渗透压对痢疾志贺菌水通道蛋白glpF基因表达的影响

目的探讨细菌水-甘油通道蛋白（GlpF）的生理功能及其对生长繁殖的影响。方法将用简并PCR发现表达水通道蛋白glpF基因的痢疾志贺菌接种于不同渗透压的液体培养基和添加了GlpF功能抑制剂的相应培养基中,培养不同时间后取培养液检测其生长繁殖量,RT-PCR分析其GlpF的表达。结果痢疾志贺菌GlpF的表达随培养基渗透压的改变而变化,在低渗培养基中的表达低于在等渗环境中;在高渗透压的培养基中,其表达显著高于在等渗培养基中。在加入Hg2＋抑制剂抑制GlpF的表达后,在低渗培养基中,未明显影响细菌的生长繁殖,但在较高渗透压的培养基中,细菌的繁殖量显著少于在未加Hg2＋抑制剂的同样渗透压培养基中。结论在非等渗环境中,细菌GlpF的表达对细菌细胞内外水分的调节,维持胞内环境稳定起到重要作用,尤其在高渗透压环境中更为明显。     

生物膜定量分析仪对不同细菌早期生物膜形成能力的比较研究

目的通过生物膜定量分析仪来观察铜绿假单胞菌(Pseudomonas aeruginosa PAO1),变形链球菌(Streptococcus mutans UA159)以及大肠埃希菌(Escherichia coli MG1655)生物膜形成能力的不同,并以各菌株的吸光度值A600为参考,对3种菌株早期生物膜形成能力进行比较。方法通过向生物膜培养悬液中加入与细菌直径相近的磁性小珠,利用这些小珠在磁场中受到生物膜的位移约束力的原理,采用生物膜定量分析仪,定量比较3种菌株在生物膜形成上的差别。结果实验发现铜绿假单胞菌PAO1和大肠埃希菌MG1655的细菌增长速度基本相同,但铜绿假单胞菌PAO1的生物膜形成明显快于大肠埃希菌MG1655。大肠埃希菌MG1655和变形链球菌UA159的生物膜形成速度基本相同,但大肠埃希菌MG1655的细菌增长速度明显高于变形链球菌UA159。结论不同细菌有各自的生物膜形成模式。生物膜定量分析仪作为一种高效简便的检测手段,可用于生物膜早期形成的动态分析。     

Vgb在大肠埃希菌中表达及生物活性的研究

目的将vgb克隆到大肠埃希菌表达载体pQE-30中,研究vgb表达产物对细胞摄氧能力的影响。方法利用PCR和基因重组技术克隆vgb与大肠埃希菌表达质粒pQE V,大肠埃希菌转化采用CaCl2法,VHb活性分析采用CO示差光谱法。结果克隆了vgb和重组质粒pQE V,vgb基因在大肠埃希菌中获得表达,在A420 nm处达到典型吸收峰。结论 vgb在大肠埃希菌中表达产物加强了对氧的摄取能力,对解决细胞高密度发酵培养中氧需矛盾、促进代谢产物的产率具有非常重要的应用价值。     

Zonulin介导的肠道微生物对肠上皮细胞屏障功能的调节

利用Caco-2肠上皮细胞单层屏障模型研究四种肠道微生物对肠道屏障的影响。实验设置空白对照组(control)、大肠埃希菌组(Eco)、肺炎克雷伯菌组(Kpn)、粪肠球菌组(Efa)、乳酸杆菌组(Lac)。加入各组细菌共培养,结果表明大肠埃希菌、肺炎克雷伯菌、粪肠球菌均引起单层细胞跨膜电阻值(transepithelial electrical resistance,TEER)明显下降(P0.001);Eco组、Kpn组作用后细胞释放zonulin蛋白增加(P0.01),Efa组作用于Caco-2细胞单层后,zonulin释放量较对照组升高,但差异无统计学意义(P0.05);Eco组、Kpn组三种紧密连接蛋白occludin、claudin-1、ZO-1表达明显减少,Efa组、Lac组occludin、claudin-1表达与空白对照组相比无明显变化,胞浆蛋白ZO-1表达降低;细菌与细胞共培养6 h后免疫荧光观察紧密连接蛋白分布情况,Eco组、Kpn组可见荧光强度减弱,荧光不连续,甚至有缺口及裂隙,Efa组、Lac组荧光强度稍减弱,但仍沿胞膜分布,条带较清晰,与空白对照组差异不明显。肠道四种微生物中大肠埃希菌、肺炎克雷伯菌可能通过zonulin途径降低紧密连接蛋白表达、改变蛋白分布,最终导致肠道屏障功能受损,益生菌对肠道屏障无损伤作用。     

