siRNA干扰铜绿假单胞菌MexA-MexB-OprM外排泵mexB基因表达的研究

目的 探讨RNA干扰技术对铜绿假单胞菌MexA-MexB-OprM外排泵mexB基因表达及有关功能的影响.方法 设计并合成4条小分子干扰RNA(small interfering RNA,siRNA)(siRNA1、siRNA2、siRNA3和siRNA4)序列,构建干扰RNA( short hair pin RNA,shRNA)表达载体,将带有siRNA质粒电转入PAO1和临床耐药株PAO3.应用E-test方法检测PAO1和PAO3抗生素MIC的变化.用半定量RT-PCR和定量PCR方法检测mexB的表达变化.结果 经酶切鉴定siRNA表达载体构建成功,并明显提高PAO和临床耐药株PAO3对抗生素的敏感性.RT-PCR结果显示干扰后的PAO1和临床耐药株PAO3 mexB基因表达明显下降(P＜0.05).结论 siRNA成功干扰MexA-MexB-OprM外排泵mexB基因的表达,提高铜绿假单胞菌对抗生素的敏感性,为临床上治疗铜绿假单胞菌感染,降低其耐药性提供了新的思路.     

pvdQ基因对铜绿假单胞菌群集运动的作用

目的 探讨铜绿假单胞菌pvdQ(PA2385)基因对群集运动的作用.方法 构建pvdQ基因表达质粒pME6032-pvdQ并鉴定,采用电穿孔法将带有pvdQ基因的质粒转入铜绿假单胞菌野生株PAO1中,构建pvdQ-高表达株.同时将空质粒pME6032采用电穿孔法转入PAO1中,构建pME6032空质粒株.选择含有反向筛选基因sacB的pEX18Gm质粒作为自杀载体,构建缺失pvdQ基因的铜绿假单胞菌无痕缺失突变株,溶菌肉汤(LB)液过夜培养,观察菌株的直径变化.统计学处理采用单因素方差分析.结果 经鉴定,成功构建pvdQ-高表达株和pvdQ-突变株,比较4株菌的群集运动直径变化:PAO1为(20.52±1.80)mm,pME6032空质粒株为(19.39±2.10)mm,pvdQ-高表达株为(51.20±2.16)mm,pvdQ-突变株为(3.30±0.55)mm.pME6032空质粒株与野生株PAO1相比,直径无显著变化(t=-0.1493,P>0.05),pvdQ-突变株与野生株PAO1相比,直径减小(t=2.8525,P<0.05),pvdQ-高表达株与野生株PAO1相比,直径增大(t=1.4230,P<0.05).结论 pvdQ基因参与调控铜绿假单胞菌群集运动,且能促进铜绿假单胞菌群集运动.

Abstract：

Objective To investigate the effect of pvdQ(PA2385)gene on Pseudomonas aeruginosa swarming motility.Methods The plasmid pME6032 with pvdQ gene was constructed and identified,then transformed into Pseudomonas aeruginosa PAO1 using the eleetroporation to build pvdQ-overexpression strain.The pME6032-PAO1 strain was constructed with the same method.The cloning plasmid pEX18Gm containing sacB was successfully used to construct unmarked deletion mutant of pvdQ gene and pvdQ mutant strain. Bacteria were inoculated in LB and were cultured overnight.The clones were measured for the diameter of the swarming zone.The statistical analysis was done using one-factor ANOVA.Results Strains of pvdQ-overexpression and pvdQ-mutant were successfully constructed and confirmed by polymerase chain reaction(PCR).The four strains were compared for the swarming motility by changes in diameter:PAO1(20.52±1.80)mm,pME6032-PAO1 strain(19.39±2.10)mm,pvdQ-overexpression strain(51.20±2.16)mm,pvdQ-mutant strain(3.30±0.55)mm.The diameter of pME6032-PAO1 strain was not significantly different from that of wild strain PAO1(t=-0.1493,P>0.05).However,the diameter of pvdQ(Q-mutant strain was significantly shorter than that of wild strain PAO1(t=2.8525,P<0.05).while the diameter of pvdQ-overexpression strain was longer than that of the wild strain PAO1(t=1.4230,P<0.05).Conclusions pvdQ gene may be involved in regulating the swarming motility of Pseudomonas aeruginosa,which can promote Pseudomonas aeruginosa swarming motility.     

