表皮葡萄球菌耐药性与生物被膜的相关性

目的分析表皮葡萄球菌耐药性与生物被膜的相关性。方法采用刚果红平板法检测表皮葡萄球菌胞间多糖黏附素(PIA);半定量生物被膜形成试验检测生物被膜表型;采用二倍稀释法检测各药物对该菌的最低抑菌浓度(MIC)。结果42株表皮葡萄球菌中,9株为PIA+/生物被膜表型+,4株为PIA+/生物被膜表型-,29株为PIA-/生物被膜表型-;3组菌对苯唑西林、克林霉素、阿米卡星、左氧氟沙星和阿莫西林/克林霉素的耐药性差异有统计学意义;万古霉素和阿莫西林/克拉维酸对3组菌的MIC低于NCCLS给出的敏感值,青霉素、苯唑西林、克林霉素、红霉素和头孢西丁对3组菌的MIC则远远高于NCCLS给出的敏感值,且β-内酰胺类的3种药物对3组菌的MIC差异有统计学意义。结论表皮葡萄球菌生物被膜形成是多基因调控过程,在细菌耐药中具有重要作用。     

原子力显微镜技术研究重组人内皮抑素对内皮细胞的作用

目的应用原子力显微镜(atomic force microscope,AFM)技术研究重组人内皮抑素(recombinant human endostatin,rhES)对内皮细胞的作用。方法采用MTT法检测不同浓度的rhES(0.05~2.4μg/ml)对人脐静脉内皮细胞ECV304增殖活力的影响;分别用0.8和2μg/ml的rhES处理ECV304细胞,应用AFM观察内皮细胞整体形貌的变化,SPI 3800 New DFM动力显微镜观察ECV304细胞表面局部形貌的变化。结果 rhES可明显抑制ECV304细胞增殖,且呈剂量效应(P0.001);rhES可降低贴壁的ECV304细胞的厚度,且呈剂量依赖效应,使较光滑的细胞表面变粗糙,产生了一些微小的突起;经rhES处理的ECV304细胞表面结构呈现不规则的变化。结论 AFM技术具有样品制备简便和分辨率较高等优点,适合贴壁培养细胞的原位观察。     

整合素αvβ_3拮抗剂-苯丁酸氮芥偶联毒素的合成及其体外抗肿瘤活性

目的合成整合素(Integrin)αvβ3拮抗剂-苯丁酸氮芥偶联毒素,并检测其体外抗肿瘤活性。方法化学合成In-tegrinαvβ3拮抗剂,并与苯丁酸氮芥通过酰胺键偶联,MTT法检测偶联毒素对人脐静脉内皮细胞ECV304和肝癌细胞HepG2的抑制作用。结果合成的偶联毒素经核磁共振和质谱分析鉴定,表明结构正确,纯度达90%以上,对HepG2细胞的抑制效果弱于苯丁酸氮芥,但对ECV304细胞的抑制特异性较好。结论Integrinαvβ3拮抗剂-苯丁酸氮芥偶联毒素可抑制HepG2和ECV304细胞的增殖,为癌症治疗提供了一个新途径。     

十二烷基羟丙基磺基甜菜碱对产膜表皮葡萄球菌粘附作用的影响

通过表面活性剂十二烷基羟丙基磺基甜菜碱(DSB)对表皮葡萄球菌生物被膜菌粘附的抑制作用研究,为抗由表皮葡萄球菌生物被膜菌引起的相关感染提供新的探索方向和治疗途径。以万古霉素为阳性对照,利用XTT减低法评价DSB对表皮葡萄球菌初始粘附的影响。发现质量浓度为64 mg/L的DSB对表皮葡萄球菌初始粘附有显著的抑制作用;用质量浓度为512,256和128 mg/L的DSB对实验材料预处理后,对表皮葡萄球菌初始粘附的抑制作用明显。结果表明,DSB对表皮葡萄球菌形成生物被膜的初始粘附过程具有显著的抑制作用。     

