麦冬不同提取物对过氧化氢损伤人血管内皮细胞ICAM-1、VEGF、Bcl-2表达的影响

目的：观察麦冬不同提取物对过氧化氢诱导的人脐静脉内皮细胞（HUVEC）间黏附分子-1（ICAM-1）和VEGF、Bcl-2表达的影响。方法：体外培养HUVEC,用过氧化氢（H2O2）制造HUVEC损伤模型。以四甲基偶氮唑蓝（MTT）比色法检测细胞存活数量,用流式细胞仪检测HUVEC表面ICAM-1的表达量;免疫细胞化学方法检测HUVEC的VEGF、Bcl-2的分布情况。结果：模型组较正常对照组细胞增殖活性明显降低（P〈0．01）。与模型组相比,经麦冬水提物、正丁醇提取物处理组细胞增殖活性明显增加（P〈0．05,P〈0．01）。流式细胞仪检测显示正丁醇提取物可降低过氧化氢增加的ICAM-1基因的表达。Bcl-2的表达,模型组明显低于正常对照组,而正丁醇组表达明显高于模型组（P〈0．01）。VEGF的表达,模型组明显高于正常对照组,麦冬水提物、正丁醇提取物处理组高于模型纽（P〈0．05,P〈0．01）。结论：麦冬提取物具有抗凋亡、促增殖、降低细胞间黏附分子-1表达的作用,尤以正丁醇提取物效果更为显著。     

两种根管冲洗液对狗牙根管内细菌抗菌作用的实验研究

目的观察2%洗必泰溶液和3%双氧水2种根管冲洗液对狗牙根管内粪肠球菌、溶血链球菌、微小消化链球菌、中间普氏菌及具核酸杆菌的影响,为临床根管治疗提供参考。方法选择成年健康杂种狗3只,共有24个实验牙,48个实验牙根。于狗牙根管内接种粪肠球菌、溶血链球菌、微小消化链球菌、中间普氏菌及具核酸杆菌。对狗牙完成根管治疗。于治疗后12个月拍摄根尖X线片,并记录牙齿和根尖周组织的临床表现。将患牙随机分为3组,每组7颗患牙,去除根管内充填物并进行根管预备,实验一组用2%洗必泰溶液冲洗根管,实验二组用3%双氧水冲洗根管,对照组用0.9%NaC l溶液冲洗根管。分别在根管预备前及根管预备冲洗后对根管内细菌取样,培养,鉴定并记录细菌菌落数量,测定根管内细菌变化情况。结果根管预备冲洗后3组根管内的细菌量均较根管预备前显著下降（P〈0.05）。2%洗必泰溶液和3%双氧水2种根管冲洗液的杀菌效果明显好于0.9%NaC l溶液（P〈0.05）,2%洗必泰溶液明显好于3%双氧水（P〈0.05）。结论2%洗必泰溶液是有效的根管冲洗药物,可明显减少狗牙根管内粪肠球菌、溶血链球菌、微小消化链球菌、中间普氏菌及具核酸杆菌的数量,但不能完全清除根管内的细菌。     

肺炎链球菌溶血素黏膜免疫抗定植保护作用的研究

目的原核表达△A146Ply蛋白,评价△A146Ply黏膜免疫对肺炎链球菌（Streptococcuspneumon—ioe,5．Pn）在宿主鼻咽部定植的保护作用。方法IPTG诱导、Ni—NTA树脂纯化获得纯化的△A146Ply蛋白,经黏膜免疫BALB／C小鼠,制备其特异性抗血清;进行体内抗定植实验,观察小鼠鼻咽部灌洗液和肺部残存的细菌数量,检测△A146Ply黏膜免疫对19F型肺炎链球菌在鼻咽部定植的保护作用。为验证该保护作用是否具有广谱性,培养血清型14型、3型、6型和2型S．pn,经鼻腔感染免疫后小鼠,评价△A146Ply蛋白黏膜免疫对多株肺炎链球菌定植的保护作用。进行体外抗黏附实验,检测△A146Ply蛋白及其抗血清是否对无荚膜的肺炎链球菌R6黏附A549细胞具有抑制作用。结果获得了纯度〉90％的目的蛋白;体内实验结果显示,△A146Ply黏膜免疫可以显著降低肺炎链球菌19F在宿主鼻咽部和肺部残存的细菌数量（P〈0．01）;14型和3型肺炎链球菌在免疫后小鼠鼻咽部及肺部定植的数量均显著下降（P〈0．05）,2型肺炎链球菌在免疫后小鼠肺部定植的数量显著下降（P〈0．05）,6B型肺炎链球菌在免疫组与对照组小鼠鼻咽部及肺部均无显著差异（P〉0．05）;△A146Ply特异性抗血清△A146Ply蛋白对R6黏附A549细胞的抑制效应呈剂量依赖性。结论△A146Ply蛋白经黏膜免疫BALB／C小鼠可以对多种血清型的肺炎链球菌在宿主鼻咽部及肺部的定植提供显著保护作用,为Ply作为肺炎链球菌疫苗候选蛋白的应用提供了实验依据。     