实时荧光定量PCR检测非酒精性脂肪性肝炎新西兰兔肠道内菌群的变化

目的 应用实时荧光定量-聚合酶链反应法(fluorescent real-time polymerase chain reaction,RT-PCR)法对高脂喂养诱导非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)新西兰兔的粪便中大肠埃希菌、乳酸杆菌及双歧杆菌属的数量进行定量检测分析.方法 14只雄性新西兰兔随机分为模型组和对照组,分别用普通饲料和高脂饲料喂养12周,取粪便提取细菌组DNA,根据细菌的16S rDNA基因序列设计大肠埃希菌、乳酸杆菌及双歧杆菌属的种属特异性引物,进行实时荧光定量PCR反应,定量分析不同细菌的数量.结果 模型组动物病理学检测肝细胞脂肪变性,提示模型成功.对照组与模型组动物粪便中细菌数量分别为大肠埃希菌(11.48 ±1.09,7.39 ±0.81)、乳酸杆菌(4.94±0.95,5.65 ±0.91)、双歧杆菌属(4.07 ±0.97,6.45±0.90),经统计学分析表明模型组较对照组大肠埃希菌数量明显增多(t=-3.282,P =0.013),而乳酸杆菌、双歧杆菌属数量变化无统计学意义(t1=-1.204,P1=0.268;t2=0.423,P2=0.683).结论 NASH模型组粪便内大肠埃希菌数量较正常组增多,提示大肠埃希菌数量与非酒精性脂肪性肝病(Nonalcoholic fatty liver disease,NAFLD)有关,而乳酸杆菌及双歧杆菌数量无明显变化,其与NAFLD的关系有待进一步研究.     

党参多糖对双歧杆菌和大肠埃希菌体外生长的影响

目的探讨党参多糖体外对双歧杆菌和大肠埃希菌生长的影响。方法每隔12 h采用分光光度法测600 nm细菌培养液A值,气相色谱法测培养48 h后的双歧杆菌培养液中乙酸含量。结果党参多糖体外对大肠埃希菌没有促进或抑制生长的作用,对双歧杆菌有促进生长的作用,在中药作用下,双歧杆菌代谢的乙酸含量与其数量呈正相关关系。结论党参多糖能够通过促进双歧杆菌的生长,从而增加乙酸的代谢,增强双歧杆菌的定植抗力,对肠道一些致病菌发挥生物拮抗作用。     

白花丹素对E.coli藻酸盐合成的抑制作用

细菌分泌的胞外多糖在生物被膜的形成和发展过程中发挥着重要作用。通过测定白花丹素对大肠埃希菌10389菌株(E.coli 10389)藻酸盐合成的影响及其对rse A和rpo E基因表达量的影响,探讨白花丹素对大肠埃希菌生物被膜(biofilm,BF)形成的抑制作用及机制。研究结果显示,白花丹素能抑制E.coli 10389生物被膜的形成,其抑杀E.coli 10389的最低抑菌浓度(minimum inhibitory concentration,MIC)和最低杀菌浓度(minimal bactericidal concentration,MBC)分别为16和64μg/mL。白花丹素对成熟BF内的细菌也有抑制和杀灭作用,其抑杀E.coli 10389成熟BF内细菌的MIC和MBC分别为64和128μg/mL。白花丹素能够抑制E.coli 10389藻酸盐的合成,其中1/2MIC的白花丹素作用E.coli 10389 24 h后,与对照组比,藻酸盐的合成量降低了34.83%(P0.01)。白花丹素可显著影响E.coli 10389 rse A和rpo E基因的相对表达量,其中1/2MIC的白花丹素作用E.coli 10389 24 h后,与对照组相比,rse A的表达量上调了17.43%,rpo E的表达量降低了12.8%(P0.05)。结果表明,白花丹素能够抑制E.coli 10389 BF的形成,其作用机制可通过影响rse A和rpo E的基因表达量,进而抑制藻酸盐的合成来抑制大肠埃希菌生物被膜的形成。     

