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    Editorial
    How to conquer bacteria in post-antibiotic era?
    GUO Xiao-kui
    2012, 32 (11):  1401. 
    doi: 10.3969/j.issn.1674-8115.2012.11.001

    Abstract ( 902 )   PDF (250KB) ( 1279 )  

    The occurrence of antibiotic resistant bacteria, especially pan-drug resistant bacteria may make no antibiotics available for the treatment of infectious diseases, which will lead to the emerge of post-antibiotic era. It is imperative to find new antibacterial drugs or strategies to solve the increasingly serious problem of drug resistance. The invention status and application prospect of newtype antibiotics, antimicrobial peptide, bacteriophage and traditional Chinese medicine preparation are introduced in this paper, which may provide reference for the exploration of antibacterial drugs to conquer bacteria in post-antibiotic era.

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    Applications of Mycobacterium tuberculosis genome microevolution
    YAO Yu-feng
    2012, 32 (11):  1404. 
    doi: 10.3969/j.issn.1674-8115.2012.11.002

    Abstract ( 1177 )   PDF (224KB) ( 1127 )  

    Mycobacterium tuberculosis has been a serious threat to human health globally. In the past 10 years, more achievements on the study of tuberculosis by Chinese scientists have gained worldwide acceptance. However, the diagnosis, treatment, prevention and increasing drug resistance remain to be major problems for tuberculosis control in China. Currently, we have to put efforts on the translation of the tuberculosis basic research and focus on the important clinical issues. Based on the progress of the genome sequencing, it has been found that all genetic events occurring in the Mycobacterium tuberculosis genome evolution play an important role in all aspects including biological properties, pathogenicity and drug resistance. For example, a pyrophosphohydrolase mutation identified by the genome comparison revealed the loss of virulence of the avirulent strain H37Ra. Analysis of Mycobacterium tuberculosis genome microevolution will be beneficial to the diagnosis, treatment and prevention of tuberculosis.

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    Monographic report (Pathogenic microbiology)
    Gene fusion and expression of trpA and yfp mediated by λ-Red recombinant system in Escherichia coli
    CHEN Xing, YAO Yu-feng, ZHOU Ai-ping, et al
    2012, 32 (11):  1406. 
    doi: 10.3969/j.issn.1674-8115.2012.11.003

    Abstract ( 1417 )   PDF (546KB) ( 863 )  

    Objective To investigate the expression of tryptophan synthase α subunit-encoding gene (trpA) of Escherichia coli (E. coli), fusion fragment of trpA, yellow fluorescent protein gene (yfp) and chloramphenicol-encoding gene (cat) was knocked into E.coli strain MG1655 genome using λ-Red system. Methods Target genes trpA, yfp and cat were amplified by PCR, and the recombinant fragment was obtained by fusion PCR. The recombinant fragment was transformed into E.coli strain MG1655 harboring genes of λ-Red system in plasmid pKD46. After PCR checking and sequencing confirmation, the fusion protein expression was observed by fluorescence microscopy. Results The recombinant fragment trpA-yfp-cat was successfully obtained and transformed into MG1655, and the positive clones were screened out and confirmed by sequencing. The yellow fluorescence was observed throughout the bacterial cytoplasm, indicating that trpA-yfp fusion protein was successfully expressed in MG1655. Conclusion The trpA gene could be replaced by trpA-yfp fusion fragment in situ using λ-Red system, and its expression could be reflected indirectly by observing the expression of yellow fluorescent protein. The function of trpA and bacterial growth have not been changed, which lays foundation for the further study of E.coli tryptophan synthesis.

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    Evaluation of in vitro bactericidal activity of recombinant lysostaphin against Staphylococcus aureus isolated from burn wounds
    CHEN Xu, WANG Wen-kui, MENG Zhi-min, et al
    2012, 32 (11):  1411. 
    doi: 10.3969/j.issn.1674-8115.2012.11.004

    Abstract ( 1041 )   PDF (307KB) ( 933 )  

    Objective To evaluate the in vitro bactericidal activity of recombinant lysostaphin against Staphylococcus aureus isolated from burn wounds. Methods One hundred and seventy-one Staphylococcus aureus strains were isolated from burn wounds. The minimum inhibitory concentrations (MIC) of vancomycin, erythromycin, clindamycin, cefoxitin and sulfamethoxazole, which were common drugs used for burns in clinics against Staphylococcus aureus were measured by agar dilution method. Methicillin-resistant Staphylococcus aureus (MRSA) and methicillinsensitive Staphylococcus aureus (MSSA) were divided according to the MIC of cefoxitin. Drug resistance analysis of Staphylococcus aureus was conducted according to the results of antimicrobial susceptibility tests of vancomycin, erythromycin, clindamycin and sulfamethoxazole. The MIC of recombinant lysostaphin against Staphylococcus aureus was measured by micro-broth dilution method, and 50% minimun inhibitory concentration (MIC50) and 90% minimun inhibitory concentration (MIC90) were calculated. Results According to the results of antimicrobial susceptibility tests, the resistance rates of Staphylococcus aureus to sulfamethoxazole, erythromycin, clindamycin and vancomycin were 99.4%, 88.3%, 87.1% and 0 respectively, and the prevalence of MRSA was 85.4%(146 strains). The MIC of recombinant lysostaphin against Staphylococcus aureus ranged from 0.015 to 0.5 U/mL, with MIC50 of <0.015 U/mL and MIC90 of 0.03 U/mL. Conclusion Recombinant lysostaphin has favorable in vitro bactericidal activity against Staphylococcus aureus. With the increasing resistance of Staphylococcus aureus to clinical drugs, recombinant lysostaphin may potentially be a potent drug for Staphylococcus aureus infection of the burn wounds.