氨溴索对铜绿假单胞菌生物膜早期黏附及胞外多糖的影响

目的研究氨溴索对铜绿假单胞菌PA01菌株BF早期黏附及胞外多糖复合物（Extracellular Pdymeric Substances,EPS）的影响。方法利用荧光多功能酶标仪检测各组不同时间点96孔板中pGFPuv转化PA01菌株的荧光强度,计算黏附率以表示干预对不同时间点细菌黏附的影响;利用罗丹明标记的麦胚凝集素（WGA）特异性结合细菌EPS,荧光显微镜下定性观察各组EPS的变化;利用硫酸-苯酚法定量各组细菌EPS的产量。结果8h组,氨溴索高浓度干预后细菌的黏附率由0．72±0．17下降至0．49±0．08,t=4．03,P〈0．05,与克拉霉素阳性对照组黏附率（0．50±0．06）相比,t=-1．19,P〉0．05;氨溴索低浓度干预后黏附率也有下降,但不及高浓度组明显;其余时间组趋势与8h组大致相似。罗丹明标记WGA可使胞外多糖显色,在荧光显微镜下观察可见氨溴索干预后EPS减少,稀薄;EPS定量实验,EPS总量（μg）／细菌干重（g）氨溴索干预组（477．82±7．90）较生理盐水对照组（523．76±10．12）有明显降低,t=8．76,P〈0．05。结论氨溴索可显著减少PA01菌株黏附及产EPS的能力。     

KI对l染铅近曲肾小管上皮细胞I A 表达及凋亡的影响

目的：研究碘化钾对染铅肾小管上皮细胞XIAP的表达及凋亡的影响,探讨其对抵抗铅导致的近曲肾小管上皮细胞损伤的可能机制。方法：应用流式细胞术检测染铅HK-2细胞及对照组细胞中XIAP的表达及凋亡情况。结果：染铅HK-2细胞中XIAP的表达明显低于对照组,且虽然铅浓度的增高而降低（P〈0．05／P〈0．01）,碘化钾能够减弱上述变化（P〈0．05／P〈0．01）;染铅HK-2细胞的凋亡率明显高于对照组,且虽然铅浓度的增高而升高（P〈0．05／P〈0．01）,碘化钾能够降低染铅细胞的凋亡率（P〈0．05／P〈0．01）。结论：碘化钾能够上调染铅HK-2细胞中抗凋亡蛋白XIAP的表达,降低凋亡率,在一定程度上降低铅对细胞的损伤。     

S1P对心肌细胞的保护作用研究

目的：探究1-磷酸鞘氨醇（S1P）对缺氧／复氧乳鼠心肌细胞的保护作用及其分子机制。方法：在大鼠乳鼠心肌细胞原代培养基础上,应用液体石蜡覆盖法制备心肌细胞缺氧／复氧模型,采用流式细胞术PI染色法和流式细胞术罗丹明123染色法检测S1P对缺氧／复氧心肌细胞的细胞凋亡和线粒体膜电位的影响;Western Blot分析法检测S1P作用后的心肌细胞p-Akt1蛋白水平变化,并且观察PI3K（磷脂酰肌醇3-激酶）阻断剂渥曼青霉素（wommamin）对S1P上述作用的影响。结果：在S1P的影响下,缺氧／复氧心肌细胞的凋亡率显著下降（P〈0．01）,线粒体膜电位的去极化被明显抑制（P〈0．05）,p-Akt1水平明显升高（P〈0．01）,wormannin能够部分阻断S1P的上述效应。结论：S1P能够显著抑制缺氧／复氧引起的心肌细胞凋亡,其机制可能与S1P激活PDK-Akt信号通路进而稳定线粒体膜电位有关。     

