[摘 要] 目的：检测miR-452-5p在食管鳞状细胞癌（ESCC）中的表达，并探讨其异常表达对食管癌KYSE-150细胞增殖、侵袭能力和EMT进程的影响及其分子机制。方法：收集2012年3月至2015年12月在河北医科大学第四医院就诊的86名ESCC患者的癌组织样本和对应的癌旁组织，用qPCR法检测miR-452-5p及其他相关基因在ESCC组织和细胞中的表达；向KYSE-150细胞中分别转染miR-452-5p mimic或pcDNA3.1-SOX7构建过表达的细胞株。分析miR-452-5p表达与ESCC病理特征和患者5年OS的关系。用MTS、Tanswell法检测miR-452-5p过表达对食管癌KYSE-150细胞增殖、侵袭能力和EMT进程的影响；用双荧光素酶报告基因实验及TOP/FOP报告基因系统检测miR-452-5p与SRY盒转录因子（SOX7）3'UTR区的结合作用及对Wnt/β-catenin通路活化水平的影响。结果：miR-452-5p在ESCC组织中呈明显高表达（P<0.01），并与ESCC患者的淋巴结转移、TNM分期及5年OS密切相关（均P<0.01）。miR-452-5p过表达明显促进食管癌KYSE-150细胞的增殖、侵袭能力及EMT进程（P<0.05或P<0.01）。SOX7是miR-452-5p的直接靶基因，miR-452-5p通过对SOX7的负向调控影响了Wnt通路活化水平（P<0.05或P<0.01），同时，miR-452-5p表达也受Wnt通路活化水平的影响（P<0.05或P<0.01），其可能为Wnt通路下游靶基因。结论：miR-452-5p通过miR-452-5p/SOX7/Wnt/miR-452-5p正反馈环路提高Wnt/β-catenin通路活化水平，进而促进ESCC KYSE-150细胞的增殖、侵袭能力及EMT进程，miR-452-5p有望成为ESCC患者靶向治疗的潜在靶点及预后评估的新型分子标志物。

Objective To study the therapeutic effect of phages on extensively drug-resistant Acinetobacter baumannii-induced sepsis in mice.Methods (1) Sixty BALB/c mice were divided into blank control group,sepsis control group,antibiotics treatment group,phage treatment group,and phage control group according to the random number table,with 12 mice in each group.Mice in blank control group were intraperitoneally (the same injection position below) injected with 1 mL normal saline.Mice in sepsis control group,antibiotics treatment group,and phage treatment group were injected with 1 mL extensively drug-resistant Acinetobacter baumannii (the strain was isolated from the blood of a severely burned patient hospitalized in our unit) in the concentration of 5 × 107 colony-forming unit/mL to reproduce sepsis model.Two hours later,mice in sepsis control group,antibiotics treatment group,and phage treatment group were injected with 1 mL saline,1 mg/mL imipenem/cilastatin,and 1 × 108 plaque-forming unit (PFU)/mL phages screened based on above-mentioned Acinetobacter baumannii (the same phages below) respectively.Mice in phage control group were injected with 1 mL phages in the titer of 1 × 108 PFU/mL.The injection was performed continuously for 7 days in each living mouse,and the survival situation of mice was observed each day to calculate the survival ratio in one week.(2) Another 60 BALB/c mice were grouped and treated as in experiment (1),and the injection was performed continuously for 5 days in each living mouse.On experiment day 2,4,and 6,3 mice from each group were selected (if the number of survived mouse in any group was less than 3 at sample collecting,all the survived mice were selected),and blood was drawn to determine white blood cell count (WBC,with 3 samples at each time point in each group).On experiment day 2,blood was drawn from the mice that had their blood taken earlier for bacterial culture,and lung,liver,kidney,and spleen tissue was collected from the same mice.The tissue samples were added to the LB solid medium after being homogenized and diluted for bacterial culture.The content of bacteria was calculated after the bacterial colony number was counted.Data were processed Wilcoxon rank sum test,one-way analysis of variance,LSD test and Kruskal-Wallis rank sum test.Results (1) On experiment day 7,there were 12,8,10,and 12 mice survived in blank control group,antibiotics treatment group,phage treatment group,and phage control group respectively,while no mouse survived in sepsis control group.Compared with that in sepsis control group,the survival ratio of mice was significantly higher in the other four groups (with Z values from 55.635 to 106.593,P values below 0.05).The survival ratio of mice in phage treatment group was slightly higher than that in antibiotics treatment group,without statistically significant difference (Z =2.797,P ＞0.05).