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    Research on relationship between polymorphisms of Mtub-39 and expression of downstream genes of Mycobacterium tuberculosis
    ZHOU Ai-ping, XU Zhi-hong, SUN Qing, et al
    2012, 32 (11):  1415. 
    doi: 10.3969/j.issn.1674-8115.2012.11.005

    Abstract ( 1057 )   PDF (628KB) ( 1007 )  

    Objective To analyse the 5 MIRU loci of Mycobacterium tuberculosis, and investigate the relationship between polymorphisms of MIRU loci and expression of downstream genes. Methods Bioinformatics method was used to predict the downstream genes promoter regions of 5 MIRU loci (ETR-C, Mtub-30, Mtub-39, MIRU-27 and MIRU-40). Promoter sequence was amplified by PCR, and was cloned into mycobacterial promoterless probe vector pMC210 to generate the recombinants. After confirmation by restriction endonuclease digestion and sequence analysis, the recombinant plasmids were transformed into mycobacterium smegmatis mc2155 by electroporation. The transcriptional level of reporter gene lacZ was evaluated by Real-Time PCR, and the influence of polymorphisms of MIRU loci on the expression of downstream genes was examined. Results Mtub-39 locus core region contained the promoter of the downstream gene. The recombinant plasmids harboring Mtub-39 locus with 1, 3 or 5 copy numbers were constructed. Real-Time PCR revealed that Mtub-39 locus with polymorphisms (pMC210-Mtub-39-N158, pMC210-Mtub-39-N139 and pMC210-Mtub-39-N146) had significant differences in the transcriptions of reporter gene lacZ (P=0.006 5). Conclusion The polymorphisms of Mtub-39 can significantly affect the promotor activity of the downstream genes, and sequentially regulate the expression of the gene.

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    In vitro inhibitory effect of quaternized chitosan against Klebsiella pneumoniae
    HUANG Jie-wen, CHEN Zhi-jun, SHEN Jun-fei, et al
    2012, 32 (11):  1421. 
    doi: 10.3969/j.issn.1674-8115.2012.11.006

    Abstract ( 1240 )   PDF (355KB) ( 1285 )  

    Objective To investigate the in vitro inhibitory effect of quaternized chitosan against Klebsiella pneumoniae. Methods Plating serial dilutions were used to detect the direct inhibitory effects of chitosan and quaternized chitosan against Klebsiella pneumoniae. Real-time PCR was employed to detect the expression of important biofilm formation related genes lsrR, lsrK and gmd. Crystal violet staining was utilized to examine the biofilm formation of Klebsiella pneumoniae. Results There were significant decrease in the live bacteria counts in chitosan and quaternized chitosan wells from 6 h after treatment (P<0.05), and the live bacteria counts in quaternized chitosan wells were significantly lower than those in chitosan wells at 12 h after treatment, with the 12h inhibitory ratios of chitosan and quaternized chitosan of (50.2±9.3)% and (72.6±8.0)% respectively (P<0.05). Chitosan and quaternized chitosan increased the expression of lsrR gene, quaternized chitosan decreased the expression of lsrK gene, and there was also a downward trend of expression of gmd gene after treatment with quaternized chitosan. Chitosan and quaternized chitosan achieved an inhibition on the biofilm formation of Klebsiella pneumoniae from 6 h after treatment (P<0.05), while there was no significant difference in the biofilm formation inhibition ability between chitosan and quaternized chitosan (P>0.05). Conclusion Chitosan and quaternized chitosan can effectively inhibit the proliferation and biofilm formation of Klebsiella pneumoniae. The antibacterial activity against Klebsiella pneumoniae from quaternized chitosan is better than that of chitosan, and the expression of multiple biofilm formation related genes is influenced by quaternized chitosan.

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    Recombination, preparation and immunological evaluation of Mycobacterium tuberculosis specific antigen ESXO
    FENG Li, YE Juan, WANG Feng-ping, et al
    2012, 32 (11):  1426. 
    doi: 10.3969/j.issn.1674-8115.2012.11.007

    Abstract ( 1637 )   PDF (470KB) ( 995 )  

    Objective To determine the expression of recombinant Mycobacterium tuberculosis (MTB) specific antigen ESXO in E.coli, and evaluate its immunogenicity. Methods Recombinant ESXO protein of MTB was obtained with conventional molecular cloning method. The serum antibody against ESXO was detected by enzyme linked immunosorbent assay (ELISA) in 84 patients with tuberculosis (TB) and 48 healthy controls, the serum immunogenicity was evaluated, and the results were compared with findings in detection of ESAT-6 and CFP-10 protein for serodiagnosis of TB. Results The recombinant specific antigen ESXO was expressed as soluble and inclusion body protein in E.coli BL21 plysS (DE3), with the relative molecular weight of 27 300, which was in line with expectations. The immunogenic evaluation of recombinant ESXO protein indicated that its specificity and sensitivity reached 94.0% (45/48) and 32.1% (27/84) respectively, which was equal to ESAT-6 and CFP-10 in specificity and higher than ESAT-6 in sensitivity. Conclusion The specific antigen ESXO is expected to be a novel candidate antigen for the diagnosis and vaccine design in TB.

