黄素的抑菌作用机制. 利用电导率、生物大分子分析、呼吸代谢抑制检测等方法,验证大黄素的药效作用. 实验结果显示,大黄素作用金黄色葡萄球菌后,培养基溶液中电导率比对照组增加了2.23%,DNA和RNA大分子的含量比对照组增加了67.36%,大黄素作用金黄色葡萄球菌16 h后,菌体可溶性蛋白总量比对照组减少了28.3%;大黄素能抑制金黄色葡萄球菌物质代谢中的2种关键酶的活性,其中琥珀酸脱氢酶活性抑制率为53.8%,苹果酸脱氢酶的活性抑制率为25.5%.上述结果表明,大黄素可以破坏细菌细胞膜的通透性,抑制菌体内的蛋白质合成,通过抑制代谢关键酶的活性发挥杀菌作用.     

5-氯水杨醛缩2,4-二羟基苯酰肼的抗菌抗氧化活性研究

目的考察5-氯水杨醛缩2,4-二羟基苯酰肼对金黄色葡萄球菌的抗菌活性及体外抗氧化活性。方法采用试管法和平板法测定其对金黄色葡萄球菌最小抑菌浓度(MIC)、最小杀菌浓度(MBC),绘制杀菌曲线,并通过扫描电镜研究其作用机制,采用DPPH法测定抗氧化活性。结果该化合物对金黄色葡萄球菌的MIC为2.5 mg/m L,MBC为5 mg/m L。杀菌曲线结果表明该化合物对金黄色葡萄球菌的杀菌作用表现出明显的浓度依赖性。扫描电镜结果表明该化合物的作用机制可能与破坏菌体细胞壁、改变其通透性有关。同时该化合物具有较强的清除自由基的能力。结论 5-氯水杨醛缩2,4-二羟基苯酰肼具有显著的抗金黄色葡萄球菌作用,同时具有较好的体外抗氧化活性。     

稀土离子(Nd3+)对金黄色葡萄球菌细胞壁结构的影响

本文的研究目的是探索稀土离子Nd^3＋对金黄色葡萄球菌细胞壁结构的影响作用。采用透射电镜、氨基酸自动分析仪和红外光普法等检测手段,提出了Nd^3＋可使金黄色葡萄球菌细胞壁的形态和结构发生改变;低于抑菌浓度的NdCl3对金黄色葡萄球菌细胞壁的古成具有促进作用：高浓度（指杀菌浓度和抑菌浓度）Nd^3＋可断裂细胞壁中肽聚糖的大部分肽键和氢键,致使细胞壁中肽聚糖的交联网状结构被破坏。     

Survival of Staphylococcus aureus inside neutrophils contributes to infection

Neutrophils have long been regarded as essential for host defense against Staphylococcus aureus infection. However, survival of the pathogen inside various cells, including phagocytes, has been proposed as a mechanism for persistence of this microorganism in certain infections. Therefore, we investigated whether survival of the pathogen inside polymorphonuclear neutrophils (PMN) contributes to the pathogenesis of S. aureus infection. Our data demonstrate that PMN isolated from the site of infection contain viable intracellular organisms and that these infected PMN are sufficient to establish infection in a naive animal. In addition, we show that limiting, but not ablating, PMN migration into the site of infection enhances host defense and that repletion of PMN, as well as promoting PMN influx by CXC chemokine administration, leads to decreased survival of the mice and an increased bacterial burden. Moreover, a global regulator mutant of S. aureus (sar-) that lacks the expression of several virulence factors is less able to survive and/or avoid clearance in the presence of PMN. These data suggest that the ability of S. aureus to exploit the inflammatory response of the host by surviving inside PMN is a virulence mechanism for this pathogen and that modulation of the inflammatory response is sufficient to significantly alter morbidity and mortality induced by S. aureus infection.     

Synergy between extracellular group IIA phospholipase A2 and phagocyte NADPH oxidase in digestion of phospholipids of Staphylococcus aureus ingested by human neutrophils

Acute inflammatory responses to invading bacteria such as Staphylococcus aureus include mobilization of polymorphonuclear leukocytes (PMN) and extracellular group IIA phospholipase A2 (gIIA-PLA2). Although accumulating coincidentally, the in vitro anti-staphylococcal activities of PMN and gIIA-PLA2 have thus far been studied separately. We now show that degradation of S. aureus phospholipids during and after phagocytosis by human PMN requires the presence of extracellular gIIA-PLA2. The concentration of extracellular gIIA-PLA2 required to produce bacterial digestion was reduced 10-fold by PMN. The effects of added gIIA-PLA2 were greater when present before phagocytosis but even apparent when added after S. aureus were ingested by PMN. Related group V and X PLA2, which are present within PMN granules, do not contribute to bacterial phospholipid degradation during and after phagocytosis even when added at concentrations 30-fold higher than that needed for action of the gIIA-PLA2. The action of added gIIA-PLA2 required catalytically active gIIA-PLA2 and, in PMN, a functional NADPH oxidase but not myeloperoxidase. These findings reveal a novel collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.     