人血管内皮生长因子受体-Fc在DG44细胞中的表达及其生物活性

目的在中国仓鼠卵巢细胞DG44中表达人血管内皮生长因子受体(vascular endothelial growth factor receptor,VEGFR)-Fc,并检测其生物活性。方法化学合成人VEGFR1的第2免疫球蛋白结构域(VEGFR1D2)基因和人VEGFR2的第3免疫球蛋白结构域(VEGFR2D3)基因,通过重叠PCR(Overlap PCR)将VEGFR1D2-VEGFR2D3和人IgG1Fc拼接形成VEGFR-Fc融合基因,插入真核表达载体pD2中,构建重组表达质粒pD2-VEGFR-Fc,在FreeStyleTMMAX Reagent和OptiPROTMSFM介导下转染至DG44细胞中,MTX加压筛选稳定表达VEGFR-Fc蛋白的细胞株,SDS-PAGE、Western blot和ELISA法检测细胞培养上清中VEGFR-Fc蛋白的表达。表达的VEGFR-Fc蛋白经HiTrapTMProteinA FF柱纯化后,利用显微镜观察法和血管内皮细胞ECV304模型检测其生物活性。结果 VEGFR-Fc基因扩增产物大小为1 377 bp;重组表达质粒pD2-VEGFR-Fc经双酶切和测序证明构建正确;质粒pD2-VEGFR-Fc转染的DG44细胞培养上清中含VEGFR-Fc蛋白,表达量为0.5 g/L;细胞培养上清经HiTrapTMProteinA FF柱纯化后,杂蛋白去除效果较好;纯化的VEGFR-Fc能与VEGF特异性结合,抑制ECV304生长。结论成功在DG44细胞中表达了具有生物活性的VEGFR-Fc,为进一步研究其在抑制血管生成和抗肿瘤中的作用奠定了基础。     

基于MAP的一种新型低毒仿生粘合材料

为了提高人工粘合材料的黏附与生物相容性能,基于生物来源的粘合基材MAP构建仿生合成粘合材料,进行防水、快速粘合相关结构设计,合成得到一种MAP型低毒粘合材料。通过1H NMR归属官能团和氢信息,以GPC表征合成材料的相对分子质量分布,以热分析与细胞毒性实验进行材料热稳定性与毒性评价。结果表明,合成得到的聚合材料结构符合预期设计,质量分布均匀、黏均相对分子质量为2662,表现出良好的热稳定性与低毒性,对ECV304细胞的24h增殖抑制作用低于30%,具有进一步应用于生物医学领域、进行快速粘合修复的良好前景。     

贻贝黏附蛋白型聚氨酯黏合材料的制备

基于生物来源黏合材料贻贝黏附蛋白的结构、进行材料结构设计,经由扩链、偶联的分步合成,得到一种MAP型聚氨酯黏合材料。以热失重分析、细胞毒性实验进行该黏合材料的初步性能评价,结果表明,该黏合材料聚合分布均匀、黏均相对分子质量为955,表现出良好的热稳定性以及对体外活细胞的低毒性,黏合材料质量浓度为4 000μg/L时、对ECV304细胞的24 h增殖抑制作用低于30%,具有进一步应用于生物医学领域、进行快速黏合修复的良好前景。     

人血管抑制因子Vasostatin(120～180aa)的真核表达、纯化及活性鉴定

目的利用毕赤酵母表达系统表达人血管抑制因子Vasostatin(120～180aa),经纯化后,观察其抑制血管内皮细胞增殖的活性。方法采用PCR技术扩增出人血管抑制因子Vasostatin(120～180aa)的全长cDNA序列,将其克隆至真核表达载体pPIC9K中,转化毕赤酵母KM71,以甲醇诱导表达,并进行Western blot检测及金属螯合层析纯化。采用MTT法检测纯化的血管抑制因子对血管内皮细胞增殖的抑制作用。结果重组表达质粒pPIC9K-Vasostatin(120～180aa)经菌落PCR、酶切和测序鉴定,证明构建正确。经Western blot检测可见一条特异条带,纯化后的蛋白纯度达88.76%,浓度为300μg/ml,在体外可抑制人脐静脉血管内皮细胞ECV304的增殖。结论人血管抑制因子Vasostatin(120～180aa)可在毕赤酵母中以分泌形式表达,并具有抑制血管内皮细胞增殖的活性。     

聚乳酸-聚乙醇酸-聚乙二醇膜片与血管平滑肌细胞相容性研究

目的探讨生物可降解材料聚乳酸-聚乙醇酸-聚乙二醇(PLGA-PEG)与血管平滑肌的细胞相容性,为可降解血管支架材料的研究提供依据。方法采用体外培养的兔血管平滑肌细胞种植在PLGA-PEG膜片上,用相差显微镜观察细胞的生长情况,每天计数,绘制细胞生长曲线,用MTT法测定细胞增殖指数,流式细胞仪检测细胞周期分布。结果兔血管平滑肌细胞在PLGA-PEG膜片上生长良好,与对照组差异无显著意义;MTT法检测细胞增殖指数,与对照组差异无显著意义;流式细胞仪检测细胞周期显示对细胞增殖活性无明显影响。结论PLGA-PEG与兔血管平滑肌细胞具有良好的细胞相容性,可以用于可降解血管支架的制备。     