FISH-FCM方法检测酵母-细菌二元体系中微生物数量

以废水生物处理系统和生物发酵系统出现的酵母-细菌二元体系作为研究对象,探讨了二元体系生物样品的最佳超声分散条件以及同时检测酵母和细菌数量时荧光原位杂交（FISH）-流式细胞术（FCM）技术的测量精度。染色-FCM检测表明：同时适用于混合酵母样品（酵母假菌丝）和混合细菌样品（活性污泥絮体）的最佳超声分散条件为100W、60～90s,但过强超声条件（120s）对酵母Candida tropicalis纯培养物（或细菌Escherichia coli纯培养物）的超声粉碎效果完全不同于混合酵母样品（或混合细菌样品）。在此基础上,以C．tropicalis和E．coli分别作为酵母和细菌的模式微生物,采用双探针杂交的FISH—FCM技术检测了背景微生物（C．tropicalis或Ecoll）浓度为10^7个／ml时二元体系中目标微生物（10^2～10^7个／ml）的数量。流式细胞仪能够明显区分噪音和二元体系中的酵母和细菌,因而一次进样时能够同时分析两种微生物数量,并且目标微生物浓度可以精确到10^4个／ml,此时微生物含量仅为0．1％,其中细菌浓度甚至可以精确到10^3个／ml（相应含量仅为0．01％）。然而,当二元体系中目标微生物浓度过低时（〈10^4个酵母／ml或〈10^4个细菌／ml）,背景微生物的存在会严重影响FISH—FCM技术的测量准确性。     

细胞渗透性神经酰胺可抑制大鼠离体黄体细胞甾体激素生成并诱导细胞凋亡

本文旨在观察神经酰胺对离体孵育的大鼠黄体细胞孕酮分泌及细胞凋亡的影响,以PMSG－hCG处理的雌性Wistar大鼠为模型,分离制备黄体细胞,将外源性细胞渗透性神经酰胺与黄体细胞共同孵育,分别用放免法和流式细胞仪分析神经酰胺对黄体细胞孕酮生成和凋亡的影响,同时还检测了一氧化氮合酶（NOS）活性和一氧化氮（NO）水平的变化,结果显示,神经酰胺可以剂量相关方式抑制hCG－诱导的孕酮分泌,而对基础孕酮没有显著影响,离体孵育12h的大鼠黄体细胞存在自发性凋亡,5umol/L神经酰胺能显著增加亡率（P＜0．05）,流式细胞仪分析可见增强的凋亡蜂,实验还发现,50umol/L神经酰胺能明显促进NOS活性（P＜0．01）和NO生成（P＜0．01）,结果提示,神经酰胺可能通过调节甾体激素生成和细胞凋亡而作为一种重要的信息分子参与黄体退化等卵巢的生理过程。     

不同临床类型乙型病毒感染者外周血T细胞亚群与血清HBVDNA载量及HBeAg滴度相关性分析

目的：探讨慢性乙型肝炎病毒（HBV）感染患者外周血T细胞亚群与血清HBVDNA载量及HbeAg滴度的关系。方法：选取103名HBV感染患者和20名健康者为研究对象。流式细胞术检测外周血T细胞亚群,聚合酶链式反应及酶免疫分析法分别检测血清HBVDNA载量及HbeAg滴度。结果：慢性乙型肝炎患者和慢性HBV携带者外周血CD3可、CD4T淋巴细胞亚群百分数低于健康对照组,结果有统计学意义（P〈0．05或0．01;而CD8＋T细胞亚群则呈现相反趋势,结果亦有统计学意义（P〈0．05或0．01）。HBeAg阴性组中,HBVDNA水平与CD8T细胞亚群百分数呈正相关（r=0．567,P〈0．01）,与CD47CD8＋T细胞亚群百分数比值呈负相关（r=-0．601,P〈0．01）,而与CD3＋T、CD4＋T细胞亚群百分数无相关性。HBeAg阳性组中,HBVDNA水平及HbeAg滴度与cD3＋1r、cD41、CD8叮细胞百分数及CD47CD8＋T细胞百分数均无相关性（P〉0．05）。结论：不同临床类型的慢性乙型肝炎病毒感染患者外周血T细胞亚群存在不同程度细胞免疫功能降低和细胞免疫调节异常。HbeAg阴性的HBV感染患者,其血清HBVDNA水平与外周血T淋巴细胞免疫存在相关性。     