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    Research on molecular epidemiology and resistance mechanism of carbapenems resistant Acinetobacter baumannii
    LI Yong-li, YING Chun-mei
    2012, 32 (11):  1430. 
    doi: 10.3969/j.issn.1674-8115.2012.11.008

    Abstract ( 1600 )   PDF (593KB) ( 1129 )  

    Objective To investigate the gene homology of Acinetobacter baumannii clinical isolates in hospital, and explore the main carbapenems resistance mechanism of Acinetobacter baumannii. Methods The minimal inhibitory concentrations of 12 antimicrobial agents against 81 Acinetobacter baumannii clinical isolates were determined by agar dilution method. Pulsedfield gel electrophoresis (PFGE) was employed to type 81 Acinetobacter baumannii isolates. PCR was used to detect the genes of β-lactamases and main structural genes of adeABC, adeIJK and adeFGH efflux pump system, and the distributions of these genes in imipenem resistant Acinetobacter baumannii group and imipenem sensitive Acinetobacter baumannii group were compared. Results Eighty-one Acinetobacter baumannii clinical isolates were commonly resistant to 12 antimicrobial agents, with the lowest resistance rate of 30.9% for polymyxin B, the second lowest resistance rate of 53.1% for imipenem, and resistance rates over 60% for the other antimicrobial agents. Eighty-one Acinetobacter baumannii strains were classified into 7 types based on PFGE pulsotypes, named type A, B, C, D, E, F and G. Isolates of type A were the main epidemic strains. All the strains carried OXA-51 gene, and OXA-24, OXA-58, VIM-1 and VIM-2 genes were not detected. The detection rates of AmpC, OXA-23 and IMP-1 of β-lactamase genes were 83.9%(68/81), 71.6% (51/81) and 54.3%(44/81) respectively, and those of adeB, adeJ, and adeG of efflux pump genes were 77.8% (63/81), 92.6% (75/81) and 90.1% (73/81) respectively. Statistical analysis revealed that the distribution rates of AmpC (χ2=8.9, P<0.05), OXA-23 (χ2=28.05, P<0.05), adeB (χ2=9.5, P<0.05) and adeG (χ2=5.20, P<0.05) in imipenem resistant Acinetobacter baumannii group were significantly different from those in imipenem sensitive Acinetobacter baumannii group. Conclusion There has been clonal spread of Acinetobacter baumannii stains among patients in hospital, which are mainly type A strain. β-lactamases genes Amp-C and OXA-23 and efflux pump systems adeABC and adeFGH may play an important role in the carbapenems resistance mechanism of Acinetobacter baumannii.

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    Toxin detection and ribotyping of nosocomial Clostridium difficile strains
    ZHANG Li-hua, DONG Dan-feng, JIANG Cen, et al
    2012, 32 (11):  1436. 
    doi: 10.3969/j.issn.1674-8115.2012.11.009

    Abstract ( 1319 )   PDF (437KB) ( 1279 )  

    Objective To investigate the virulence of Clostridium difficile clinical isolates, and conduct an epidemiologic study by PCR-ribotyping for the strains. Methods The unformed stool samples from hospitalized patients with diarrhea were collected, and were inoculated onto CDMN selective culture media after pretreatment with dehydrated alcohol for the culture of Clostridium difficile. Isolates were identified by Gram staining, oxygen tolerance test and agglutination assay. Bacterial genome DNA was extracted from the Clostridium difficile strains, and toxin gene tcdA and tcdB were amplified by PCR with specific primers. Meanwhile, PCR-ribotyping was performed through amplification of the genomic 16S-23S rDNA intergenic spacer region sequence, followed by electrophoresis to distinguish different ribotypes according to the polymorphism of the bands. Results Forty-four Clostridium difficile strains were divided into 3 toxin types: A+B+strains, A-B+strains and A-B-strains, which accounted for 57%, 34% and 9%, respectively. All the strains belonged to 18 ribotypes, mainly ribotype R8 (20%) and ribotype R4 (18%). Conclusion In the study, the Clostridium difficile clinical isolates were mainly A+B+ strains, and there existed relatively predominant ribotypes, but no evidence suggested nosocomial outbreak of Clostridium difficile infection.

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    Research progress of bacterial pan-genome
    ZHUANG Xu-ran, ZHU Yong-zhang
    2012, 32 (11):  1440. 
    doi: 10.3969/j.issn.1674-8115.2012.11.010

    Abstract ( 1660 )   PDF (327KB) ( 1669 )  

    Bacterium, one of the most ancient organisms, has great diversity and obvious differentiation in phenotype among different strains and even in different lines of one strain. The hereditary basis of differentiation is due to the genomic genetic information difference among different strains. In order to illustrate the individual genetic diversity and explore the hereditary basis of individual phylogenesis and phenotype difference, the concept of pan-genome is put forward. The causes of hereditary diversity of bacteria, the research strategy of pan-genome and its application in bacterial research are reviewed in this paper.

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    Cloning, expression and immune response in mice of specific antigen Rv3117 from M. tuberculosis
    TANG Jun-ming, CHEN Cui-cui, WANG Xue-cai, et al
    2012, 32 (11):  1444. 
    doi: 10.3969/j.issn.1674-8115.2012.11.011

    Abstract ( 948 )   PDF (372KB) ( 890 )  

    Objective To clone recombinant protein Rv3117 from M. tuberculosis, and investigate its immune response in mice. Methods The gene encoding Rv3117 protein was amplified by PCR from genome DNA of M. tuberculosis, and was then cloned into corresponding site of the expression vector pET-32a. The recombinant plasmid pET32a-Rv3117 was transformed into E.coli BL21 plysS (DE3), induced with isopropyl-β-D-thiogalactopyranoside (IPTG), and purified by Ni-NTA purification system. The expression of recombinant protein was identified by SDS-PAGE analysis. Western blotting was performed on sera from Rv3117-immunized C57BL/6 mice. Results The prokaryotic expression vector pET32a-Rv3117 was successfully constructed. The target protein was expressed in soluble form in E.coli BL21 plysS (DE3), with the relative molecular weight of 48 100, which was in line with the expectations. Western blotting revealed a positive stripe at the destination. Conclusion The recombinant protein Rv3117 has been successfully cloned from M. tuberculosis, which could induce immune response in mice.

