C1q, as a LAIR-1 ligand, maintains monocytes quiescence and possess immunosuppressive properties. To understand the roles and molecular mechanisms, C1q mediated inflammation cytokines and several pivotal proteins in THP-1 cells after H. pylori infection were detected. The results showed that the expression of IL-8, IL-10, LAIR-1, phosphorylated/total JNK, phosphorylated/total p38-MAPK, phosphorylated/total AKT and phosphorylated/total NF-κB were up-regulated significantly in THP-1 cells after H. pylori infection. There was significant upregulation in IL-10 concentration, phosphorylated/total p38-MAPK and phosphorylated/total AKT, and downregulation in phosphorylated/total JNK in non-H. pylori infected THP-1 cells pretreated with C1q. C1q was also able to increase IL-8 and IL-10 production, and reduce LAIR-1 and phosphorylated/total p38-MAPK expression in pretreatment-C1q THP-1 cells after H. pylori infection. These results together indicated that H. pylori might induce IL-8 and IL-10 production through JNK, p38-MAPK, PI3K/AKT and NF-κB signaling pathway. C1q manipulate LAIR-1 to regulation IL-8 and IL-10 secretion in THP-1 cells after H. pylori infection through the p38-MAPK signaling pathway. This information is helpful to further understand the role and mechanisms of C1q on inflammation cytokines secretion in monocytes after H. pylori infection.

Objective To analyze the current situation of reform on medical insurance payment mode in county-level public hospitals,discover problems on payment reform and propose recommendations for county-level public hospitals.Methods Questionnaires and field research are used to collect data,and soft SPSS19.0 are used to make data analysis.Results Among these county-level public hospitals in Shandong Province participating in the reform of first and second batches,there are 160(92.5％) hospitals carrying out health insurance payment reform,and health insurance payment is in still a single way.26.2％ and 11.4％ disease categories are over 50 diseases in disease limited price payment and disease quota payment hospitals.63.8％ medical institutions' assessment results are related with medical payment.Conclusion According to the results,it is necessary to promote the method of multiplex payment,and increase the number of diseases payment according to the disease categories.During the process of reforming of medical payment,it is necessary to overcome tripartite resistance coming from "medical,insurance,and suffering".This will play a positive role on multiple forms of payment of medical institutions.

Many virulence genes have been reported to play important roles in Helicobacter pylori pathogenesis. However the detailed mechanisms of many of them have not been completely clear. In this study, we found gene hp0169, encoding a putative collagenase (HpPrtC), was involved in pathogenesis of H. pylori. Recombinant HpPrtC shows activities to both native and heat-denatured collagens. This result indicated that HpPrtC may act as a virulence factor to help the bacterium colonize in their host stomach by degrading surrounding collagens. hp0169 was deleted by homologous recombination to study its function in bacterium-host cell interaction. For the pathogenic functions on the host cells, the hp0169 mutant exhibits no significant changes on inducing apoptosis of GES-1 cells. However, the viability and proliferation rate of GES-1 cells infected with mutant strain were higher than the cells infected with wild-type strain. These results indicated that except for its collagenolytic activity, HpPrtC might participate in H. pylori pathogenesis through an additional pathway. Functional studies on hp0169 involved in pathogenesis would shed light on deep understanding of the pathogenic mechanism of H. pylori.

Objective: Helicobacter pylori is a Gram-negative, microaerophilic bacteria usually found in the stomach, which may evade its host's immune system and present long-term symptoms in affected individuals. This study aimed to evaluate the functional role of leukocyte-associated immunoglobulin (Ig) like receptor-1 (LAIR-1) in the strategies and underlying molecular mechanisms by which H. pylori escapes the host's immune responses.Methods: LAIR-1 knockdown THP-1 cells were used to detect cell apoptosis, cell proliferation, interleukin-8 (IL-8), IL-10, and activation of intracellular signaling induced by H. pylori.Results: Cell apoptosis, cell proliferation, IL-8, and IL-10 were increased in THP-1 cells after 24 h of H. pylori infection. Functional analysis indicated LAIR-1 silencing obviously inhibited the phosphorylation of IKB alpha, eIF2 alpha, JNK, and Smad2 in the THP-1 after H. pylori infection. In addition, there were no significant differences in proliferation rates between control siRNA group and LAIR-1 siRNA group regardless of whether THP-1 cells were infected by H. pylori.Conclusion: These results together indicated that LAIR-1 modulated cell apoptosis and inflammatory cytokines secretion in THP-1 cells, which might help sustain inflammation and prevent removal of the bacteria by the immune responses. (C) 2017 Elsevier Ltd. All rights reserved.