大蒜素对铜绿假单胞菌生物膜早期黏附及胞外多糖的影响

目的研究大蒜素对铜绿假单胞菌PA01菌株生物膜（biofilm,BF）早期黏附及胞外多糖复合物（Extracellular Polymeric Substances,EPS）的影响。方法利用荧光多功能酶标仪检测各组不同时间点96孔板中pGF-Puv转化PA01菌株的荧光强度,计算黏附率以表示干预对不同时间点细菌黏附的影响,利用荧光显微镜下定性观察细菌的黏附量;应用异硫氰酸标记的刀豆蛋白A（FITC conjugated concanavalin A,FITC-conA）特异性结合细菌EPS,荧光显微镜下定性观察各组EPS的变化;利用硫酸-苯酚法定量各组细菌EPS的产量。结果6h组,大蒜素高浓度干预后细菌的黏附率由0．70±0．03下降至0．50±0．01,t=15．014,P〈0．05,大蒜素低浓度干预后黏附率也有下降,但不及高浓度组明显;除了9h组其他时间组趋势与6h组大致相似,可能与大蒜素含量下降有关。荧光显微镜观察可见生理盐水对照组细菌菌落分布,干预组黏附的细菌稀疏散在分布,以高浓度为甚。FITC-conA可使胞外多糖显色,在荧光显微镜下观察可见大蒜素干预后EPS减少,稀薄;EPS定量实验,EPS总量大蒜素高浓度干预组（181．19±1．59）μg较生理盐水对照组（602．66±21．94）μg有明显降低,t=60．589,P〈0．05。结论大蒜素可显著减少PA01菌株黏附及产EPS的能力。     

甲型链球菌SN-34和SN-35体外抑菌作用的研究

目的观察甲型链球菌SN-34和SN-35的抑菌效果。方法在固体培养基上用复层琼脂法观测甲型链球菌SN-34和SN-35对化脓性链球菌32309-2和金黄色葡萄球菌26001的抑菌环大小;在液体培养基中,甲型链球菌SN-34和SN-35分别与化脓性链球菌32309-2和金黄色葡萄球菌26001共同培养,然后观察一定间隔（2h）细菌的数量变化,并绘制生长曲线。结果化脓性链球菌32309-2的生长明显受到抑制。结论仅甲型链球菌SN-34具有抑菌作用。     

Streptococcus pyogenes induces oncosis in macrophages through the activation of an inflammatory programmed cell death pathway

Macrophages are crucial components of the host defence against Streptococcus pyogenes . Here, we demonstrate the ability of S. pyogenes to kill macrophages through the activation of an inflammatory programmed cell death pathway. Macrophages exposed to S. pyogenes exhibited extensive cytoplasmic vacuolization, cellular and organelle swelling and rupture of the plasma membrane typical of oncosis. The cytotoxic effect of S. pyogenes on macrophages is mediated by the streptococcal cytolysins streptolysin S and streptolysin O and does not require bacterial internalization. S. pyogenes -induced death of macrophages was not affected by the addition of osmoprotectant, implicating the activation of an orchestrated cell death pathway rather than a simple osmotic lysis. This programme cell death pathway involves the loss of mitochondria transmembrane potential (Δ ψ _m) and was inhibited by the addition of exogenous glycine, which has been shown to prevent necrotic cell death by blocking the opening of death channels in the plasma membrane. The production of reactive oxygen species and activation of calpains were identified as mediators of the cell death process. We conclude that activation of the inflammatory programmed cell death pathway in macrophages could constitute an important pathogenic mechanism by which S. pyogenes evades host immune defences and causes disease.     