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    Original article (Basic research)
    Effects of p38 MAPK signaling pathway on B7H1-Ig fusion protein-initiated differentiation of Tr1 cells
    SHAO Xiao-yi, ZHOU Yun, LU Li-ming, et al
    2012, 32 (11):  1448. 
    doi: 10.3969/j.issn.1674-8115.2012.11.012

    Abstract ( 943 )   PDF (709KB) ( 940 )  

    Objective To investigate the effects of mitogen-activated protein kinase (MAPK) signaling pathway on B7H1-Ig fusion protein-initiated differentiation of type 1 regulatory T cells (Tr1). Methods Fresh isolated naive CD4+CD62L+T cells of C57BL/6 mice were stimulated with immobilized B7H1-Ig fusion protein plus anti-CD3 monoclonal antibody to induce Tr1 cells. The activities of three major MAPK (ERK1/2, p38MAPK, JNK) signaling pathways during the process of Tr1 cells differentiation were analyzed by Western blotting. In experiments, the specific inhibitors of ERK1/2, p38 and JNK (PD98059, SB203580 and SP600125) were added separately at the time of culture initiation, and the influences on cytokines secretion, immune function and Foxp3 expression of B7H1-Igstimulated CD4+T cells were detected respectively by ELISA, mixed lymphocyte reaction (MLR), flow cytometry and Western blotting. Results B7H1-Ig-plus-anti-CD3 stimulation activated and induced the generation of a subset of IL-10+IFN-γ+IL-5+IL-4low/-IL-2low/-Foxp3- Tr1 cells, which played immunosuppressive roles via secreting IL-10. Western blotting indicated that B7H1-Ig activated the p38 MAPK signaling pathway in CD4+T cells, while had no significant effect on ERK1/2 and JNK signaling pathways. Blocking p38 MAPK activity could significantly inhibit the production of IL-10 and IL-5 induced by B7H1-Ig in CD4+T cells, weaken the immunosuppressive function of B7H1-Ig-stimulated CD4+T cells and promote them to differentiate into CD25+Foxp3+Tregs. Conclusion The activation or inhibition of p38 MAPK signaling pathway is one of the important molecule mechanisms to control B7H1-Ig-induced Tr1 cells differentiation and transformation between Tr1 cells and CD4+CD25+Foxp3+Tregs.

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    DNA methylation of promoter of apoptosis-related FADD and APAF1 genes in rats with adriamycin nephropathy
    LIU Jian, ZHONG Fang, XU Li-li, et al
    2012, 32 (11):  1455. 
    doi: 10.3969/j.issn.1674-8115.2012.11.013

    Abstract ( 1158 )   PDF (554KB) ( 955 )  

    Objective To investigate the relationship between DNA methylation of apoptosis-related genes in renal tissues and disease progression in rats with adriamycin nephropathy. Methods Twelve male SD rats were randomly divided into adriamycin nephropathy group and normal control group, with 6 rats in each group. Adriamycin nephropathy model was established in adriamycin nephropathy group by injection of 5 mg/kg adriamycin on the first day and 2.5 mg/kg adriamycin one week later through penis vein. Blood samples were collected via abdominal aorta 8 weeks after model establishment, then rats were sacrificed, and renal tissues were harvested. The biochemical parameters such as serum creatinine (Crea), blood urea nitrogen (BUN), serum albumin (ALB) and urinary albumin to creatinine ratio (UACR) were measured. The pathological changes of renal tissues were observed with Masson staining. The apoptosis of renal tubular cells and glomerular cells was detected by TUNEL staining. The DNA methylation in the promoter of Fas-association protein with death (FADD) and apoptotic protease activating factor-1 (APAF1) genes was determined by high-resolution melting (HRM) analysis, and the expression of FADD mRNA was detected by Real-Time PCR. Results Crea, BUN and UACR in adriamycin nephropathy group were significantly higher than those in normal control group (P<0.05 or P<0.01), while ALB in adriamycin nephropathy group was significantly lower than that in normal control group (P<0.01). Pathological examination revealed that compared with normal control group, there were increased glomerular mesangial cell proliferation, swelling of renal tubular epithelial cells and inflammatory cell infiltration in adriamycin nephropathy group. TUNEL assay indicated that the number of apoptotic tubular cells and glomerular cells in adriamycin nephropathy group was significantly higher than that in normal control group (P<0.01). HRM analysis and Real-Time PCR demonstrated that compared with normal control group, the promoter of FADD was relatively hypermethylated, and the expression of FADD mRNA was significantly up-regulated in adriamycin nephropathy group (P<0.05). Conclusion In rats with adriamycin nephropathy, the DNA in the promoter of FADD is hypermethylated, but it may not be the major factor affecting the expression of FADD mRNA.