Objectives To create an hp0788-deleted strain (Helicobacter pylori ATCC26695△0788km) and to functionally study the role of hp0788 in H.pylori pathogenesis.Methods A knockout plasmid was constructed based on pSJHK.Upstream and downstream sequences (800-1100bp) of the hp0788 gene were amplified as upstream and downstream homologous arms.These homologous arms were digested with restriction enzymes and ligated into pSJHK,yielding the plasmid pSJHK-0788.This plasmid was transformed into H.pylori ATCC26695 by electroporation to generate an hp0788-deleted strain.The adherence of FITC-labeled the H.pylori to GES-1 was measured using flow cytometry.The viability of GES-1 cells and the rate of GES-1 cell apoptosis were determined in an H.pylori ATCC26695 or H.pylori ATCC26695△0788km and GES-1 co-culturing system.Results The plasmid pSJHK-0788 was transformed into H.pylori ATCC26695 by electroporation to generate an hp0788-deleted strain.FITC-labeled H.pylori ATCC26695 adhered to GES-1 cells at a rate of 100％ and H.pylori ATCC26695△0788km adhered to GES-1 cells at a rate of 90.40％ (t=2.80,P＜0.05).After infection with H.pylori ATCC26695 and co-culturing for 8 h,the rate of GES-1 cell apoptosis increased (15.73 ± 7.84) ％;after infection and co-culturing for 16 h,the rate of GES-1 cell apoptosis increased (25.26±5.81) ％.After infection with H.pylori ATCC26695△0788km and co-culturing for 8 h,the rate of GES-1 cell apoptosis increased (12.46 ±12.30) ％;after infection and co-culturing for 16 h,the rate of GES-1 cell apoptosis increased (21.13±10.09) ％.After infection with H.pylori ATCC26695 and co-culturing for 8 h,the viability of GES-1 cells decreased 53％;after infection and co-culturing for 16 h,the viability of GES-1 cells decreased 42％.After infection with H.pylori ATCC26695△0788km and co-culturing for 8 h,the viability of GES-1 cells decreased 66％;after infection and co-culturing for 16 h,the viability of GES-1 cells decreased 50％ (t=3.30,2.80,-2.93,-2.76,P＜0.05).Conelusion The rate at which H.pylori ATCC26695 adhered to GES-1 cells decreased as a result of the deletion of hp0788 and the apoptosis of GES-1 cells also decreased,but cell viability increased.These results suggested that hp0788 plays an important role in H.pylori pathogenesis.

Objectives To culture Helicobacter pylori in a serum and brain heart infusion broth and in a β-cyclodextrin and brain heart infusion broth and to observe the growth of H.pylori in these two types of culture media Methods H.pylori was inoculated in the serum and brain heart infusion broth and in the β-cyclodextrin and brain heart infusion broth and shaken at a speed of 120 r/min in 37 ℃ microaerophilic environment.Live bacteria were counted using dilution plate counting,and a growth curve was generated.Results The lag phase of H.pylori in these two types of culture media was 10 hours.The log phase of H.pylori in the serum and brain heart infusion broth was 45 hours while that in the β-cyclodextrin and brain heart infusion broth was 40 hours.The specific growth rate was respectively 0.058/h and 0.043/h in the mid-log phase.The stationary phase was 15 hours and 20 hours,respectively.The highest concentration of H.pylori was 2.067 × 108 CFU/ml and 1.378× 108 CFU/ml,respectively.Conclusion H.pylori grew well in the serum and brain heart infusion broth and in the β-cyclodextrin and brain heart infusion broth.However,H.pylori cultured in the serum and brain heart infusion broth grew at a higher density and had a longer log phase.