Flow cytometric evaluation of adhesion of Streptococcus pyogenes to epithelial cells

The precise roles of various surface molecules in the attachment of Streptococcus pyogenes to host epithelia are currently unclear. A flow cytometry assay that facilitates the analysis of the kinetics of S. pyogenes adhesion to epithelial cells was developed. Dose- and time-dependent adhesion isotherms with both buccal epithelial cells (BECs) and Hep-2 cells as substrata were obtained. Although binding equilibrium is reached within 2 h on both cell types, saturation of binding sites on BECs is not achieved within a wide range of experimental conditions. This indicates a high degree of non-specific attachment to that cell type. Since no rinsing step is necessary when using flow cytometry to analyze adhesion, low-affinity associations were observable. This was confirmed by determining bacterial desorption rates early and late in the adsorption process. Binding irregularities were also easily detected since the cytometer records and displays data for up to 10,000 epithelial cells per time point. It is proposed to use this methodology to assign roles to particular surface molecules/characteristics during distinct phases of adhesion.     

Experimental evaluation of the bacteriotoxic phototherapy of inflammatory diseases of the oral cavity

The combined use of laser radiation and Radachlorine revealed the presence of correlation between the number of viable Streptococcus pyogenes cells and the concentration of photosensitizer (r=-0.62), time of irradiation (r=-0.75), as well as energy density (r=-0.80). The death of 100% of suspended S. pyogenes cells was observed at the concentration of Radachlorine reaching 14.4 mM and radiation power reaching 1 W, irrespective of energy density. Pulse and superpulse laser radiation, carried out simultaneously with the use of Radachlorine at a concentration of 1.8 microM, resulted in the death of 100% of S. pyogenes cells in vitro. On the experimental model of inflammation the application of 0.1% Radachlorine gel demonstrated higher effectiveness (in comparison with 0.35% Radachlorine solution) when used simultaneously with laser radiation, both continuous and superpulse (with energy density being 400 j/cm2 and power being 1 W). The use of these parameters in the treatment of the experimental foci of inflammation in the animals led to the complete elimination of S. pyogenes from infected tissues.     

Influenza A virus-infected hosts boost an invasive type of Streptococcus pyogenes infection in mice

The apparent worldwide resurgence of invasive Streptococcus pyogenes infection in the last two decades remains unexplained. At present, animal models in which toxic shock-like syndrome or necrotizing fasciitis is induced after S. pyogenes infection are not well developed. We demonstrate here that infection with a nonlethal dose of influenza A virus 2 days before intranasal infection with a nonlethal dose of S. pyogenes strains led to a death rate of more than 90% in mice, 10% of which showed necrotizing fasciitis. Infection of lung alveolar epithelial cells by the influenza A virus resulted in viral hemagglutinin expression on the cell surface and promoted internalization of S. pyogenes. However, treatment with monoclonal antibodies to hemagglutinin markedly decreased this internalization. Our results indicate that prior infection with influenza A virus induces a lethal synergism, resulting in the induction of invasive S. pyogenes infection in mice.     

Assessment of cell viability by flow cytometric analysis using DNase exclusion

A new method was developed for selective measurement of DNA distributions in viable cell populations. The method is based on the fact that non-viable cells lose membrane integrity and treatment of such cells with DNase should remove their DNA. The DNase-treated cells were stained with DNA fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) in the presence of Triton X-100. DNA distribution was measured by flow cytometry prior to and after treatment with DNase. Percentage of cells stained after DNase treatment was considered as an index of cell viability. Optimal conditions for DNase treatment and application of DNase exclusion test for the analysis of spontaneous cell death, selective death of cells arrested in S/G2 phases, instant cell disintegration induced by cytotoxic compounds and cell death induced by hyperthermia are described.     