1株枯草芽胞杆菌体外拮抗6种肠道致病菌的研究

目的研究枯草杆菌BS-3株对大肠埃希菌等6种肠道致病菌的拮抗作用。方法通过在体外BS-3菌株分别与大肠埃希菌等6种致病菌混合培养后,观察不同时间内各菌的菌量变化。结果BS-3菌株与6种肠道致病菌混合培养24、48、72和96h,其菌量逐渐增加;6种致病菌的菌量随着培养时间延续逐渐减少,其中产毒性大肠埃希菌、致病性大肠埃希菌和宋内志贺菌与对照组比较差异更明显。结论BS3菌株在培养生长过程中,可抑制大肠埃希菌等6种肠道致病菌的生长。     

人乳头瘤病毒18型L1蛋白在大肠埃希菌中的可溶性表达及病毒样颗粒的形成

目的利用大肠埃希菌系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,纯化和重组装获得HPV18病毒样颗粒(VLPs),为进一步研制HPV18基因工程疫苗奠定基础。方法首先按大肠埃希菌密码子偏好进行HPV18L1全基因合成,经PCR扩增出截短的HPV18L1基因,构建重组表达载体PET30a-L1,通过优化表达在大肠埃希菌BL21中可溶性表达L1蛋白,其次采用硫酸铵沉淀、离子交换层析、疏水层析后,获得高纯度的的L1蛋白,再通过解聚和重聚获得VLPs。结果全基因优化并截短的HPV18L1蛋白在大肠埃希菌系统中以可溶形式表达,纯化后的蛋白纯度达到90%以上,电镜下观察到直径为60 nm的VLPs颗粒。结论利用大肠埃希菌系统可溶性表达非融合HPV18L1蛋白,并获得均一的VLPs颗粒,为疫苗的开发奠定基础。     

小鼠肠道菌群的多样性不因大肠埃希菌O157:H7感染而降低

目的探讨大肠埃希菌O157∶H7感染对小鼠肠道菌群的影响。方法采用大肠埃希菌O157∶H7菌悬液,灌胃制备感染小鼠。随后测定小鼠的生长情况和死亡率;收集肠道菌群,DGGE检测小鼠个体间肠道菌群的变化,高通量测序检测组间感染小鼠肠道菌群的变化。结果大肠埃希菌O157∶H7感染小鼠与正常对照组小鼠相比,小鼠体重、死亡率差异无统计学意义。DGGE结果显示,在不同小鼠个体之间,它们的肠道细菌的种类和数量存在明显差异。高通量测序结果显示,大肠埃希菌O157∶H7感染小鼠肠道菌群的多样性高于对照组,但优势菌的丰度有显著变化。结论大肠埃希菌O157∶H7感染,尽管极大改变了小鼠肠道优势菌群的比例,但没有降低其多样性。另外,小鼠个体间肠道菌群多样性的差异在微生态研究中不容忽视。     

Osmotic modification of thermal damage in Escherichia coli bacteria at various pH values of the media

A study was made of the influence of media with different osmotic pressure on cell survival and on optic density of supernatants from Escherichia coli B/r and E. coli Bs-1 cell suspensions heated under different pH values of media. Hyperthermia induced cell death accompanied with the loss of optically active (lambda = 260 nm) material. Both cell damage effects were increased in acid and alkaline conditions, compared to neutral condition of heating. Hypertonic media results in a decrease in thermic cell death and loss of cell substances. Under this condition, the protection influence of high osmotic pressure was seen to increase significantly in acid and alkaline conditions of heating, compared to neutral condition. It has been proposed that a higher thermal damage of microorganisms in acid and alkaline beating conditions and protection influence of hypertonic media, especially expressed in acid and alkaline medium, is caused to a great extent by the status of osmotic cell homeostasis.     

