阿司匹林与两性霉素B对白色念珠菌生物膜的体外联合作用研究

目的：研究解热镇痛药药理学浓度的阿司匹林与抗真菌药两性霉素B对白色念珠菌生物膜在体外的联合作用.方法：采用96孔板培养真菌生物被膜,银染法鉴定生物被膜,结晶紫染色法定量测定.结果两性霉素B(125μg.ml-1)组与相应的阿司匹林高剂量(200μg.ml-1)联合用药组比较,有显著性差异,p<0.05 结论：生理浓度的阿司匹林(200μg.ml-1)可增强两性霉素B(125μg.ml-1)对白色念珠菌生物膜的抑制作用。 关键词 生物被膜,白色念珠菌,阿司匹林,两性霉素B ,体外     

头孢哌酮钠/舒巴坦钠与阿奇霉素对腹膜透析管大肠杆菌生物被膜的影响

目的体外建立腹膜透析管大肠杆菌生物被膜模型,探讨头孢哌酮钠/舒巴坦钠、阿奇霉素对大肠杆菌生物被膜的影响。方法采用腹膜透析液-腹膜透析管系统建立体外大肠杆菌生物被膜模型,将头孢哌酮钠/舒巴坦钠、阿奇霉素分别单独作用及联合作用于腹透管内大肠杆菌生物被膜,扫描电镜观察腹透管上生物被膜变化并计数存活细菌菌落数。结果用腹透液培养大肠杆菌24h后,腹透管上可形成明显的细菌生物膜;头孢哌酮钠/舒巴坦钠联合阿奇霉素组作用后,腹透管上细菌生物膜被严重破坏;与空白对照组比较,阿奇霉素组大肠杆菌生物被膜内存活细菌数无明显变化(P>0.05);头孢哌酮钠/舒巴坦钠组存活细菌数明显减少(P<0.01);头孢哌酮钠/舒巴坦钠联合阿奇霉素组较单用头孢哌酮钠/舒巴坦钠组存活细菌数明显减少(P<0.01)。结论头孢哌酮钠/舒巴坦钠与阿奇霉素联合作用可严重破坏腹膜透析管内已形成的大肠杆菌生物被膜,较单用头孢哌酮钠/舒巴坦有更强的抗菌效果。     

MTS法细胞毒试验的非线性关系研究

目的　探讨MTS法细胞毒试验的细胞数 OD关系以及药物剂量 -抑制率关系 ,为更好地应用MTS法提供提示。方法　采用HL 6 0和Raji细胞株。细胞数 OD关系试验 :96孔板接种不同浓度的细胞 ,加入MTS培养 1～ 5h后于 4 90nm波长测定OD ,用SPSS 11 0软件对数据作曲线估计。细胞毒试验 :96孔板每孔接种细胞 ,加不同剂量的药物培养 72h ,加入MTS培养 3h后于 4 90nm波长测定OD ,用SPSS11 0软件对数据作曲线估计。结果　 2种细胞 5个时间点的细胞数 OD关系以三次项曲线拟合度最好 ,r2 为 0 997～1 0 0 0 ,均大于线性模型的r2 0 938～ 0 993。 3种药物对 2种细胞的剂量 -抑制率以三次项曲线拟合度最好 ,r2 为0 998～ 1 0 0 0 ,优于对数剂量 -概率单位线性模型的r20 94 8～ 0 987。结论　三次项曲线对MTS法细胞毒试验的细胞数 OD关系有很高拟合度且优于线性曲线模型。通过药物剂量 -抑制率的三次项曲线拟合方程可望得到比常用的概率单位回归法更准确的IC50 。     

氨溴索与莫西沙星联合对铜绿假单胞菌生物膜的体外作用

目的 研究氨溴索与莫西沙星对铜绿假单胞菌生物膜的体外作用.方法 以中心静脉导管为载体培养铜绿假单胞菌生物膜,银染法鉴定生物膜,结晶紫染色法定量测定生物膜OD值并做出比较分析.结果 莫西沙星低、中、高单剂量组生物被膜OD值与低中、高联合用药组两两比较,差异均有统计学意义（P〈0.01）;氨溴索组与联合用药组相比,差异也有统计学意义（P〈0.01）.结论 氨溴索可以增强莫西沙星对铜绿假单胞菌生物膜的抑制作用,可作为临床上治疗BF相关感染辅助用药.     