细菌L型对化学因素敏感性研究

本文研究了伤寒沙门氏菌、葡萄球菌及白喉棒状杆菌L型对化学消毒剂、细菌生长抑制剂、酸和碱的敏感性,并与其亲代细菌型作了比较。结果表明:细菌L型对表面活性剂、酚剂、重金属盐、脱水剂、卤素及氧化剂的敏感性明显(P<0.05)或不明显(P>0.05)增加,伤寒沙门氏菌L型对龙胆紫、玫瑰色酸、胆盐及酸的敏感性增高,葡萄球菌及白喉棒状杆菌L型对龙胆紫、玫瑰色酸、胆盐及酸的敏感性降低,伤寒沙门氏菌及白喉棒状杆菌L型对碱敏感性降低。     

4-PA对BEL-7402细胞的生长调控和诱导凋亡作用的研究

应用ＭＴＴ、流式细胞术研究了新型肿瘤诱导分化剂４－ＰＡ对人肝癌细胞系ＢＥＬ－７４０２细胞的生长调控、诱导调亡作用。结果表明：４－ＰＡ对ＢＥＬ－７４０２细胞增殖抑制的ＩＣ５０为４－９ｍｍｏｌ／Ｌ,随着作用时间的增加,ＩＣ５０增加。作用效果迅速,在０．５小时就有效地抑制了细胞增殖,抑制率随４－ＰＡ浓度的增加和作用时间的处长而增加,在达到最大抑制率后出现饱和。流式细胞仪对细胞周期和细胞调亡的分析发现：４     

4-PA对BEL-7402细胞的生长调控和诱导凋亡作用的研究

应用ＭＴＴ、流式细胞术研究了新型肿瘤诱导分化剂４－ ＰＡ 对人肝癌细胞系ＢＥＬ－ ７４０２ 细胞的生长调控、诱导凋亡作用。结果表明： ４ － ＰＡ 对ＢＥＬ－ ７４０２ 细胞增殖抑制的ＩＣ５０ 为４ －９ｍ ｍｏｌ／Ｌ,随着作用时间的增加,ＩＣ５０ 增加。作用效果迅速,在０ ．５ 小时就有效地抑制了细胞增殖,抑制率随４－ ＰＡ 浓度的增加和作用时间的延长而增加,在达到最大抑制率后出现饱和。流式细胞仪对细胞周期和细胞凋亡的分析发现：４ － ＰＡ 对细胞增殖期（Ｓ期、Ｇ２ 期） 有较强的抑制和逆转作用,使细胞群截止在Ｇ１ 期。随着作用时间的延长和４ － ＰＡ 浓度的增加,细胞凋亡的比例增加,凋亡细胞多来自于细胞增殖期（Ｓ 期、Ｇ２ 期） 。诱导凋亡和使细胞截止于细胞静息期是４ － ＰＡ 抑制细胞增殖的主要途径。     

Organism-specific neutrophil-endothelial cell interactions in response to Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus

The recruitment of polymorphonuclear leukocytes (PMNs) from the vascular space into the lung interstitium and airspace is an early step in the host innate immune response to bacterial invasion of these sites. To determine the ability of intact bacteria to directly elicit PMN migration across an endothelial monolayer, we studied in vitro migration of PMNs across a monolayer of human pulmonary microvascular endothelial cells in response to Streptococcus pneumoniae, Staphylococcus aureus, and Escherichia coli, as well as to purified E. coli LPS. Bacterial induction of PMN migration was dose dependent and elicited by > or =10(4) bacteria/ml of each of the species tested. Pretreatment of PMNs with blocking Abs to CD18 significantly inhibited migration of PMN in response to all stimuli tested, but had the most profound effect on migration to S. pneumoniae and S. aureus. Intact E. coli were 10 times more potent in inducing transmigration of PMNs than a corresponding amount of purified LPS. Bacterial induction of PMN migration did not correlate with up-regulation of surface endothelial ICAM-1 expression (purified LPS > intact E. coli > S. aureus and S. pneumoniae) nor up-regulation of VCAM-1 and E-selectin. Neutralizing Ab to ICAM-1 had no effect on PMN migration to any of the bacteria or to purified LPS. These findings demonstrate that diverse bacterial pathogens induce PMN migration across a pulmonary microvascular endothelial cell monolayer in a fashion that appears to be organism specific. In addition, intact bacteria elicit PMN-endothelial cell interactions distinct from those seen when purified bacterial products are used as agonists.     