Effects of salinity manipulations on blood pressures in an osmoconforming chordate,the hagfish, <Emphasis Type="Italic">Eptatretus cirrhatus</Emphasis>

Arterial and venous pressures were measured in hagfishes subjected to acute changes in salinity. The osmotic pressure of the seawater (SW) was increased or decreased by approximately 10%. Sixty minutes after the change in medium osmolarity the osmotic pressure of the blood corresponded with that of the medium. Following transfer to 90% SW all measured parameters changed as predicted for a passive increase in blood volume, apart from the pressure in the posterior cardinal vein (PCV) which fell. By 2 h dorsal aortic (DA) pressure and pressure in the PCV and supraintestinal vein had returned to pre-change values. In contrast, following exposure to 110% SW, pressures fell and apart from the supraintestinal vein they remained low at 120 min. At 24 h, DA pressure was lower than pre-change values for both groups. The data are consistent with the concept of central venous tone being regulated in hagfishes, which cope better with volume expansion than volume depletion.     

大肠埃希菌生物被膜体外模型的建立及黄连水提物对其抑制作用的初步研究

建立大肠埃希菌生物被膜（biofilm,BF）在Ф30培养皿和96孔板表面形成的体外模型,并开展黄连水煎液对BF抑制作用的初步研究。选取临床分离的大肠埃希菌菌株,在Ф30培养皿中采用LB（Luria-Bertani medium）培养基系统复制体外BF模型,经银染后利用显微摄影系统观察BF形态;在96孔板中采用LB培养基系统复制体外BF模型,采用MTT法利用酶标仪测定OD值。将黄连水煎液作用于大肠埃希菌生物被膜体外模型,分别采用MTT法和银染法考察黄连水提物对大肠埃希菌生物被膜的影响。Ф30培养皿表面可以观察到黑染呈棉絮状的膜样物而空白组没有此样物质;96孔板中,模型组的OD值为4．191,空白组的OD值为0．069;药物作用24h后黄连组的BF明显少于空白对照组;80mg／mL的黄连水煎液即开始对大肠埃希菌生物被膜有抑制作用,抑制率为20．8％,生药浓度达到180mg／mL时为最佳抑制浓度,抑制率为70．23％。Ф30培养皿和96孔板表面可以形成大肠埃希菌生物被膜,黄连水煎液可以抑制和破坏早期及成熟BF,且其抑制作用表现出了一定的量效关系,此方法对黄连水煎液作用于大肠埃希菌生物被膜是可行且稳定的,为应用于临床试验奠定基础。     

The effect of media tonicity on Escherichia coli resistance to heating with different gradient

The influence of NaCl water solutions and glycerine hypertonic concentration on the survival of bacteria Escherichia coli B/r heated with different values of heat drop was investigated. It was shown that the transfer of cell suspensions from isotonic conditions to media with raised osmotic pressure, preliminarily heated up to 60 degrees C, and the following heating at this temperature inhibited differences in cell sensitivity to heating at different heat drop. Unlike, it was found that the transfer of cell suspensions from isotonic conditions to hypertonic media before and after heating at 60 degrees C increased differences in resistance of these microorganisms to heating at different heat drop. It is proposed that different resistance of bacteria to damaging action of hyperthermia at different heat drop, and a modified influence of hypertonic solutions on these differences may be due to heat induced destabilization of cell osmotic homeostasis. The extent of expression of this destabilization may be determined by a quantitative ratio of osmotic pressure values in the cell-suspension medium system in particular temperature and tonic environmental conditions.     