氨基胍对人翼状胬肉成纤维细胞增殖影响的体外试验研究

目的:观察氨基胍(AG)对体外培养的人翼状胬肉成纤维细胞增殖的影响。方法:取人翼状胬肉标本进行体外贴壁细胞常规培养,传代至第3～5代细胞用于试验,以每孔1×104个细胞接种于培养板中,加入不同浓度(250、500、750、1000μmol.L-1)的AG作为AG组,另设不加AG的为对照组,检测作用24、48、72h后各孔的吸光度,并计算细胞增殖抑制率,每个时间点、每个浓度均设6个复孔。结果:人翼状胬肉成纤维细胞增殖抑制率随AG浓度的增大和作用时间的延长明显升高(P<0.05)。结论:AG在受试浓度和受试时间范围内,对体外培养的人翼状胬肉成纤维细胞具有抑制作用且呈浓度、时间依赖性。     

PI3K/AKT信号通路特异性抑制剂LY294002对Tca8113细胞增殖活性的影响

目的探讨PI3K/AKT通路特异性抑制剂LY294002对人舌鳞癌Tca8113细胞株增殖活性的影响。方法用不同药物浓度的LY294002(5μmol/L,10μmol/L,20μmol/L,40μmol/L)处理Tca8113细胞,MTT法检测对照组和实验组在不同的作用时间(24h、48h、72h、96h)下,Tca8113细胞的OD值,计算增殖抑制率,绘制Tca8113细胞浓度-抑制率曲线及时间-抑制率曲线。结果随药物浓度的增加及作用时间的延长,Tca8113细胞的OD值逐渐减小,增殖抑制率逐渐增大(P<0.01)。结论LY294002对体外培养的人舌鳞癌Tca8113细胞增殖有抑制作用,其抑制作用与LY294002的药物浓度及作用时间呈正相关。     

PI3K／AKT信号通路特异性抑制剂LY294002对Tea8113细胞增殖活性的影响

目的探讨PI3K／AKT通路特异性抑制剂LY294002对人舌鳞癌Tea8113细胞株增殖活性的影响。方法用不同药物浓度的LY294002（5μmol／L,10μmol／L,20μmol／L,40μmol／L）处理Tea8113细胞,MTT法检测对照组和实验组在不同的作用时间（24h、48h、72h、96h）下,Tca8113细胞的OD值,计算增殖抑制率,绘制Tea8113细胞浓度-抑制率曲线及时间-抑制率曲线。结果随药物浓度的增加及作用时间的延长,Tea8113细胞的OD值逐渐减小,增殖抑制率逐渐增大（P〈0．01）。结论LY294002对体外培养的人舌鳞癌Tca8113细胞增殖有抑制作用,其抑制作用与LY294002的药物浓度及作用时间呈正相关。     

消癌平对人膀胱癌细胞体外生长作用的实验研究

目的探讨不同浓度的消癌平注射液对人膀胱癌细胞(EJ细胞)体外生长的抑制作用。方法通过四甲基偶氮唑蓝(MTT)法检测不同浓度消癌平注射液对EJ细胞体外生长的抑制率,从而筛选出能够抑制EJ细胞体外生长的药物浓度。结果消癌平注射液浓度为30、40、50、60μl/ml时对EJ细胞生长具有明显抑制作用,实验组OD值均低于对照组,差异有统计学意义(P<0.01)。实验组生长抑制率随药液浓度的升高而升高,计算出的半抑制率(IC)=54.17μl/ml。结论消癌平注射液浓度在≥30μl/ml时对EJ细胞体外生长有较好的抑制作用。     

双黄连联合万古霉素对耐甲氧西林金黄色葡萄球菌生物被膜抑制作用的研究

目的研究双黄连联合万古霉素对MRSA生物被膜的影响。方法平板培养法培养MRSA生物被膜,银染法鉴定。96孔平板培养,分为四组:空白对照组(A组)、双黄连组(B组)、万古霉素组(C组)、双黄连+万古霉素组(D组)。经上述不同药物对生物被膜处理后,比色法测定藻酸盐含量,MTT法测定各组活菌计数。结果 1 B、C、D组作用后藻酸盐含量明显低于A组(P<0.05),D组分别较B组及C组低(P<0.05);2 B、C、D组作用后活菌计数明显低于A组(P<0.05),D组活菌计数亦分别较B组及C组低(P<0.05)。结论双黄连可抑制MRSA生物被膜合成,并有显著抑菌作用,与万古霉素有协同作用。     