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    Effects of prenatal exposure to brominated diphenyl ethers-209 on reproductive development of female offspring rats
    LIANG Chen, HE Xiao-wen, XIE Xin, et al
    2012, 32 (11):  1461. 
    doi: 10.3969/j.issn.1674-8115.2012.11.014

    Abstract ( 879 )   PDF (619KB) ( 940 )  

    Objective To investigate the effects of brominated diphenyl ethers-209 (BDE-209) on the reproductive development of female offspring rats. Methods Twenty-four pregnant SD rats were randomly divided into low-dose BDE-209 group (exposure to 100 mg·kg-1·d-1 BDE-209), medium-dose BDE-209 group (exposure to 300 mg·kg-1·d-1 BDE-209), high-dose BDE-209 group (exposure to 900 mg·kg-1·d-1 BDE-209) and control group (exposure to corn oil), with 6 rats in each group. The body weight, height and tail length were obtained on postnatal day (PND) 4, PND10, PND16, PND21, PND30 and PND40, and anogenital distance was measured on PND16 and PND21 in female offspring rats. Serum estradiol levels were determined on PND21 and PND40 after sacrifice of 1 female offspring rat respectively. Organ coefficients of ovary, liver and kidney were calculated on PND40, and the weight and pathological changes of ovary were observed. Results The body weight, height and tail length in high-dose BDE-209 group were significantly lower than those in control group in part of the development period (P<0.05). The anogenital distance in low-dose BDE-209 group was significantly longer than that in control group on PND21 (P<0.05), the number of secondary follicles in medium-dose BDE-209 group was significantly larger than that in control group on PND40 (P<0.05), and there was no significant difference in serum estradiol levels among groups on PND21 and PND40 (P>0.05). Conclusion Exposure to BDE-209 during pregnancy can impair the reproductive development of female offspring rats.

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    Transcriptional inhibition of GADD45A by PML/RARα
    RAO Yu-qing, WANG Kan-kan, ZHANG Ji
    2012, 32 (11):  1466. 
    doi: 10.3969/j.issn.1674-8115.2012.11.015

    Abstract ( 1302 )   PDF (493KB) ( 1031 )  

    Objective To investigate the mechanism by which the aberrant fusion protein, PML/RARα, transcriptionally regulates GADD45A in acute promyelocytic leukemia (APL) cells. Methods ChIP-PCR was employed to study the enrichment of PML/RARα at the intron 3 of GADD45A. Real-Time PCR was conducted to investigate whether the ectopic expression of PML/RARα could impact the mRNA level of GADD45A gene in PR9 cell line. Eucaryotic expression vector was utilized to overexpress PML/RARα and p53 protein in H1299 cells, in order to investigate the co-regulation relationship between PML/RARα and p53 on GADD45A gene. Results ChIP-PCR revealed that the specific PML/RARα antibody could enrich the DNA segment of intron 3 of GADD45A gene compared to the negtive results of non-specific normal IgG, which indicated that PML/RARα could bind to the intron 3 of GADD45A. In PR9 cells, the expression of PML/RARα exhibited significant negtive correlation with GADD45A mRNA expression. PML/RARα could inhibit the transcriptional activity of p53 on GADD45A in H1299 cells (P<0.01). Conclusion PML/RARα can bind to the intron 3 of GADD45A gene. GADD45A is also the target of p53 protein, and PML/RARα can inhibit GADD45A expression and interrupt p53 dependent transcriptional effect of GADD45A gene.

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    Effects of glutamine on secretion suppression of RAW 264.7 cells induced by mesenteric lymph in rats with sepsis
    LIANG Meng-fan, WANG Xue-min, XUE Ying, et al
    2012, 32 (11):  1472. 
    doi: 10.3969/j.issn.1674-8115.2012.11.015

    Abstract ( 973 )   PDF (278KB) ( 976 )  

    Objective To investigate the effects of intravenous administration of glutamine (Gln) on secretion suppression of RAW 264.7 cells induced by mesenteric lymph in rats with sepsis. Methods SD rats were randomized into sham-operation group (Sham group), sepsis group (CLP group) and Gln group, with 10 rats in each group. Sepsis model was established by cecal ligation and puncture in CLP group and Gln group. Within 10 minutes after model establishment, 0.75 g/kg Gln was administered via single tail-vein injection in Gln group, while the same amount of normal saline was administered in Sham group and CLP group. Mesenteric lymph was obtained from 5 rats in each group, and blood samples were taken from the other rats 6 h after model establishment in each group. RAW264.7 cells were incubated with lymph for 4 h in each group, and the supernatants were harvested. The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in mesenteric lymph and supernatants were measured by ELISA, and the concentrations of Gln in mesenteric lymph and D-lactate in plasma were determined by spectrophotometric assay. Results The concentrations of TNF-α and IL-6 in mesenteric lymph in CLP group and GLN group were significantly higher than those in Sham group (P<0.05). The concentrations of TNF-α and IL-6 in supernatants in CLP group were significantly lower than those in sham group (P<0.05), while the concentration of TNF-α in supernatants in Gln group was significantly higher than that in CLP group (P<0.05). There was no significant difference in the concentrations of Gln in mesenteric lymph among three groups (P>0.05). The concentration of D-lactate in plasma in Gln group was significantly lower than that in CLP group (P<0.05). Conclusion Secretion of RAW264.7 cells activated by LPS can be suppressed by mesenteric lymph in rats with sepsis, which can be partially alleviated by Gln treatment.

