Growth of uropathogenic Escherichia coli strains at solid surfaces

The adhesion and growth of two catheter-associated (O2K2 and O83K?) and two non catheter-associated (O111K58 and 0157K-) uropathogenic Escherichia coli strains on glass, poly(methyl methacrylate) (PMMA), a negatively charged copolymer of MMA and methacrylic acid (MAA) and a positively charged copolymer of MMA and trimethylaminoethyl methacrylate chloride (TMAEMA-Cl) were studied. The solid surfaces were placed in a parallel plate perfusion system. After preadhesion of the bacteria onto the surfaces, growth was initiated by perfusing the system with MacConkey broth. Growth was measured by counting adherent bacteria as a function of time. Bacterial strains were characterized by means of water contact angle, microbial adhesion to hydrocarbon (MATH), anion exchange resin retention (ARR) and zeta potential measurements. Solid surfaces were characterized by means of water contact angle and zeta potential measurements. The catheter-associated strains had significantly higher water contact angles, zeta potentials and ARR values than the non catheter-associated strains. Non catheter-associated strains did not grow at the surfaces used. Catheter-associated strains did not grow at the positively charged surface but exhibited growth at the other surfaces. Strains grew more rapidly at surfaces with a relatively high negative zeta potential and a low water contact angle than at surfaces with a relatively low negative zeta potential and a high water contact angle. The growth of strain O2K2 on glass was significantly reduced when urine instead of MacConkey broth was used as perfusion medium.     

Hemocompatibility studies of surface-treated polyurethane-based chronic indwelling catheters

The objectives of this research were to evaluate and compare the interactions of several polyurethane-based central venous catheter materials with blood. Specifically, measurements of fibrinogen adsorption, platelet adhesion, kallikrein generation, and fibrinopeptide A (FPA) release were performed. The catheter materials examined in this study included: platinum-cured, 50 shore A durometer, barium sulfate-filled, silicone (SI); Tecoflex EG85A-B20 polyurethane (PU); PU catheters whose outer surface had been impregnated with ion beam-deposited silver atoms (AgI and AgII); PU catheters coated with a hydrophilic, polyacrylic acid polymer (UC); PU catheters coated with an air-cured PTFE emulsion (CS); and PU catheters coated with an aminofunctional dimethylsiloxane copolymer (JG). The time course of fibrinogen adsorption from plasma to the SI, JG, PU, and CS materials was similar, with CS exhibiting the least amount of adsorbed fibrinogen after 1 h (65±4.7 ng cm^-2) and PU the greatest (144±16.5 ng cm^-2). After 90 min of contact, AgI and AgII exhibited the greatest number of adherent platelets, levels that were approximately two to three times higher than those on the other catheter materials. With the exception of UC and PU, which caused kallikrein generation levels approximately half that of the positive (glass) control, little kallikrein formation was observed for any of the materials relative to the negative control. Finally, FPA generation was greatest using the SI, CS, and PU materials, with the latter causing the production of almost four times the amount of FPA as the negative control. This preliminary assessment of the hemocompatibility of the various catheters suggests that the surface treatments did not adversely affect their interactions with blood components; further investigations of these materials are therefore warranted in order to completely characterize their behavior prior to use in clinical situations.     

神经细胞黏附分子通过非Wnt依赖的β-catenin途径促进小鼠黑色素瘤细胞增殖

 目的：研究神经细胞黏附分子（neural cell adhesion molecule, NCAM）对小鼠黑色素瘤B16-F0细胞增殖的影响及其分子机制。方法：采用RNA干扰在B16-F0细胞中基因沉默NCAM,通过MTT和软琼脂克隆形成实验考察细胞增殖能力的变化;通过小鼠皮下肿瘤移植实验考察体内黑色素瘤生长的变化;使用免疫印迹筛选出受NCAM影响的信号分子,在此基础上使用RNA干扰和高表达实验确定介导NCAM调控增殖的主要信号分子。结果：NCAM基因沉默后B16-F0细胞的增殖能力和克隆形成能力明显降低,细胞在小鼠皮下形成的黑色素瘤的生长也明显受到抑制。其中,β-catenin介导了NCAM对于B16-F0细胞增殖的调控,但NCAM对于β-catenin的调控不依赖于经典的Wnt通路。结论：NCAM通过Wnt非依赖性的β-catenin通路促进小鼠黑色素瘤细胞的增殖。     