白头翁皂苷D联合索拉非尼对人肝癌细胞侵袭与转移的影响

[摘要]目的 探讨白头翁皂苷D联合索拉非尼对人肝癌细胞株侵袭与转移的影响。 方法 将人肝癌细胞BEL-7402分为白头翁皂苷D单药处理组、索拉非尼单药处理组及两药联合处理组,观察比较三种处理方式对肝癌细胞侵袭和转移的影响。结果 检测分析各组黏附抑制率,作用3h,索拉非尼组显著高于白头翁皂苷D组（P＜0.05或＜0.01）,联合用药组显著高于索拉非尼组和白头翁皂苷D组（P＜0.01）;作用5h,白头翁皂苷D组黏附抑制率显著高于索拉非尼组（P＜0.01）,并且高于白头翁皂苷D组作用3h（P＜0.01）,说明两药联合对肝癌细胞的黏附抑制率具有协同作用（Q=2.33＞1.15）;检测分析各组迁移抑制率,索拉非尼单药组和两药联合组对肝癌细胞的迁移抑制率均显著高于白头翁皂苷D单药组（P＜0.01）,并且两药联合组的抑制率显著高于索拉非尼单药组（P＜0.01）,说明两药联合对肝癌细胞的迁移抑制作用具有协同作用（Q=1.39＞1.15）;检测分析侵袭抑制率,索拉非尼单药组和两药联合组均显著高于白头翁皂苷D组（P＜0.01）,说明两药联合对肝癌细胞的侵袭抑制作用具有拮抗作用（Q=0.68＜0.85）。各组的VEGF-C、MMP-9表达情况：白头翁皂苷D单药组的MMP-9表达水平显著下降,索拉非尼组和两药联合组的VEGF-C、MMP-9及表达水平均显著下降（P＜0.05）;与白头翁皂苷D单药组比较,两药联合组的VEGF-C表达水平显著下降;两药联合组较白头翁皂苷D单药组VEGF-C水平显著下降（P＜0.05）。结论 白头翁皂苷D、索拉非尼单药以及两药联合对肝癌细胞BEL-7402细胞株的黏附、迁移、侵袭具有一定的抑制作用,且二者对肝癌细胞的黏附、迁移的抑制具有协同作用。     

丛生形盔珊瑚共附生真菌的分离及其抗菌活性的筛选

目的本试验旨在研究珊瑚共附生真菌抗菌活性。方法采用96孔板培养法结合酶标仪测定。以6种常见的致病或致食品腐败的细菌为指示菌,对其共附生真菌次级代谢产物进行抗菌活性筛选,对抗菌能力及抗菌谱进行研究。结果结果表明:共分离丛生形盔珊瑚共附生真菌19株,均具有不同程度的抗菌活性。高活性菌株占52.63%,其中4-4、4-5两菌株表现出良好的抗菌活性。结论丛生形盔共附生真菌是潜在的抗菌资源,具有较高的应用潜力和综合开发前景。     

右美托咪定对小鼠树突状细胞免疫功能的影响

目的研究右美托咪定对小鼠树突状细胞功能的影响及作用机制。方法分离获取小鼠骨髓原性树突状细胞鉴定成功后,体外分组培养,以正常培养基作为空白组,实验组为含有10μmol·L-1右美托咪定的正常培养基,对照1组和对照2组分别为含有10μmol·L-1酚苄明及同时含有10μmol·L-1右美托咪定和酚苄明的正常培养基,分别于96孔板培养24 h、Transwell 24孔板培养4 h和6孔板培养24 h。测定实验药物对小鼠树突状细胞活力、细胞迁移数量及肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)的影响情况。结果空白组、实验组、对照1组和对照2组的树突状细胞活力分别为(99.96±0.19)%,(62.61±0.08)%,(98.74±0.45)%和(79.25±0.23)%,树突状细胞迁移细胞数量分别为(3684.68±38.36),(1836.37±21.38),(3659.90±27.92)和(2670.06±27.61)个,树突状细胞培养上清液的TNF-α浓度分别为(67.26±1.51),(353.81±6.29),(67.92±1.45)和(169.63±2.89)pg·mL-1,IL-6浓度分别为(29.64±1.20),(511.29±12.97),(29.38±1.88)和(230.17±2.19)pg·mL-1,IL-1β浓度分别为(46.51±2.43),(436.97±5.86),(46.19±1.29)和(147.30±3.57)pg·mL-1,实验组与其他组比较,差异均有统计学意义(均P<0.05)。结论右美托咪定能够影响小鼠树突状细胞的活力、迁移能力以及促炎性细胞因子的分泌,其作用机制跟α肾上腺素受体的激活有关。     