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    Experimental study of 3.0T diffusion weighted imaging and diffusion tensor imaging in diagnosis and outcome prediction of cerebral blast injury
    WU Peng, LV Guo-shi, HAN Feng, et al
    2012, 32 (11):  1476. 
    doi: 10.3969/j.issn.1674-8115.2012.11.017

    Abstract ( 964 )   PDF (724KB) ( 999 )  

    Objective To investigate the value of diffusion weighted imaging (DWI) and diffusion tensor imaging (DTI) in diagnosis of non-hemorrhagic foci and white matter fiber injury early after blast in rabbits, and explore their role in outcome prediction. Methods Thirty New Zealand rabbits were selected, and model of cerebral blast injury was established. After injury, routine CT, magnetic resonance imaging (MRI), DWI and DTI were performed, and quantified analysis of region of interest was conducted with Functool 2 postprocessing technology. The cerebral histopathological changes were observed, and contrast analysis was carried out with findings from DWI and DTI of the same plane. Results Of the 30 rabbits, there was no abnormal cerebral imaging findings in 6 rabbits by CT, T1WI and T2WI, and there was no abnormal low signal in 2 of the 6 rabbits by DWI. Among the other 24 rabbits, CT only revealed pneumatosis in brain, MRI routine subsequence demonstrated non-hemorrhagic foci, which were characterised by low or slightly lower signal on T1WI and high signal on T2WI, and DWI indicated bright-white high signal. DWI illustrated 189 non-hemorrhagic foci, which exhibited punctiform (28.5%), lamellar (54.0%) and slinar (17.5%) high signal, with sharp border. The number of non-hemorrhagic foci detected by DWI was significantly larger than those revealed by conventional T1WI and T2WI (P<0.01). DTI demonstrated decrease in apparent diffusion coefficient (ADC) and fiber tractography (FA) of region of interest, especially for the inner capsule (P<0.01). There was linear correlation of decrease in ADC and FA values of cerebral white matter and brain stem with time of survival of rabbits (r=0.53,P=0.05;r=0.13,P=0.03;r=0.25,P=0.04;r=0.27,P=0.02). Conclusion DWI may provide higher detection rate for non-hemorrhagic foci in rabbits with cerebral blast injury, especially for small lamellar non-hemorrhagic foci. DTI may play a role in outcome prediction through measurement of decrease in ADC and FA values.

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    Original article (Clinical research)
    Clinical features of infective endocarditis with positive antineutrophil cytoplasmic antibodies
    YAO Dong-ting, YANG Cheng-de, DING Hui-hua, et al
    2012, 32 (11):  1482. 
    doi: 10.3969/j.issn.1674-8115.2012.11.018

    Abstract ( 1000 )   PDF (356KB) ( 902 )  

    Objective To investigate the clinical features of infective endocarditis (IE) with positive or negative antineutrophil cytoplasmic antibodies (ANCA). Methods Thirty-nine patients with IE were were divided into ANCA positive group and ANCA negative group according to the serum ANCA findings by ELISA. The clinical features of patients such as general conditions, susceptible factors, clinical manifestations, laboratory indexes and involved valves were retrospectively analysed, and the parameters were compared between two groups. Results ANCA was positive in 13 of the 39 patients (33.3%), and all were PR3-ANCA positive. Native valve endocarditis was diagnosed in 37 patients, and organic heart disease was present in 34 of them, while no basic heart disease was found in the other 3 patients. There was no significant difference in the prevalence of susceptible factors between ANCA-positive group and ANCA-negative group (P>0.05). The major clinical manifestations of patients included anemia (66.7%), splenomegaly (38.5%), nephropathy (28.2%), arthralgia (23.1%) and edema of lower extremity (17.9%). The prevalence of edema of lower extremity in ANCApositive group was significantly higher than that in ANCA-negative group (38.5% vs 7.7%)(P<0.05). As for laboratory findings, higher C-reactive protein (CRP) values were found in all the patients, higher erythrocyte sedimentation rate (ESR) occurred in 89.7% patients, while lower concentration of haemoglobin (Hb) was detected in 66.6% patients. Besides, 50.0% patients had hematuria, and 53.8% patients had proteinuria. However, there was no significant difference in all the laboratory indexes between two groups (P>0.05). Blood cultures were performed in 37 patients, and a causative microorganism was identified in 15 (40.5%) patients. The positive rate of blood culture in ANCA-positive group was significantly higher than that in ANCA-negative group (69.2% vs 25.0%)(P<0.05). Seventeen strains of pathogenic bacteria were isolated, among which 14 were Streptococcus. Three patients in ANCA-positive group died, among whom 2 were misdiagnosed as ANCA associated small vessel vasculitis (AAV). There was no case of death in ANCA-negative group. Conclusion There may exist cases of ANCA-positive IE, which may be misdiagnosed as AAV. ANCA-positive IE may be attached great importance in clinics.

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    Comparison of activated coagulation time and thromboelastography in evaluation of effect of unfractionated heparin during PCI
    HOU Xu-min, DAI Jin-jie, HAN Wen-zheng, et al
    2012, 32 (11):  1486. 
    doi: 10.3969/j.issn.1674-8115.2012.11.019

    Abstract ( 1260 )   PDF (370KB) ( 1229 )  

    Objective To determine the coagulation function before, during and after selective percutaneous coronary intervention (PCI) with activated coagulation (ACT) and thromboelastography (TEG), and evaluate the sensitivity and association between these two methods. Methods Thirty-six patients with stable angina undergoing selective PCI were enrolled, and unfractionated heparin (UFH) 100 U/kg was intravenously administered before PCI. ACT and TEG reaction time (TEG-R) were examined before PCI and 5 min and 30 min after UFH administration. The parameters of coagulation function (PT and APTT) and blood routine parameters (PLT, RBC and Hb) were measured before PCI and 6 h, 24 h and 72 h after PCI. Results ACT and TEG-R 5 min after UFH administration were significantly longer than those of baseline (P<0.001), while ACT significantly shortened (P<0.001) and TEG-R still maintained at a higher level (P>0.05) 30 min after UFH administration. There were significant differences between PT, APTT, RBC and Hb 6 h after PCI and those of baseline (P<0.05 or P<0.001), while there was no significant difference between these parameters 24 h after PCI and those of baseline (P>0.05). There was no significant difference in PLT among these time points (P>0.05). Correlation analysis revealed that there was significant curvilinear correlation between ACT and TEG-R (P<0.001). Conclusion TEG-R is more sensitive than TEG-R in detection of residual UFH during PCI, which indicates that TEG may be a useful tool for monitoring of UFH therapy during PCI.