(2) On experiment day 2,WBC data of mice in blank control group,phage treatment group,and phage control group were close [respectively (5.60 ± 0.94) × 109/L,(5.16 ±0.36) × 109/L,and (5.26 ± 1.89) × 109/L],all significantly lower than the datum in sepsis control group [(8.64 ±0.64) × 109/L,P ＜0.05 orP ＜0.01],and the WBC data in the latter two groups were significantly lower than the datum in antibiotics treatment group [(7.80 ± 1.76) × 109/L,with P values below 0.05].On experiment day 4,WBC data of mice in antibiotics treatment group,phage treatment group,and phage control group were close,all significantly lower than the datum in blank control group (P ＜ 0.05 or P ＜ 0.01),and WBC data in the above-mentioned four groups were all lower than the datum in sepsis control group (with P values below 0.01).On experiment day 6,there was no statistically significant difference in WBC among blank control group,antibiotics treatment group,phage treatment group,and phage control group (x2 =4.128,P ＞0.05).On experiment day 2,respectively 12,7,and 2 mice were detected as blood bacterial culture-positive in sepsis control group,antibiotics treatment group,and phage treatment group,while no positive result was detected in the other two groups.Positive ratios of blood bacterial culture of mice in blank control group,phage treatment group,phage control group were significantly lower than the ratio in sepsis control group (with x 2 values from-30.000 to 30.000,P values below 0.01).Positive ratio of blood bacterial culture of mice in antibiotics treatment group was significantly higher than that in blank control group or phage control group (withx 2 values respectively 17.500 and-17.500,P values below 0.05).On experiment day 2,except for the kidney tissue of mice in phage treatment group,the bacteria load in each viscus of mice in blank control group,phage treatment group,and phage control group was significantly lower than that in sepsis control group (withx 2 values from-9.000 to 9.000,P values below 0.01).The bacteria load in kidney of mice in antibiotics treatment group was significantly higher than that in blank control group or phage control group (withx2 values respectively-7.500 and 7.500,P values below 0.05).Conclusions Phages can significantly improve survival ratio,control inflammation response,and effectively clean bacteria in lung,liver,spleen,and kidney in treating extensively drug-resistant Acinetobacter baumannii-induced sepsis in mice.

Objective To analyze the distribution and drug resistance of pathogen isolated from severely burned patients with bloodstream infection,so as to provide reference for the clinical treatment of these patients.Methods Blood samples of 162 severely burned patients (including 120 patients with extremely severe burn) with bloodstream infection admitted into our burn ICU from January 2011 to December 2014 were collected.Pathogens were cultured by fully automatic blood culture system,and API bacteria identification panels were used to identify pathogen.Kirby-Bauer paper disk diffusion method was used to detect the drug resistance of major Gram-negative and-positive bacteria to 37 antibiotics including ampicillin,piperacillin and teicoplanin,etc.(resistance to vancomycin was detected by E test),and drug resistance of fungi to 5 antibiotics including voriconazole and amphotericin B,etc.Modified Hodge test was used to further identify imipenem and meropenem resistant Klebsiella pneumonia.D test was used to detect erythromycin-induced clindamycin resistant Staphylococcus aureus.The pathogen distribution and drug resistance rate were analyzed by WHONET 5.5.Mortality rate and infected pathogens of patients with extremely severe burn and patients with non-extremely severe burn were recorded.Data were processed with Wilcoxon rank sum test.Results (1) Totally 1 658 blood samples were collected during the four years,and 339 (20.4％) strains of pathogens were isolated.The isolation rate of Gram-negative bacteria,Gram-positive bacteria,and fungi were 68.4％ (232/339),24.5％ (83/339),and 7.1％ (24/339),respectively.The top three pathogens with isolation rate from high to low were Acinetobacter baumannii,Staphylococcus aureus,and Pseudomonas aeruginosa in turn.