化妆品美白原料β-熊果苷稳定性研究

通过高效液相色谱法检测β-熊果苷降解产物氢醌,考察温度、含量、pH、放置时间以及表皮葡萄球菌、表皮细胞等因素对β-熊果苷稳定性的影响。结果表明,β-熊果苷水溶液在pH=5,温度为40 ℃时相对比较稳定,pH=3时相对不稳定,且随着放置时间的延长,β-熊果苷分解产生的氢醌越多。同时研究发现将表皮葡萄球菌在不同含量的β-熊果苷培养液中培养24 h,β-熊果苷均未分解生成氢醌,说明表皮葡萄球菌不会导致β-熊果苷分解产生氢醌。将表皮细胞放在不同含量的β-熊果苷培养液中培养24 h,有氢醌生成,说明表皮细胞会促进β-熊果苷的分解。进一步研究发现,β-熊果苷原料在不同温度、湿度下均很稳定。在β-熊果苷中加入维生素C、珍珠粉,β-熊果苷的稳定性会下降。     

Low Concentration of the Neutrophil Proteases Cathepsin G,Cathepsin B,Proteinase-3 and Metalloproteinase-9 Induce Biofilm Formation in Non-Biofilm-Forming Staphylococcus epidermidis Isolates

Neutrophils play a crucial role in eliminating bacteria that invade the human body; however, cathepsin G can induce biofilm formation in a non-biofilm-forming Staphylococcus epidermidis 1457 strain, suggesting that neutrophil proteases may be involved in biofilm formation. Cathepsin G, cathepsin B, proteinase-3, and metalloproteinase-9 (MMP-9) from neutrophils were tested on the biofilm induction in commensal (skin isolated) and clinical non-biofilm-forming S. epidermidis isolates. From 81 isolates, 53 (74%) were aap⁺, icaA⁻, icaD⁻ genotype, and without the capacity of biofilm formation under conditions of 1% glucose, 4% ethanol or 4% NaCl, but these 53 non-biofilm-forming isolates induced biofilm by the use of different neutrophil proteases. Of these, 62.3% induced biofilm with proteinase-3, 15% with cathepsin G, 10% with cathepsin B and 5% with MMP -9, where most of the protease-induced biofilm isolates were commensal strains (skin). In the biofilm formation kinetics analysis, the addition of phenylmethylsulfonyl fluoride (PMSF; a proteinase-3 inhibitor) showed that proteinase-3 participates in the cell aggregation stage of biofilm formation. A biofilm induced with proteinase-3 and DNAse-treated significantly reduced biofilm formation at an early time (initial adhesion stage of biofilm formation) compared to untreated proteinase-3-induced biofilm (p < 0.05). A catheter inoculated with a commensal (skin) non-biofilm-forming S. epidermidis isolate treated with proteinase-3 and another one without the enzyme were inserted into the back of a mouse. After 7 days of incubation period, the catheters were recovered and the number of grown bacteria was quantified, finding a higher amount of adhered proteinase-3-treated bacteria in the catheter than non-proteinase-3-treated bacteria (p < 0.05). Commensal non-biofilm-forming S. epidermidis in the presence of neutrophil cells significantly induced the biofilm formation when multiplicity of infection (MOI) 1:0.01 (neutrophil:bacteria) was used, but the addition of a cocktail of protease inhibitors impeded biofilm formation. A neutrophil:bacteria assay did not induce neutrophil extracellular traps (NETs). Our results suggest that neutrophils, in the presence of commensal non-biofilm-forming S. epidermidis, do not generate NETs formation. The effect of neutrophils is the production of proteases, and proteinase-3 releases bacterial DNA at the initial adhesion, favoring cell aggregation and subsequently leading to biofilm formation.     