硝酸镓对耐碳青霉烯肺炎克雷伯菌生长及其生物膜的抑制作用

目的 检测新型抗菌剂硝酸镓(gallium nitrate, GaN)对耐碳青霉烯肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumonia, CRKP)生长及其生物膜(biofilm, BF)的抑制作用。方法 收集2015-2016年安徽省地区34家医院临床分离的CRKP共82株，采用VITEK-2 Compact全自动微生物分析系统进行细菌鉴定，琼脂倍比稀释法进行药敏试验；微量肉汤稀释法测定GaN对CRKP的最低抑菌浓度(minimal inhibitory concentration, MIC)；96孔板结晶紫染色法检测82株实验菌株BF的A570值，筛选出BF强阳性菌株；检测1/2MIC的GaN对BF强阳性菌株的BF形成的抑制作用。比较GaN与庆大霉素(gentamicin, GEN)分别对CRKP的BF形成的抑制作用。激光共聚焦扫描显微镜观察GaN作用下的菌株生物膜三维立体结构。结果 临床分离的CRKP呈多重耐药。GaN对82株CRKP的MIC范围为0.0625~2μmol/mL，其中MIC50、MIC90分别为0.25和0.5μmol/mL。BF强阳性菌株所占比例为37.8%(31/82)。三维立体图片显示CRKP在不同GaN浓度作用下BF形成的量不同。1/2MIC的GaN可显著抑制CRKP的BF形成。GaN对CRKP的BF形成的抑制作用优于GEN。结论 GaN对CRKP生长及其BF有明显抑制作用。     

大黄等四种中草药体外抑菌活性比较

目的：研究大黄、金银花、黄连和鱼腥草四种中药材的体外抑菌活性。方法：采用琼脂扩散法和菌落计数法测定其对金黄色葡萄球菌和大肠杆菌的体外抑菌情况。结果：中药材对金黄色葡萄球菌和大肠杆菌均有不同程度的抑菌作用,其中大黄抑菌效果最好,其次为黄连和金银花等。结论：大黄等中药材可作为抑菌剂来开发,更好地应用于医疗与食品工业等方面。     

Cytotoxicity Assessment in Cultures of Differentiating Rodent Embryonic Cells

Several techniques for assessing the cytotoxicity of chemical agents in the micromass cell culture systems for rat embryo midbrain (CNS) and limb bud (LB) cells were compared. Cultures were treated with the antitubulin agent alben-dazole (ABZ) and its sulfoxide derivative (SOABZ) and cultured in Primaria 35 mm dishes, Primaria 96-well plates, or collagen-coated 96-well plates. Three assays for cytotoxicity were employed: staining with the vital dye neutral red (NR), mitochondrial reduction of MTT, and total culture protein levels. Comparison of the cytotoxicity IC₅₀ values was also made to the IC₅₀ value for differentiation. The IC₅₀ values for cytotoxicity and differentiation in CNS cultures were statistically equivalent regardless of the procedure employed. However, in LB cultures, the IC₅₀'s for cytotoxicity generated by NR staining of fixed cells in Primaria 96-well plates were significantly different from those for differentiation and total culture protein content. With a single exception (NR staining of unfixed ABZ-treated LBs in collagen-coated 96-well plates), no other cytotoxicity procedures yielded IC₅₀ values that differed significantly from those for differentiation. We conclude that the NR cytotoxicity procedure should be applied to micromass culture with caution due to the sensitivity of this assay to cell density, cell type, and the surface on which the cells are plated. Assaying for total culture protein content yields important supplementary information.     

Equilibrium drug solubility measurements in 96-well plates reveal similar drug solubilities in phosphate buffer pH 6.8 and human intestinal fluid

This study was conducted to develop a high throughput screening (HTS) method for the assessment of equilibrium solubility of drugs. Solid-state compounds were precipitated from methanol in 96-well plates, in order to eliminate the effect of co-solvent. Solubility of twenty model drugs was analyzed in water and aqueous solutions (pH 1.2 and 6.8) in 96-well plates and in shake-flasks (UV detection). The results obtained with the 96-well plate method correlated well (R(2)=0.93) between the shake-flask and 96-well plates over the wide concentration scale of 0.002-169.2mg/ml. Thereafter, the solubility tests in 96-well plates were performed using fasted state human intestinal fluid (HIF) from duodenum of healthy volunteers. The values of solubility were similar in phosphate buffer solution (pH 6.8) and HIF over the solubility range of 10(2)-10(5)μg/ml. The new 96-well plate method is useful for the screening of equilibrium drug solubility during the drug discovery process and it also allows the use of human intestinal fluid in solubility screening.     