(2) Except for the low drug resistance rate to polymyxin B and minocycline,drug resistance rate ofAcinetobacter baumannii to the other antibiotics were relatively high (81.0％-100.0％).Pseudomonas aeruginosa was sensitive to polymyxin B but highly resistant to other antibiotics (57.7％-100.0％).Enterobacter cloacae was sensitive to imipenem and meropenem,while its drug resistance rates to ciprofloxacin,levofloxacin,cefoperazone/sulbactam,cefepime,piperacillin/tazobactam were 25.0％-49.0％,and those to the other antibiotics were 66.7％-100.0％.Drug resistance rates of Klebsiella pneumoniae to cefoperazone/sulbactam,imipenem,and meropenem were low (5.9％-15.6％,two imipenem-and meropenem-resistant strains were identified by modified Hodge test),while its drug resistance rates to amoxicillin/clavulanic acid,piperacillin/tazobactam,cefepime,eefoxitin,amikacin,levofloxacin were 35.3％-47.1％,and those to the other antibiotics were 50.0％-100.0％.(3) Drug resistance rates of methicillin-resistant Staphylococcus aureus (MRSA) to most of the antibiotics were higher than those of the methicillin-sensitive Staphylococcus aureus (MSSA).MRSA was sensitive to linezolid,vancomycin,and teicoplanin,while its drug resistance rates to compound sulfamethoxazole,clindamycin,minocycline,and erythromycin were 5.3％-31.6％,and those to the other antibiotics were 81.6％-100.0％.Except for totally resistant to penicillin G and tetracycline,MSSA was sensitive to the other antibiotics.Fourteen Staphylococcus aureus strains were resistant to erythromycin-induced clindamycin.Enterococcus was sensitive to vancomycin and teicoplanin,while its drug resistance rates to linezolid,chloramphenicol,nitrofurantoin,and high unit gentamicin were low (10.0％-30.0％),and those to ciprofloxacin,erythromycin,minocycline,and ampicillin were high (60.0％-80.0％).Enterococcus was fully resistant to rifampicin.(4) Fungi was sensitive to amphotericin B,and drug resistance rates of fungi to voriconazole,fluconazole,itraconazole,and ketoconazole were 7.2％-12.5％.(5) The mortality of patients with extremely severe burn was higher than that of patients with non-extremely severe burn.The variety of infected pathogens in patients with extremely severe burn significantly outnumbered that in patients with non-extremely severe burn (Z =-2.985,P =0.005).Conclusions The variety of pathogen in severely burned patients with bloodstream infection is wide,with the main pathogens as Acinetobacter baumannii,Staphylococcus aureus,and Pseudomonas aeruginosa,and the drug resistance situation is grim.The types of infected pathogen in patients with extremely severe burn are more complex,and the mortality of these patients is higher when compared with that of patients with non-extremely severe burn.

[摘 要] 目的： 探讨长链非编码RNA（lncRNA）LINC01140在食管鳞状细胞癌（esophageal squamous cell carcinoma，ESCC）组织及细胞中的表达及其对Eca109细胞增殖与侵袭的影响及其分子机制。方法：选取2012年3月至2015年5月河北医科大学第四医院收治的133例ESCC患者的临床资料和GEPIA数据库中收集的182例ESCC组织及286例食管正常黏膜组织的LINC01140表达数据，以及ESCC细胞系Kyse150、Eca109和TE13。用qPCR法检测癌组织和细胞中LINC01140的表达水平，分析其表达水平与患者临床病理特征及预后的关系。分别将pcDNA3.1-LINC01140、阴性对照（pcDNA3.1-NC）或miR-452-5p mimic及阴性对照（miR-NC）转染到Eca109细胞，MTS、Transwell实验分别检测细胞的增殖与侵袭能力。用双荧光报告基因实验及TOP/FOP报告基因系统检测LINC01140与miR-452-5p的靶向结合作用及LINC01140对Wnt/β-catenin通路活化水平的影响。结果：LINC01140在ESCC组织和细胞中表达均显著下调（均P<0.01），LINC01140低表达与ESCC患者年龄、淋巴结转移、TNM分期及OS密切相关（均P<0.05）。LINC01140过表达明显抑制Eca109细胞的增殖及侵袭能力（均P<0.01）。机制研究表明，LINC01140可能通过竞争结合miR-452-5p影响Wnt/β-catenin信号通路的活化水平继而调控Eca109细胞的恶性生物学行为。结论：LINC01140通过靶向miR-452-5p/Wnt/β-catenin轴促进ESCC细胞的增殖与侵袭能力，其有望成为ESCC患者靶向治疗的潜在靶点及预后评估的标志物。