Relevance of Cell Wall and Extracellular Matrix Proteins to Staphylococcus Epidermidis Adhesion and Biofilm Formation

In the present study, the protein profiles of the cell wall (CW) extracts of eight Staphylococcus epidermidis strains were analysed. The protein pattern of the extracellular matrix (EM) of four S. epidermidis good biofilm producer strains was also obtained. The main goal was to relate these protein patterns with their bacterial adhesion and biofilm forming ability. For this purpose, 2 h adhesion and 8 d-old biofilm assays were carried out, with the adhered cells and biomass quantified by microscopic observation and crystal violet quantification, respectively. CW and EM proteins were visualized by SDS–PAGE. According to the results, a significant percentage of the CW proteins detected was common to all the strains studied. However, the most adhering strains expressed a high number of proteins associated with the initial adhesion process, and the strongest biofilm producers expressed proteins that were absent in the protein profile of the strains that produced lower amounts of biofilm. Therefore, the present results demonstrate the importance of such proteins in adhesion and biofilm formation processes. Some of the CW proteins were also present in the EM protein pattern, though to a smaller amount, as well as several exoenzymes important for the host immune system invasiveness.     

Hyperoside Downregulates the Receptor for Advanced Glycation End Products (RAGE) and Promotes Proliferation in ECV304 Cells via the c-Jun N-Terminal Kinases (JNK) Pathway Following Stimulation by Advanced Glycation End-Products In Vitro

Hyperoside is a major active constituent in many medicinal plants which are traditionally used in Chinese medicines for their neuroprotective, anti-inflammatory and antioxidative effects. The molecular mechanisms underlying these effects are unknown. In this study, quiescent ECV304 cells were treated in vitro with advanced glycation end products (AGEs) in the presence or absence of hyperoside. The results demonstrated that AGEs induced c-Jun N-terminal kinases (JNK) activation and apoptosis in ECV304 cells. Hyperoside inhibited these effects and promoted ECV304 cell proliferation. Furthermore, hyperoside significantly inhibited RAGE expression in AGE-stimulated ECV304 cells, whereas knockdown of RAGE inhibited AGE-induced JNK activation. These results suggested that AGEs may promote JNK activation, leading to viability inhibition of ECV304 cells via the RAGE signaling pathway. These effects could be inhibited by hyperoside. Our findings suggest a novel role for hyperoside in the treatment and prevention of diabetes.     

Trans fatty acids influence the oxidation of LDL in ECV304 cells

The objectives of this study are to explore whether separate sources of trans fatty acids (TFA) have different effects on ECV304 cell line and to further elucidate the oxidation mechanism induced by TFA. ECV304 cells are used in the study because they display many endothelial features. Cell apoptosis rates increased in a dose‐dependent manner following 24‐h treatment with TFA from separate sources. Additionally, TFA stimulated human alpha‐defensin 1 (HNP‐1) expression and resulted in a significant increase in both malondialdehyde (MDA) and ROS levels. MDA levels reach their peak at 18 h. HNP‐1 expression levels increase at 2 h and then reach their peak at 10 h. At the same time, the protein carbonyl (PCO) value declines slightly. After 10 h of TFA co‐culture, the cells were washed and fresh low‐density lipoprotein (LDL) was added. MDA generation significantly increased after 6 h and it could be inhibited by 4‐aminobenzoic acid hydrazide (ABAH) or sodium ferulate. However, after the TFA co‐culture for 2 h, adding LDL for 6 h just caused slight MDA generation change and the MDA generation could be inhibited by verapamil or sodium ferulate. TFA from different sources did not have different effects on ECV304. HNP‐1 mediates the oxidation induced by TFA by activating ROS. Furthermore, TFA can stimulate the oxidation of LDL in ECV304 cells through both passive and active pathway. In the oxidation induced by linoelaidic acid, ABAH can decrease the MDA generation in active oxidation pathway and verapamil can decrease the MDA generation in passive oxidation pathway.     

Effect of pH on distribution and adhesion of Staphylococcus aureus to glass

The adhesion of Staphylococcus aureus ATCC 25923 to glass at different pH values was investigated using scanning electron microscopy (SEM) and image analysis with the Mathlab^® program. The images obtained by SEM show that the adhesion behaviour of Staphylococcus aureus ATCC 25923 depends on the pH of the suspending medium. At highly acidic (pH 2, pH 3) and alkaline pH, the cells deposited in aggregate forms, while at pH 5 the aggregation phenomenon was absent. The quantitative adhesion (number of adhering cells to glass surface) showed that cells adhered strongly in the pH range 4 to 6 and weakly at highly acidic (pH 2, pH 3) and alkaline pH. Moreover, the surface properties of the cells were characterized by the microbial adhesion to solvents (MATS) method. A good correlation was obtained between physicochemical properties (hydrophobicity or electron donor electron acceptor character) of Staphylococcus aureus ATCC 25923 and the number of adhering cells to glass. These results show that the adhesion of Staphylococcus aureus ATCC 25923 to glass is controlled by both acid–base and hydrophobic interactions.