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    Analysis of clinical features and prognosis in 126 patients with peripheral primitive neuroectodermal tumor
    ZHANG Feng-chun, TANG Lei, MA Yue, et al
    2012, 32 (11):  1490. 
    doi: 10.3969/j.issn.1674-8115.2012.11.020

    Abstract ( 1022 )   PDF (711KB) ( 862 )  

    Objective To investigate the clinical characteristics and factors of prognosis for peripheral primitive neuroectodermal tumors (pPNET). Methods The clinical data of 9 patients in clinics and medical records of 117 patients with pPNET reported by literatures were collected. The data of imaging, histopathology, immunohistochemistry, clinical features and treatment of patients with pPNET were analysed. Kaplan-Meier method was employed to calculate the 1-year, 3-year and 5-year survival rates of patients, and the factors of prognosis were analysed. Log-rank tests were used for univariate analysis, and Cox proportional hazards was adopted for multivariate analysis. Results There were 65 males and 61 females, with the average age of (29.90±16.06) years. There were 35 cases (27.8%) with stage I, and the percentages of stageⅡ, Ⅲ, and Ⅳ were 42.1%, 8.7% and 21.4%, respectively. One hundred and ten patients received surgical treatment, radical surgery was performed in 88 patients, and 12 patients underwent surgery after neoadjuvant chemotherapy. Thirty-two patients (25.4%) received adjuvant radiotherapy, and 61 patients (48.4%) underwent adjuvant chemotherapy. Forty-seven patients received first-line chemotherapy, among whom 37 received second-line or more than second-line salvage therapy. The mean follow-up duration was 19.0 months. Relapse occurred in 52 patients, and there were 38 cases of death. The 1-year, 3-year and 5-year survival rates were 54.8%, 15.9% and 3.2%, respectively. Univariate analysis revealed that tumor size, lymph node status, distant metastasis and stage were significant prognostic factors, and multivariate analysis indicated that complete surgery was an independent prognostic factor. Conclusion Comprehensive therapy is the main treatment for pPNET, and complete surgery is an important prognosis factor.

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    Clinical analysis of 837 cases of laparoscopic surgery for acute cholecystitis
    GUO Zheng-hua, ZHANG Yu-cheng, LOU Xiao-lou
    2012, 32 (11):  1497. 
    doi: 10.3969/j.issn.1674-8115.2012.11.021

    Abstract ( 991 )   PDF (277KB) ( 1020 )  

    Objective To investigate the rationality and timing of laparoscopic cholecystectomy (LC) for acute cholecystitis. Methods The clinical data of 837 patients with acute cholecystitis undergoing LC (LC group) and 185 patients receiving open cholecystectomy (OC) for acute cholecystitis (OC group) were retrospectively analysed. According to the time of disease onset, patients in LC group were subdivided into acute group (n=679, time of disease onset≤72 h) and subacute group (n=158, time of disease onset>72 h). The mean time of operation, volumes of bleeding during operation, rates of transference to open abdominal surgery, scores of visual analogue scale (VAS) after operation, prevalences of complications after operation and duration of hospitalization were compared between LC group and OC group and between acute group and subacute group. Results Compared with OC group, the time of operation and duration of hospitalization were shorter, the volume of bleeding during operation was smaller and VAS was lower in LC group, and there were significant differences in these parameters between two groups (P<0.05). There was no significant difference in the prevalences of complications after operation between OC group and LC group (P>0.05). Compared with subacute group, the time of operation and duration of hospitalization were shorter, the volume of bleeding during operation was smaller and the rate of transference to open abdominal surgery was lower in acute group, and there were significant differences in these parameters between two groups (P<0.05). Conclusion LC is feasible and safe for patients with acute cholecystitis, and surgical intervention within 72 h of disease onset may yield higher success rate and safety.

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    Impact of unilateral or bilateral cataract surgery on vision health related quality of life
    ZUO Lei, ZHANG Jian-hong, ZOU Hai-dong, et al
    2012, 32 (11):  1501. 
    doi: 10.3969/j.issn.1674-8115.2012.11.022

    Abstract ( 1078 )   PDF (336KB) ( 1053 )  

    Objective To investigate the impact of unilateral or bilateral cataract surgery on vision health related quality of life. Methods Two hundred and fifty-eight patients with cataract undergoing phacoemulsification with intraocular lens implantation were selected, with the mean age of (73.16±10.79) years. The quality of life was investigated in patients undergoing unilateral cataract surgery (groupⅠ, n=136) or bilateral cataract surgery (groupⅡ, n=122) with Chinese-version low vision quality-of-life questionnaire (CLVQOL) before surgery and 3 months after surgery, the scores of vision health related quality of life (VRQOL) were calculated and compared between groups, and the influential factors were analysed. Results There was no significant difference in the scores of VRQOL, weighted average logarithm of minimum angle of resolution (logMAR) visual acuity, age, time of education, gender, systemic or ocular comorbidities before operation between two groups (P>0.05), and there was also no significant difference in the prevalences of surgical complications between two groups (P>0.05). Three months after surgery, there were significant differences in the improvement of weighted average logMAR visual acuity between groupⅠ and groupⅡ (0.42±0.38 vs 0.63±0.53, P=0.04), while there was no significant difference in the improvement of VRQOL scores between groupⅠand groupⅡ (P=0.112). Multivariate analysis indicated that ways of surgery (unilateral or bilateral surgery) was less important factor to the improvement of scores of VRQOL (P=0.054). Conclusion In elder patients with high preoperative disease burden, either unilateral or bilateral cataract surgery could bring improvement to vision health related quality of life, and there is no significant difference between these two ways of surgery.