左卡尼汀通过抑制钙/钙调素依赖蛋白激酶Ⅱ信号通路抑制过氧化氢诱导的大鼠心肌细胞凋亡

 目的： 观察左卡尼汀对过氧化氢（H2O2）诱导的大鼠心肌细胞凋亡的保护作用及其机制。方法：利用200 μmol/L H2O2刺激12 h,建立体外原代培养新生乳鼠心肌细胞凋亡模型。Ca2+螯合剂1,2-双(2-氨基苯氧基)乙烷-N, N, N′, N′-四乙酸(BAPTA)、钙调素依赖蛋白激酶II (CaMKII)特异性抑制剂KN93及左卡尼汀分别于加入H2O2前30 min或1 h加入,以检测这3种药物对H2O2刺激下心肌细胞活力、细胞凋亡、细胞内静息钙浓度（［Ca2+］i）及磷酸化CaMKII (p-CaMKII)表达的影响。利用MTT比色法检测心肌细胞活力;流式细胞仪检测细胞凋亡率;利用激光共聚焦扫描检测［Ca2+］i;蛋白质免疫印迹法检测cleaved caspase-3及p-CaMKII的表达。结果：模型组经200 μmol/L H2O2作用12 h后,细胞活力显著下降,细胞凋亡率显著增加。BAPTA、KN93及左卡尼汀预处理显著抑制上述细胞损伤。进一步研究发现,H2O2 诱导的 ［Ca2+］i水平升高、cleaved caspase-3及p-CaMKII的表达增加均可被上述3种药物不同程度地抑制。结论：左卡尼汀可抑制H2O2所致的心肌细胞凋亡,该心肌保护作用可能与其抑制Ca2+/CaMKⅡ信号通路有关。     

SB203580对急性肺栓塞后肺动脉高压及MCP-1表达的影响

 目的：观察单核细胞趋化蛋白1(monocyte chemoattractant protein-1, MCP-1)与急性肺栓塞（pulmonary thromboembolism, PTE）后肺动脉高压形成的关系;探讨p38丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）特异性抑制剂SB203580对急性PTE后肺动脉高压及MCP-1表达的影响。方法：采用自体血栓回输法复制Sprague-Dawley大鼠急性PTE模型;将大鼠随机分成5组,每组观察1 h、4 h和8 h 3个时点;急性PTE模型复制前1 h,分别对MCP-1中和抗体C1142组及SB203580组进行药物预处理;在1 h、4 h和8 h检测各组肺动脉平均压力（mean pulmonary artery pressure, MPAP）和MCP-1 mRNA及蛋白表达。结果：（1）在相同时点,急性PTE组MPAP和MCP-1 mRNA及蛋白表达均较溶剂对照组显著升高（P<005）;（2）在相同时点,C1142组及SB203580组MPAP和MCP-1 mRNA及蛋白表达均较急性PTE组显著降低（P<005）。结论：（1）急性PTE后MCP-1的大量表达参与急性PTE性肺动脉高压的形成;（2）SB203580可能通过p38 MAPK信号转导通路,下调MCP-1表达,降低急性PTE肺动脉压力。     

RXR激动剂通过抑制PKC激活对抗高糖诱导的大鼠血管平滑肌细胞增殖

 目的:探讨视黄醇类X受体 (RXR)激动剂对高糖诱导的大鼠主动脉平滑肌细胞(RASMCs)增殖的影响及其作用机制。方法:体外组织块干涸法培养RASMCs,以25 mmol/L葡萄糖干预,模拟糖尿病患者体内环境,通过WST-1法检测细胞增殖活性,BrdU插入法测定细胞DNA合成,流式细胞术检测细胞周期进程。用免疫印迹杂交方法检测细胞周期蛋白依赖激酶2(CDK2)、细胞周期蛋白依赖激酶抑制物p27Kip1的蛋白表达及蛋白激酶C (PKC)的磷酸化水平。结果:(1) 在高糖环境(葡萄糖终浓度为25 mmol/L)下,RASMCs的增殖活性、DNA合成速率及其在细胞周期S期的分布比例均显著增加;(2) 高糖显著增加RASMCs内CDK2蛋白的表达,但明显降低p27Kip1的蛋白表达水平;(3) RXR天然配体9-顺式维甲酸(9-cis-RA)可显著抑制高糖诱导的RASMCs增殖活性增强、DNA合成加速及RASMCs在细胞周期S期分布比例的增加幅度,且具有浓度依赖性;10-7mol/L浓度的SR11237(RXR特异性配体)与等浓度的9-cis-RA具有相似的抑制效应;(4) 9-cis-RA 和SR11237均可显著抑制高糖诱导的CDK2蛋白表达水平的增加幅度,同时上调高糖环境下p27Kip1蛋白的表达;(5) PKC抑制剂(PKC inhibitor peptide, 20 μmol/L)显著抑制高糖环境下RASMCs的增殖活性和CDK2蛋白的表达,但明显增加高糖条件下p27Kip1蛋白的表达;(6) 9-cis-RA 和SR11237可抑制高糖诱导的PKC蛋白磷酸化。结论: PKC的活化参与了高糖诱导下RASMCs的增殖过程。RXR激动剂通过抑制PKC活化对抗高糖诱导的血管平滑肌细胞增殖。     