Helicobacter pylori is a Gram-negative, microaerophilic bacterium associated with human gastric diseases. Further investigations on virulence genes are still required to clarify the pathogenic mechanism of H. pylori and the heterogeneous problem of infection. In order to develop an efficient and accurate method to study gene functions in H. pylori pathogenesis, an unmarked deletion method for both a single gene and a large fragment was established based on the FLP-FRT recombination system. Using this method, the gene hp0788, encoding an outer membrane protein (HofF), was deleted. Deletion of hp0788 did not affect growth or motility of H. pylori, but reduced the adherence of the bacteria to gastric epithelial cells. The apoptosis of GES-1 cells caused by H. pylori infection was also reduced by the defection of hp0788. These suggest that hp0788 takes part in the bacterium-host interaction and plays an important role in H. pylori infection. Furthermore, a large genomic fragment deletion from hp0541 to hp0547 in cag pathogenicity island was also successfully achieved using FLP-FRT method. The innovative application of the FLP-FRT recombination system in H. pylori to construct unmarked deletion would provide a helpful tool for further function research of putative pathogenic genes and contribute to the understanding of H. pylori pathogenesis.

Only a small percentage of people infected with Helicobacter pylori (H. pylori) will develop overt chronic gastric diseases. To understand the pathological mechanism, the action of H. pylori on monocyte apoptosis was detected. H. pylori co-culturing with peripheral blood monocytes, THP-1 or U937 cells result in early apoptosis at 6, 12, and 24 h after infection. The phosphorylated Bad and JNK were increased, and Bcl-2 was declined at 6, 12, and 24 h in peripheral blood monocytes after H. pylori infection. The phosphorylated Akt was augmented at 6 and 12 h post-infection. A slow apoptotic response was induced by H. pylori via Bad and Bcl-2 regulators, activated caspase-8 and caspase-9, and JNK at 24 h in THP-1 cells. Meanwhile, only Bad and JNK were involved in regulating U937 cells apoptosis at 24 h after infection. These results supported a novel mechanism of H. pylori escaping from monocytes by upregulation of early apoptosis and inhibition of late apoptosis. The differences among the three cells may reveal why H. pylori-derived disease occurs in relatively few people and provide a pathological mechanism whereby a treatment for H. pylori-derived disease may be developed.

目的研究LAIR-1基因沉默对幽门螺杆菌（Helicobacter pylori）诱导单核细胞THP-1凋亡的影响。方法以Hp ATCC26695分别感染THP-1细胞6、12、24和48h,采用流式细胞术检测细胞凋亡及LAIR-1蛋白的表达。应用Lipofectamine RNAiMAX转染3对LAIR-1siRNAs入单核细胞THP-1中,采用半定量RT-PCR及流式细胞术检测沉默效应,筛选出的较有效的一对siRNA再转染入单核细胞THP-1,48h后加入Hp培养24h,采用流式细胞术检测细胞凋亡率。结果 Hp感染THP-1细胞6、12、24和48h后的细胞凋亡率分别为（84±1.77）%、（69.13±4.29）%、（48.3±3.37）%和（21.1±4.67）%,LAIR-1蛋白表达相对荧光强度分别为14 934±178、14 369±244、12 259±523和7438±539;转染后48h较有效的siRNA对LAIR-1mRNA及蛋白沉默效率分别为（39.1±4.67）%和（38.4±3.18）%;将筛选出的siRNA转染细胞48h后,用Hp感染24h,流式细胞仪检测显示转染组与阴性对照组细胞凋亡率分别为（56.1±9.8）%和（36.9±3.2）%,转染后感染组与空白组LAIR-1蛋白表达相对荧光强度分别为12 181±219和10 228±136。结论在Hp感染细胞THP-1 48h内,随着感染时间延长LAIR-1的表达及THP-1细胞凋亡率显著降低即LAIR-1siRNA能抑制LAIR-1的表达及THP-1细胞的凋亡。