应用siRNA干扰技术沉默铜绿假单胞菌外排泵基因mexB的蛋白表达

目的 观察siRNA干扰质粒转入铜绿假单胞菌后,MexB蛋白表达量的变化.方法 针对mexB基因设计合成特异性siRNA分子,与pGPU6/GFP/Neo载体连接,构建pGPU6/GFP/NeosiRNA重组质粒.构建重组表达质粒pET22b+/mexB,并转化大肠杆菌BL21( DE3) plysS,诱导表达MexB蛋白,蛋白纯化后免疫家兔制备多克隆抗体.siRNA质粒分别电转化铜绿假单胞菌野生株、临床耐药株及mexB基因高表达株,运用Western blot观察转化8、12、24h后MexB蛋白表达量的变化.结果 成功构建pGPU6/GFP/Neo-siRNA重组质粒.成功表达铜绿假单胞菌外排泵蛋白MexB,并制备多克隆兔抗.siRNA质粒对铜绿假单胞菌野生株、临床耐药株及mexB基因高表达株的mexB基因均具有良好的沉默效果,而且沉默效果存在时间差异性.结论 siRNA质粒转化3株铜绿假单胞菌后,在8h及12 h时均可观察到MexB蛋白表达量明显减少,而在24 h时MexB蛋白表达量无改变.     

观察铜绿假单胞菌pvdQ基因对群集游动中抗生素抗性的影响

目的 探讨铜绿假单胞菌pvdQ(PA2385)基因在群集游动中对几种常用抗生素抗性的影响.方法 构建pvdQ基因表达质粒pME6032-pvdQ并鉴定,采用电穿孔法将带有pvdQ基因的质粒转入PAO1中,构建pvdQ高表达株.同时将空质粒pME6032采用电穿孔法转入PAO1中,构建pME6032空质粒株.在不同浓度抗生素中,通过比较群集游动直径的大小,观察PAO1和pvdQ高表达株对抗生素抗性的改变.结果 经鉴定成功构建pvdQ高表达株,比较两株菌群集游动直径大小:抗生素浓度成倍增加,但群集游动直径不是成倍下降而是成不规则增加,说明PAO1和pvdQ高表达株均能提高抗生素抗性;pvdQ高表达株对头孢他啶、环丙沙星、美罗培南和多黏菌素B几种抗生素与PAO1相比抗性提高了2～4倍.结论 铜绿假单胞菌pvdQ高表达株能够提高抗生素的抗性,说明pvdQ基因在此过程中发挥重要作用,可能通过参与群集细胞的分化来提高抗生素的抗性.

Abstract：

Objective To investigate whether Pseudomonas aeruginosa pvdQ( PA2385 ) gene reveals altered antibiotic susceptibility under swarming conditions. Methods The plasmid pME6032 with pvdQ gene was constructed and identified, then transformed into Pseudomonas aeruginosa PAO1 by the electroporation, building pvdQ overexpression strain. Using the same method building pME6032-PAO1 strain.Bacteria were inoculated in LB overnight , measuring the colony diameter of the swarming zone . Results Strains of pvdQ overexpression was successfully constructed by real-time PCR. Comparison of two strains of the swarming motility of change in diameter: The result showed that PAO1 and pvdQ overexpression strains can both improve the antibiotics resistance. Swarmer cells of pvdQ overexpression strain exhibited a 2- to 4-fold increase in antibiotic resistance toward ceftazidime,ciprofloxacin, meropenem and polymyxin B compared to PAO1 on BM2-swarming agar plates. Conclusion pvdQ gene played an important role in elevating the antibiotics resistance, which through prarticipated in the swarmer cell differentiation.     

超声引导下经皮微波消融治疗原发性肝癌患者疗效分析*

目的 探讨超声引导下经皮微波消融联合胸腺素-α1治疗原发性肝癌（PLC）患者的疗效。方法 2011年3月~2013年3月我院收治的PLC患者104例,随机分为观察组52例和对照组52例。对照组采用超声引导下经皮微波消融术治疗,观察组于术后皮下注射胸腺素-α1 4周。比较治疗前和治疗后外周血T淋巴细胞亚群、肝功能、甲胎蛋白（AFP）水平变化和两组肿瘤复发或转移及3年生存情况。结果 在治疗4周时,观察组外周血CD3⁺、CD4⁺、CD4⁺/CD8⁺比值分别为71.49±6.57%、43.12±2.89%、15.89±3.24,显著高于对照组的43.21±3.74%、24.56±2.36%、5.42±2.13 （P<0.05）;观察组血清白蛋白（ALB）为39.84±2.56 g/L,显著高于对照组的34.12±1.87 g/L,丙氨酸氨基转移酶 （ALT）、天门冬氨酸氨基转移酶（AST）、AFP水平分别为34.87±3.08 U/L、43.39±2.08 U/L、85.42±10.42 μg/L,显著低于对照组的61.39±3.64 U/L、56.74±3.46 U/L、164.29±14.35 μg/L;随访3年,观察组肿瘤复发率和转移率分别为30.8%和15.4%,显著低于对照组的50.0%和32.7%（P<0.05）,观察组生存率为23.1%（12/52）,显著高于对照组的5.8%（3/52,P<0.05）。结论 超声引导下经皮微波消融后继续接受α-1胸腺素治疗PLC患者效果显著,可降低肿瘤复发或转移,延长生存时间,值得研究。