HBV X蛋白对抑癌基因p16的表达和启动子甲基化的影响

目的探讨HBV X蛋白(HBx)对抑癌基因p16的表达、p16启动子甲基化的影响,从表观遗传学的角度探讨HBx参与HBV相关性HCC发生发展的相关机制。方法以人肝母细胞瘤细胞Hep G2、表达绿色荧光蛋白(GFP)的Hep G2/GFP、稳定表达HBx蛋白的Hep G2/GFP-HBx细胞为实验系统;采用Western Blot法检测Hep G2、Hep G2/GFP、Hep G2/GFP-HBx细胞中p16蛋白的表达水平。以DNA甲基转移酶(DNMT)抑制剂5-氮杂-2'脱氧胞苷(5-Aza-2'-DC)处理Hep G2/GFP-HBx细胞,采用甲基化特异性PCR(MSP)法检测Hep G2、Hep G2/GFP细胞及药物处理和未处理的Hep G2/GFP-HBx细胞中抑癌基因p16启动子区域的甲基化情况。计量资料多组间比较采用单因素方差分析。结果 Western Blot分析示Hep G2/GFP-HBx细胞中p16蛋白相对灰度值(23.68±3.93)显著低于Hep G2(91.23±6.87)、Hep G2/GFP(94.55±8.40)细胞,差异均有统计学意义(P值分别为0.0007、0.0014),Hep G2/GFP与Hep G2细胞中p16蛋白相对灰度值差异无统计学意义(P0.05);MSP法检测示Hep G2/GFP-HBx细胞中存在p16基因启动子区域部分Cp G位点甲基化,Hep G2、Hep G2/GFP细胞中未检出其甲基化,DNMT抑制剂5-Aza-2'-DC处理的Hep G2/GFP-HBx细胞却能恢复p16基因启动子区域的未甲基化状态。结论在肝癌细胞系中,HBx通过诱导抑癌基因p16启动子甲基化而下调其表达,DNMT抑制剂5-Aza-2'-DC能恢复p16基因启动子区域的未甲基化状态,这种可逆性修饰可为HBV相关性HCC的治疗、预防提供新思路。     

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.     

Role of MexA-MexB-OprM efflux pump system in chronic Pseudomonas Aeruginosa pulmonary infection in mice

In order to investigate the role of the MexA-MexB-OprM efflux pump system in the pathogenesis of Pseudomonas aeruginosa(PA)-induced pulmonary infection,pulmonary infection models were established by intratracheal injection of K767(wild type),nalB(MexA-MexB-OprM up-regulated mutant),and △m exB(knockout) strains,separately.All mice were treated with Meropenem(intraperitoneal injection,100 mg/kg body weight,twice every day),and strain-related pathology,bacteria count,cytokine level,myeloperoxidase(MPO,indicator of neutrophil recruitment) activity,and macrophage inflammatory protein-2(MIP-2) expression were evaluated at early(3rd day post-infection) and late(7th and 14th day post-infection) stages of infection.E-test showed that △mexB was more significantly sensitive to panipenan(ETP),meropenem(MP) and imipenem(IP) than K767 and nalB strains.There was no significant difference in sensitivity to cefepime(TM) among the three stains.In contrast to the K767 and nalB groups,the △ mexB group showed decreased bacteria burden over time and less extensive pathological change.Additionally,MPO activity and levels of inflammatory cytokines(IL-1b,IL-12,and TNF-α) were increased at the early stage(day 3) and decreased at the later stage(day 14).Serum MIP-2 expression level was steadily increased in all three groups from early to late stages,but significantly higher in △m exB group than in K767 and nalB groups(P<0.05).In conclusion,the MexA-MexB-OprM efflux pump system might play an important role in PA-induced chronic pulmonary infection.High expression of the MexA-MexB-OprM efflux pump could increase antibacterial resistance and promote infection.     

宏基因组学二代测序对脊柱感染患者病原学诊断的价值

目的探讨宏基因组学二代测序(mNGS)对脊柱感染患者病原学诊断价值的效能, 为及时诊治提供参考。方法纳入2020年1月至2022年7月河南省人民医院感染科收治的40例疑似脊柱感染患者, 回顾性分析患者的病灶组织培养、组织病理学检查、组织mNGS检测结果。以临床诊断病例为标准, 分为脊柱感染组(28例)和非脊柱感染组(12例)。比较mNGS和组织培养两种检测方法对脊柱感染患者病原体检出的阳性率、灵敏度和特异度。统计学分析采用配对χ2检验。结果 40例患者中, 男23例, 女17例。mNGS检测阳性率高于组织培养阳性率[75.0%(30/40)比12.5%(5/40)], 差异有统计学意义(χ2=0.08, P<0.001)。以临床诊断病例为标准, mNGS对脊柱感染诊断的灵敏度高于组织培养(82.1%比17.9%), 差异有统计学意义(χ2=0.02, P<0.001), 而其特异度与组织培养相比(33.3%比100.0%), 差异无统计学意义(P>0.05)。结论 mNGS在脊柱感染患者的病原学诊断中具有较高的病原体检出率和灵敏度, 可为脊柱感染患者的诊治提供临床指导...