目的 分析幽门螺杆菌感染胃上皮细胞过程中整合素β1亚基表达的变化,观察整合素β1对幽门螺杆菌感染胃上皮细胞GES-1凋亡及增殖的影响.方法 将幽门螺杆菌与胃上皮细胞GES-1共培养,采用流式细胞术检测整合素β1的表达量变化;应用iRNA技术降低胃上皮细胞GES-1中β1亚基的表达;幽门螺杆菌感染胃上皮细胞及iRNA降低β1的表达后,采用流式细胞术检测细胞的凋亡,采用CCK-8法检测细胞增殖情况.结果 幽门螺杆菌感染胃上皮细胞12、24、48h后空白对照组凋亡率分别为(10.80±0.71)％、(12.23±0.06)％和(12.30±2.12)％,Hp感染组凋亡率分别为(24.20±0.14)％、(30.05±2.47)％和(26.40±1.91)％,差异均有统计学意义(t值分别为-26.28、-7.701和-12.855,P＜0.05);与幽门螺杆菌共培养6、24、48 h胃上皮细胞增殖受到抑制,抑制率分别为(35.00±3.22)％、(40.96±2.45)％和(8.00±3.33)％;幽门螺杆菌与胃上皮细胞GES-1共培养24、48 h流式细胞术检测整合素β1表达,空白对照组平均荧光强度(MFI)分别为(1616.33±24.70)和(1834.67±17.01),Hp感染组为(1484.00±60.89)和(1376.00±14.11),差异有统计学意义(t值分别为3.488和35.95,P＜0.05);应用iRNA技术降低胃上皮细胞GES-1整合素β1亚基表达24、48 h,对照组凋亡率分别为(18.75±3.59)％和(39.75±3.68)％,iRNA组凋亡率分别为(26.25±4.11)％和(55.30±5.57)％,差异有统计学意义(t值为-2.746和-4.656,P＜0.05);胃上皮细胞中β1亚基的表达降低后细胞增殖受到抑制,24、36、48、72 h抑制率分别为(6.16±1.07)％、(17.15±2.76)％、(10.84±1.34)％和(21.87±1.72)％.结论 幽门螺杆菌能降低胃上皮细胞GES-1整合素β1亚基的表达,而且可能通过降低整合素β1亚基的表达促进上皮细胞GES-1的凋亡并抑制其增殖.

目的 检测并分析幽门螺杆菌(Hp)尿素酶β亚基(UreB)与Hsp60之间的相互作用. 方法 克隆Hp 26695的尿素酶β亚基基因(UreB)融合GST标签和Hsp60基因融合His标签,分别在大肠埃希菌中进行异源表达.提取两种蛋白,采用pull-down方法检测两者间的相互作用.利用Modeller 9v2软件,以E.coli的GroEL为基础模建HpHsp60的三维结构,再与已知的UreB结构利用AutoDock 4.2软件进行分子共模拟,分析二者的相互作用面和关键氨基酸. 结果 构建了Hp Hsp60和尿素酶β亚基(UreB)的大肠埃希菌异源表达体系并获得纯化蛋白;Pull-down试验显示部分未融合GST标签的Hsp60出现在GST-UreB的洗脱液中,表明Hsp60与GST-UreB之间存在相互作用;以E.coli的GroEL为基础模建了Hp Hsp60的三维结构,利用docking技术构建UreB与Hp Hsp60的相互作用模型,发现其主要的相互作用面位于Hsp60的α9与UreB的α2之间,其可能形成氢键的关键位点为UreB的α2上的T147氨基酸与Hsp60的α9上的E237、K238. 结论 Hp Hsp60能直接与尿素酶β亚基(UreB)相互作用,Hsp60可能通过这种相互作用在UreB成熟过程中发挥分子伴侣功能.这为阐明Hp尿素酶的组装与成熟机制奠定了基础.