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    Original article (Public health administration)
    Empirical research on training quality of clinical postgraduate students
    CHEN Li, ZHANG Guo-dong, YUAN Xiao-yan, et al
    2012, 32 (11):  1506. 
    doi: 10.3969/j.issn.1674-8115.2012.11.023

    Abstract ( 1014 )   PDF (417KB) ( 813 )  

    Objective To investigate the status of training quality of clinical postgraduate students, and analyse the potential problems. Methods Cluster random sampling method was utilized to select 415 clinical postgraduate students 622 who graduated from Shanghai Jiaotong University School of Medicine between 2006 and 2009 and worked in 26 hospitals in Shanghai. The training quality of clinical postgraduate students was evaluated by department leaders or personnel staff on the aspects of ethical norms, professional ability, career development ability, health status and scientific research ability, and the results were compared with those of self-evaluation from the graduates. Results The aspects of training quality which were poorly thought of by the employers were the ability of critical thinking (21.62%), creativity (36.0%)and professional knowledge (36.04%). There were significant differences in the evaluation of training quality between employers and graduates (P<0.05). Employers emphasized on the cultivation of professional values, while graduates paid more attention to the training of professional skills. There were significant differences in the training quality among graduates with different family background, permanent residential place and undergraduate universities evaluated by the employers (P<0.05). Factor analysis yielded 4 common factors, which were professional knowledge and skills, transferable skills, research capacity and professional values. Conclusion Employers deem that the graduates lack the ability in innovation and scientific research. Employers and graduates hold different opinions with regard to the goal of clinical postgraduate training. The family background, permanent residential place and undergraduate university may influence the training quality of clinical postgraduate students. Professional knowledge and skills, transferable skills, research capacity and professional values should be considered to evaluate the training quality of clinical postgraduate students.

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    Review
    Application of radionuclide reporter gene imaging in therapeutic gene monitoring
    PAN Yu, ZHANG Yi-fan
    2012, 32 (11):  1512. 
    doi: 10.3969/j.issn.1674-8115.2012.11.024

    Abstract ( 1108 )   PDF (403KB) ( 832 )  

    As a new disease therapy method, gene therapy shows a broad application prospect. However, there are still lots of problems in gene therapy to be solved. To evaluate the efficiency of gene therapy, it is requisite to monitor and evaluate the localization and expression of the therapeutic gene in vivo. Radionuclide reporter gene imaging, with rapid development in recent years, is considered as a non-invasive and sensitive method in monitoring the expression of the therapeutic gene in vivo. This method, which has the advantage of high sensitivity and specificity and may carry on deep-tissue imaging and repeat imaging, has shown a good application prospect. The mechanism, characteristics and value of radionuclide reporter gene imaging in monitoring gene therapy are reviewed in this paper.

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    Research progress of role of microRNAs on angiogenesis
    ZHU Jian-bing, ZHANG Jun-feng
    2012, 32 (11):  1517. 
    doi: 10.3969/j.issn.1674-8115.2012.11.025

    Abstract ( 1171 )   PDF (303KB) ( 1335 )  

    MicroRNAs (miRNAs), which exert pathophysiological function by  targeting  3'-untranslated regions (3'-UTRs) as an imperfect match, regulate gene expression over 30% in human genome on the posttranscriptional level by inhibiting the translation of protein from mRNA or by promoting the degradation or expression of mRNA to control cell proliferation, differentiation and apoptosis in different types of cells. Angiogenesis is the necessary process of cardiovascular remolding. MiR-210 and miR-424 are identified as pro-angiogenic miRNAs. Endothelial specific miR-126 plays a vital role in angiogenesis, as well as regulates vascular endothelial integrity and inflammation distinctly. In contrast, miR-221, miR-222, miR-92a and miR-24 inhibit endothelial cell angiogenesis. Thus, further research of role of miRNAs on angiogenesis and novel pathways of therapeutic angiogenesis may offer new significant strategies to treat ischemic heart disease and vascular disease.

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    Relationship between exposure to high fat diet in early life and metabolic syndrome
    SHI Wen, YANG Ke-feng
    2012, 32 (11):  1521. 
    doi: 10.3969/j.issn.1674-8115.2012.11.026

    Abstract ( 1016 )   PDF (303KB) ( 1109 )  

    The exposure to high fat diet in early life may program metabolic and other systems, then causes metabolic syndrome. The animal experiment evidences about effects of high fat exposure in early life on adult insulin resistance, dyslipidemia, hypertension and adiposity are summarized in this paper.

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    Correlation of Th17 cells and regulatory T cells with maternal-fetal immune tolerance
    DONG Qian, LI Wei-ping
    2012, 32 (11):  1525. 
    doi: 10.3969/j.issn.1674-8115.2012.11.027

    Abstract ( 1360 )   PDF (285KB) ( 995 )  

    Th17 cells are identified as a new kind of helper T lymphocyte subsets, which are totally distinct from Th1 and Th2 cell subsets. The main function of Th17 cells is secreting interleukin-17 (IL-17), which may play an important role in the defensive immune response against bacterial infection, chronic inflammation as well as the pathogenesis of autoimmune disease. Studies in recent years have revealed that the balance disorder between pro-inflammatory Th17 cells and immunosuppressive regulatory T cell (Treg) plays a key role in the pathological pregnancy by breaking the maternal-fetal immune tolerance. The correlation of Th17 cells and Treg with maternal-fetal immune tolerance is reviewed in this paper.

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