Chlamydia abortus OmcB protein is essential for adhesion to host cells

Chlamydia abortus (C. abortus) is one of the most important zoonotic pathogens, causing a number of serious diseases. The adhesion of C. abortus to host cells is the first and crucial step in the process of infection. Outer membrane protein 2 (OmcB) is the second most abundant outer membrane protein. It has been shown to be an important adhesin of Chlamydia trachomatis and Chlamydia pneumoniae. In the present study, the OmcB gene of C. abortus was cloned and expressed in Escherichia coli, and the recombinant OmcB protein with His-tag was used to prepare polyclonal antibodies. Infectivity inhibition assays carried out with C. abortus in the presence of recombinant OmcB showed a considerable reduction (∼50%) in infectivity. Using anti-OmcB serum in infectivity inhibition assays resulted in a 30% reduction in infectivity. Anti-OmcB serum and recombinant OmcB protein in infection inhibition assays showed that OmcB is a surface-exposed protein that functions as an adhesin. The constructed deletion variant of the OmcB motif for infection inhibition assays showed that the first XBBXBX motif of the C. abortus OmcB protein is essential for binding to host cells.     

Rho相关激酶抑制剂Y27632对小鼠腔前卵泡体外发育的作用

目的 探讨Rho相关激酶（ROCK）抑制剂Y27632在小鼠腔前卵泡体外发育中的作用。 方法 取出生12.5 d的小鼠卵巢,机械法收集单枚腔前卵泡,采用超低吸附96孔板模拟3D环境对其进行培养。设置对照组和含有Y27632的实验组,通过形态学观察、卵泡直径的变化、成熟卵泡的数量、成熟卵母细胞纺锤体的变化等来评估卵泡的整体发育情况。采用Real-time PCR技术检测颗粒细胞中卵泡刺激素受体（FSHR）、卵母细胞中骨形态发生蛋白15（BMP15）、生长分化因子9（GDF9）等以及凋亡相关基因（Bad、Bax和Caspase-3）的表达情况,同时,用细胞存活染料检测卵泡发育过程中颗粒细胞的凋亡情况。 结果 ROCK抑制剂Y27632对卵泡直径增长和基本发育指标（存活数、成腔率和成熟率）无明显影响（P>0.05）,但对照组卵母细胞成熟后纺锤体组装出现异常。培养8 d后,与对照组相比,实验组颗粒细胞特异性基因FSHR上调;卵母细胞特异性基因BMP15和GDF9上调,而叉头框O3（FoxO3)下调;凋亡基因Bax、Bad和Caspase3表达下调,实验组卵泡内的颗粒细胞凋亡明显少于对照组。 结论 小鼠卵泡体外发育过程中,ROCK抑制剂Y27632能够抑制颗粒细胞的凋亡,防止卵母细胞纺锤体组装异常,进而提高卵泡体外发育质量。     

芦荟凝胶多糖对急性放射性皮肤损伤模型的修复作用及机制研究

目的研究芦荟凝胶多糖基于磷脂酰肌醇3-激酶(PI3K)-蛋白激酶B(Akt)-雷帕霉素靶蛋白(mTOR)信号通路对急性放射性皮肤损伤模型的修复作用。方法选取40只雄性Wistar大鼠,10只为正常组(等体积生理盐水),其余30只建立急性放射性皮肤损伤模型,随机分为模型组、低剂量组、高剂量组,每组10只。模型组和正常组均给予等体积生理盐水干预,低剂量组、高剂量组分别以单层纱布浸润0.4 mg/mL、0.8 mg/mL的芦荟凝胶多糖外敷干预。记录创面愈合时间、放射性皮肤损伤评级,检测创面微血管密度、血管内皮生长因子(VEGF)含量,病理学观察创面组织细胞形态,Western blot检测PI3K-Akt-mTOR信号通路相关蛋白表达。结果低剂量组和高剂量组创面愈合时间均短于模型组,且高剂量组创面愈合时间显著短于低剂量组(P<0.05);与模型组相比,正常组大鼠0级较多,Ⅳ级较少;低剂量组和高剂量组大鼠Ⅳ级较少(P<0.05)。模型组、低剂量组、高剂量组创面微血管密度、VEGF、PI3K、Akt蛋白表达量低于正常组,mTOR蛋白表达量高于正常组;低剂量组、高剂量组创面微血管密度、VEGF、PI3K、Akt蛋白表达量高于模型组,mTOR蛋白表达量低于模型组;高剂量组创面微血管密度、VEGF、PI3K、Akt蛋白表达量高于低剂量组,mTOR蛋白表达量低于低剂量组。结论高剂量芦荟凝胶多糖可通过调控PI3K-Akt-mTOR信号通路促进急性放射性皮肤损伤模型创面新生血管形成,加快创面修复。