不同时间服用比索洛尔对非杓犁高衄压缸压的影响

目的：观察不同给药时间分别给予比索洛尔对非杓型原发性高血压患者的降压疗效和血压节律恢复的影响。方法：选取60例非杓型高血压患者,采取随机平行对照试验,观察比索洛尔（n=30）每日早晨（8：00）给药2．5—10mg、比索洛尔（n=30）每日夜间（20：00）给药2．5—10mg治疗8周后的降压疗效。结果：两种给药方法均能降低非杓型高血压患者的全天血压（P〈0．05）。两种给药方法在白天的血压控制上无显著性差异（P〉0．05）,但在夜间血压的控制上夜间服药降压效果具有显著性差异（P〈0．05）。夜间服药在血压节律恢复方面优于早晨服药（P〈0．05）,早晨服药组有10例恢复杓型,夜间服药组有19例恢复杓型。结论：比索洛尔的两种给药方式均能安全有效的降压,但对于非杓型高血压患者夜间服药优于早晨服药,更有利于血压节律的恢复。     

An essential role of phosphatidylglycerol in the formation of the osmotically stable liposomes of Escherichia coli phospholipids

A temperature sensitive auxotroph of Escherichia coli K-12 requiring unsaturated fatty acids can grow normally at 28 degrees C, but requires an osmotic stabilizer such as a high amount of salt or sugar in the medium for the growth at 42 degrees C. Namely, the apparent osmotic stability of the cells at 28 degrees C and 42 degrees C is quite different. The osmotic properties of liposomes of the phospholipids extracted from these cells were investigated. The osmotically induced volume change of the multilamellar liposomes was examined by the turbidimetric method. The liposomes prepared from cells grown at 28 degrees C can swell and shrink under a wide range of hypo-and hypertonic conditions. However, those from cells grown at 42 degrees C could not swell under hypotonic conditions. These results exhibit a good correlation between the apparent osmotic stability of E. coli cells and the osmotic properties of the liposomes prepared from the extracted total phospholipids. To clarify the role of each phospholipid component, the osmotic properties of the liposomes reconstituted from the purified phospholipid species were further investigated. The results clearly showed that phosphatidylglycerol is the key factor that stabilizes the membranes of E. coli phospholipids against osmotic pressure.     

Effect of the Osmotic Pressure of the Growth Medium on fabB Mutants of Escherichia coli

Temperature-sensitive, unsaturated fatty acid (fabB) auxotrophs of Escherichia coli can grow at the restrictive temperature in the absence of unsaturated fatty acid in a medium with a high osmotic pressure. If a mutant culture was starved for unsaturated fatty acids and harvested just before the lysis started, the fatty acid composition of the cells was the same as that of cells grown until late log phase in a high-osmotic medium. Evidence is presented that the in vivo unsaturated fatty acid biosynthesis is significantly increased in a high osmotic medium. The increase is probably due to a partial activation of the temperature-sensitive fabB product. Besides the stimulation of the temperature-sensitive fabB product, a minimal osmotic pressure of the medium appeared to be necessary to allow growth of cells containing lipids with a changed fatty acid composition. fabA mutants are unable to grow in a high-osmotic medium in the absence of unsaturated fatty acids. No increase in the in vivo unsaturated fatty acid biosynthesis could be detected in the temperature-sensitive fabA mutants.     

The 6.9-A structure of GlpF: a basis for homology modeling of the glycerol channel from Escherichia coli

The three-dimensional structure of GlpF, the glycerol facilitator of Escherichia coli, was determined by cryo-electron microscopy. The 6.9-A density map calculated from images of two-dimensional crystals shows the GlpF helices to be similar to those of AQP1, the erythrocyte water channel. While the helix arrangement of GlpF does not reflect the larger pore diameter as seen in the projection map, additional peripheral densities observed in GlpF are compatible with the 31 additional residues in loops C and E, which accordingly do not interfere with the inner channel construction. Therefore, the atomic structure of AQP1 was used as a basis for homology modeling of the GlpF channel, which is predicted to be free of bends, wider, and more vertically oriented than the AQP1 channel. Furthermore, the residues facing the GlpF channel exhibit an amphiphilic nature, being hydrophobic on one side and hydrophilic on the other side. This property may partially explain the contradiction of glycerol diffusion but limited water permeation capacity.