Streptococcus pyogenes expressing M and M-like surface proteins are phagocytosed but survive inside human neutrophils

Strains of the Gram-positive human pathogen Streptococcus pyogenes (group A streptococcus) that express surface-associated M or M-like proteins survive and grow in non-immune fresh human blood. This is generally accepted to be caused by an antiphagocytic property of these proteins. However, in most previous studies, an inhibition of the internalization of the bacteria into host cells has not been studied or not directly demonstrated. Therefore, in the present paper, we used flow cytometry, fluorescence microscopy and electron microscopy to study phagocytosis by human neutrophils of wild-type S. pyogenes and strains deficient in expression of M protein and/or the M-like protein H. The results demonstrate that all strains of S. pyogenes tested, including the wild-type AP1 strain, induce actin polymerization and are efficiently phagocytosed by human neutrophils. In addition, using classical bactericidal assays, we show that the wild-type AP1 strain can survive inside neutrophils, whereas mutant strains are rapidly killed. We conclude that the ability of virulent S. pyogenes to survive and multiply in whole blood is most likely not possible to explain only by an antiphagocytic effect of bacterial surface components. Instead, our data suggest that bacterial evasion of host defences occurs intracellularly and that survival inside human neutrophils may contribute to the pathogenesis of S. pyogenes and the recurrence of S. pyogenes infections.     

A dual fluorescence flow cytometric analysis of bacterial adherence to mammalian host cells

Flow cytometry has provided a powerful tool for analyzing bacteria-host cell associations. Established approaches have used bacteria, labeled either directly with fluorochromes or indirectly with fluorescently conjugated antibodies, to detect these associations. Although useful, these techniques are consistently unable to include all host cells in the analysis while excluding free, aggregated bacteria. This study describes a new flow cytometry method of assessing bacterial adherence to host cells based on direct fluorescent labeling of both bacteria and host cells. Eukaryotic host cells were labeled with PKH-26, a red fluorescent dye, and bacteria were labeled with fluorescein isothiocyanate, a green fluorescent dye. The red host cells were gated and the mean green fluorescence intensity (MFI) of these red cells was determined. We used MFI values obtained from control samples (unlabeled and labeled host cells with unlabeled bacteria) to eliminate contributions due to autofluorescence. The final MFI values represent fluorescence of host cells resulting from the adherent bacteria. Because all red fluorescent cells are analyzed, this method includes all the eukaryotic cells for analysis but excludes all free or aggregated bacteria that are not bound to target cells.     

Promotion of phagocytosis of Streptococcus pyogenes in human blood by a fibrinogen-binding peptide

The binding of fibrinogen to M-related protein (Mrp) is known to contribute to the ability of Streptococcus pyogenes to evade phagocytosis by preventing the deposition of complement on the streptococcal surface. The objectives of this investigation were to map the common fibrinogen-binding domain of Mrp and to determine if this domain has a therapeutic potential to enhance phagocytosis of S. pyogenes in human blood. Using a series of recombinant, truncated proteins of Mrp, two fibrinogen-binding domains (FBD) were mapped. FBD1 was contained within amino acid residues 1-55 of Mrp and FBD2 within residues 81-138. FBD2 is found in all Mrp sequenced to date whereas FBD1 is not. Both FBD1 and FBD2 peptides but not a control peptide blocked the binding of fibrinogen to S. pyogenes and promoted phagocytosis of the streptococci in human blood. The data support the hypothesis that the binding of fibrinogen by S. pyogenes is centrally involved in their resistance to phagocytosis in human blood and suggest that treatments that interfere with the binding of fibrinogen to S. pyogenes may help in fighting infections by these organisms.     

Flow cytometric identification of microorganisms by dual staining with FITC and PI.

The identification of microorganisms by flow cytometry was evaluated by using a double staining technique with propidium iodide and fluorescein isothiocyanate and a two dimensional analysis. A diverse group of 19 different species and strains of microorganisms was tested to determine if they could be differentiated by flow cytometry. The organisms tested displayed characteristic and distinct two dimensional fluorescent patterns which allowed ready grouping and differentiation into subsets of organisms. The slopes and correlation coefficients of the histograms and the ratio of red to green signals expressed these differences quantitatively and allowed organisms to be placed into one of three groups based on these values. In some instances, as with Streptococcus pneumoniae and pyogenes and Staphylococcus aureus and epidermidis, it was possible to distinguish between species of bacteria from the same genus. The use of dual dye labeling and flow cytometry provided a rapid method of identifying selected microorganisms and may be broadly applicable for the detection and identification of many bacteria and fungi.