乙烯在绿豆下胚轴不定根形成中的作用

选择易产生不定根的绿豆下胚轴做试验材料,研究乙烯在不定根形成中的作用。试验指出,乙烯利和乙烯气体在低浓度下(1—5mgL~(-1))促进或不抑制不定根的形成,随着浓度增加,抑制作用加强。IBA促进不定根形成的作用十分显著。IBA和乙烯相结合的试验说明,两者的作用似乎是独立的。可以设想,存在一个适于生根的低内源乙烯的界限,超过这个界限,抑制不定根形成。     

Nonpurulent response to toxic shock syndrome toxin 1-producing Staphylococcus aureus. Relationship to toxin-stimulated production of tumor necrosis factor

Infection of surgical wounds with toxic shock syndrome toxin 1 (TSST-1)-producing Staphylococcus aureus does not usually elicit a purulent response from the host. Because S. aureus is normally a pyogenic pathogen, this phenomenon suggests that strains of staphylococci that produce the exotoxin are able to inhibit the migration of polymorphonuclear neutrophils (PMN) to sites of infection. We have considered that inhibition of leukocyte migration may be an effect of secreted TSST-1 and have studied direct and indirect effects of the exotoxin on migratory functions of PMN in vitro. Preincubation of PMN with TSST-1 produced no inhibition of random motility or FMLP- or C5a-stimulated chemotaxis under agarose. Supernatant fluids from mononuclear leukocytes incubated with TSST-1, however, were potently inhibitory for both PMN random and chemotactic migratory functions. The inhibitor of migration was identified as TNF based upon neutralization by anti-TNF antiserum and its presence in the culture supernatant fluids assayed in terms of cytotoxicity for murine TNF-sensitive L-929 cell line cells. Preincubation of PMN with recombinant human TNF also inhibited subsequent PMN random and chemotactic migratory functions. We propose that TSST-1 inhibits the mobilization of PMN to sites of infection by stimulation of monocyte/macrophage TNF production and suggest that TNF may also contribute to some other effects of toxic shock syndrome.     

Measuring cell viscoelastic properties using a force-spectrometer: influence of protein-cytoplasm interactions

Cell adhesive and rheological properties play a very important role in cell transmigration through the endothelial barrier, in particular in the case of inflammation (leukocytes) or cancer metastasis (cancer cells). In order to characterize cell viscoelastic properties, we have designed a force spectrometer (AFM) which can stretch cells thereby allowing measurement of their rheological properties. This custom-made force spectrometer allows two different visualizations, one lateral and one from below. It allows investigation of the effects of rheology involved during cell stretching. To test the ability of our system to characterize such viscoelastic properties, ICAM-1 transfected CHO cells were analyzed. Two forms of ICAM-1 were tested; wild type ICAM-1, which can interact with the cytoskeleton, and a mutant form which lacks the cytoplasmic domain, and is unable to associate with the cytoskeleton. Stretching experiments carried out on these cells show the formation of long filaments. Using a previous model of filament elongation, we could determine the viscoelastic properties of a single cell. As expected, different viscoelastic components were found between the wild type and the mutant, which reveal that the presence of interactions between ICAM-1 and the cytoskeleton increases the stiffness of the cell.     

Activation of NADPH oxidase and phospholipase D in permeabilized human neutrophils. Correlation between oxidase activation and phosphatidic acid production.

A major function of human neutrophils (PMN) during inflammation is formation of oxygen radicals through activation of the respiratory burst enzyme, NADPH oxidase. Stimulus-induced production of both phosphatidic acid (PA) and diglyceride (DG) has been suggested to mediate oxidase activity; however, transductional mechanisms and cofactor requirements necessary for activation are poorly defined. We have utilized PMN permeabilized with Staphylococcus aureus alpha-toxin to elucidate the signal pathway involved in eliciting oxidase activity and to investigate whether PA or DG act as second messengers. PMN were permeabilized in cytoplasmic buffer supplemented with ATP and EGTA for 15 min before addition of NADPH and various cofactors. Oxidase activation was assessed by superoxide dismutase inhibitable reduction of ferricytochrome C; PA and DG levels were measured by radiolabeled product formation or by metabolite mass formation. Both superoxide (O2-) and PA formation were initiated by 10 microM GTP gamma S; addition of cytosolic levels of calcium ions (Ca2+, 120 nM) enhanced O2- and PA formation 1.5-2 fold. DG levels showed little change during these treatments. PA formation preceded O2- production and varying GTP gamma S levels had parallel effects on O2- and PA formation. However, while PA formation and oxidase activation occurred in tandem at Ca2+ levels of < 1 microM, higher calcium enhanced PA formation but inhibited O2- production. Removal of ATP completely blocked O2- production but had little effect on PA formation; in contrast, if ATP was replaced with ATP gamma S, parallel production of PA and O2- occurred in the absence of other cofactors. Finally, while inhibition of PA production by ethanol pretreatment led to inhibition of O2- formation in PMN treated with GTP gamma S alone, in cells stimulated with a combination of GTP gamma S and Ca2+, ethanol continued to inhibit PA formation but had no effect on O2- production. Our results do not support a role for DG in the signal transduction path leading to oxidase activation and, while we show a close correlation between oxidase activation and PA production under many physiologic conditions, we also demonstrate that PA is not sufficient to induce oxidase activation and O2- formation can occur when PA production is inhibited.