黄芩素对人肝癌HepG2细胞生长的抑制作用

目的:研究黄芩素对人肝癌Hep G2细胞生长的抑制作用。方法:在培养Hep G2细胞的96孔板上加入药物,使黄芩素的终浓度分别为0、20、40、80、160μmol/L(即对照组与黄芩素1、2、3、4组)。采用MTT法检测细胞的存活情况,分光光度法检测含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-3、8、9的活性,并检测丙二醛(MDA)含量,超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性。结果:与对照组比较,培养24、48、72 h时,黄芩素2、3、4组各时间点的吸光度降低;培养24 h时,Caspase-3、8、9活性增强,MDA含量增加,SOD、GSH-Px活性减弱,差异均有统计学意义(P<0.01)。结论:黄芩素对Hep G2细胞生长具有抑制作用,其机制与增强Caspase-3、8、9活性,增加MDA含量,减弱SOD、GSH-Px活性有关。     

阿奇霉素对生物被膜菌产AmpC酶、超广谱β-内酰胺酶的影响

目的探讨生物被膜(biofilm,BF)菌的耐药机制及阿奇霉素对BF菌产AmpC酶、超广谱β-内酰胺酶(extended-spectrumβ-lactamases,ESBLS)的影响。方法应用改进的平板培养法建立大肠埃希菌BF模型,扫描电镜鉴定细菌BF。采用改良三维试验法检测ESBLS及AmpC酶。结果浮游大肠埃希菌(A组)单产AmpC,单产ESBLS酶及同时产ESBLS和AmpC酶的菌株分别占总菌株的5.0%(2/40)、20.0%(8/40)、5.0%(2/40);BF大肠埃希菌(B组)产酶菌株分别占总菌株的15.0%(6/40)、47.5%(19/40)、22.5%(9/40);阿奇霉素诱导BF菌(C组)产酶菌株分别占总菌株的7.5%(3/40)、25%(10/40)、12.5%(5/40)。对A组和B组、B组和C组的检出率两两分别进行χ2检验,结果均为P<0.05。结论阿奇霉素能够明显抑制BF大肠埃希菌产生ESBLS及AmpC酶。     

Development of a 7-Day, 96-Well Caco-2 Permeability Assay with High-Throughput Direct UV Compound Analysis

PURPOSE: The aim was to replace the traditional 21-day Caco-2 permeability protocol by a more high-throughput assay. METHODS: Caco-2 cells were seeded at high density in 96-well plates in novel cell culture boxes. After 7 days, drug permeability studies were performed. Samples were analyzed by a new UV detection method. RESULTS: With increased cell seeding density. functional Caco-2 monolayers with polarized efflux transporters were established after 7 days in 96-well polycarbonate filter plates in standard medium. For faster feeding and to eliminate medium replacement in each individual well, plates were completely submerged in medium in novel cell culture boxes, and only medium outside the plate was exchanged. For high-throughput sample analysis, a novel UV-transparent transport buffer was established that allowed direct quantification of permeated drug from its UV absorption. In vitro permeability studies analyzing 22 passively absorbed drugs in the new model correlated well with reported human permeability values (r2 = 0.8725). CONCLUSIONS: The new 7-day. 96-well Caco-2 permeability model tight to UV analysis offers considerable time, cost, and resource savings compared to the traditional model. It has a potential for automation and makes it possible to determine the permeability of passively diffusing compounds and to classify them according to the BCS in a truly medium- to high-throughput mode.     

Toxicity of chlorpyrifos against Rhyzopertha dominica and Tribolium confusum adults on different surfaces

The insecticidal efficacy of chlorpyrifos was evaluated as structural treatment on petri dish, concrete, mosaic and galvanized steel surfaces against adults of Rhyzopertha dominica F. and Tribolium confusum Jacquelin du Val. Chlorpyrifos was used at three concentrations of 0.01, 0.2 and 0.5?g AI m^?2 on the surfaces. Residual toxicity of chlorpyrifos was evaluated after 7, 15, 30 days and for petri dishes continued to 45 days postexposure. In both species, the mortality was significantly higher in treated petri dishes followed by galvanized steel and low level of mortality was noted in the concrete surfaces. The mortality of adults was extremely decreased at 30 days postexposure except for petri dishes. Results indicated that chlorpyrifos can be useful for stored product protection. However, further studies are required to evaluate the potential of chlorpyrifos in a silo-scale trial.