Helicobacter pylori infection plays an important role in the etiology of various gastroduodenal diseases. However, pathogenic mechanism of H. pylori is not clear. More potential pathogenic factors need to be further discovered and studied. In this study, two vectors for generating double-crossover recombination gene knockout plasmids in H. pylori were designed based on the backbone of plasmid pLYL03. Genes on plasmid pLYL03 were rearranged, and the redundant sequences were reduced. Erythromycin-resistant gene on pLYL03 was replaced by aphA or catGC to generate the kanamycin-resistant plasmid pSJHK or the chloramphenicol-resistant plasmid pSJHC. The sizes of pSJHK and pSJHC are 4371 and 3949 bp, respectively. Based on plasmids pSJHK and pSJHC, double-crossover recombination gene knockout plasmids targeting hp0169 and hp0788 were constructed, and deletion mutants were achieved by electroporation of the gene-targeting plasmids. The results indicated that plasmids pSJHK and pSJHC are efficient for gene deletion in H. pylori, and the transformation efficiency of pSJHC is slightly higher than pSJHK. These plasmids provide convenient genetic tools for further research of novel pathogenic factors in H. pylori. Derivative plasmids developed by changing antibiotic-resistant genes would also provide valuable tools for the study of functional genes in other bacteria.

Helicobacter pylori infection represents a key factor in the etiology of various gastro-duodenal diseases, ranging from chronic gastritis to the development of peptic ulcer disease and end-stage gastric cancer. In the present study, the 26695 and SS1 strains of H. pylori were used to study the differential functional profiles of gastric epithelial cells infected with H. pylori. The apoptosis rates in GES-1 cells were significantly increased 3, 12 and 24 h after H. pylori 26695 and SS1 infection. Moreover, apoptosis by cells infected with the H. pylori 26695 strain was significantly higher than cells infected with the SS1 strain of H. pylori. No significant changes in the proliferation rates of GES-1 cells were observed after H. pylori 26695 or SS1 infection at any time during the experimental period. Exposure to H. pylori 26695 and SS1 induced a significant decline in the adhesion rates of GES-1 cells in a time-dependent manner. Furthermore, H. pylori 26695 infection increased migration of GES-1 cells every hour during the whole experimental period compared with control cells. However, GES-1 cells infected with the H. pylori SS1 strain exhibited migration rates almost stable and comparable to those of control cells. These results indicate that the gastric epithelial cells respond differently depending on the H. pylori strains. This study indicates that the development of different gastric-related diseases may be a H. pylori strain-specific response.

OBJECTIVE:To study the role of Helicobacter pylori (H. pylori) infection in newly diagnosed childhood immune thrombocytopenia (ITP). METHODS:A total of 495 children with newly diagnosed ITP who were hospitalized for the first time between January 2011 and December 2013 were included as the case group. A total of 123 children with common respiratory tract infection (not ITP or other diseases of blood system) were randomly selected as the control group. All patients were divided into four groups by age: <1 year group, 1-3 years group, 3-7 years group, and 7-14 years group. The incidence of H. pylori infection in all age groups and the clinical outcomes of ITP children with or without H. pylori infection were retrospectively analyzed. RESULTS:The incidence rate of H. pylori infection in the case group increased with increasing age. There was no significant difference in the incidence rate of H. pylori infection between the case and the control groups among subjects of the same age (P>0.05). All the ITP patients were not given anti-H. pylori treatment and only received the treatment (glucocorticoid and/or immunoglobulin) for ITP, and their remission rate declined with increasing age. There was no significant difference in the remission rate between the ITP children with H. pylori infection and those without H. pylori infection in the same age group (P>0.05). CONCLUSIONS:H. pylori infection may not be a major cause of ITP in children, and the clinical outcomes of children with acute ITP are not affected by receiving anti-H. pylori treatment or not.