Objective To detect methylation of p16 gene in lung cancer tissues of non-small cell lung cancer patients,and to approach its clinical diagnostic value.Methods The methylation of p16 gene in DNA from 47 non-small cell lung cancer tissues and corresponding nomalignant tissues were tested with methylation-specific PCR(MSP).Results The total frequency of p16 methylation was significantly higher in lung cancer tissues than that in the corresponding malignant tissue(44.7%vs 17%)(P＜0.01).But there was no significant difference in detectiveness,clinical staging,clinical pathology type and clinical classification(P＞0.05).Conclusion The detection of methylation of p16 gene may be helpful to clinical diagnosis for non-small cell lung cancer,but its specify,sensitivity and feasibility need to be further studied.

Objective To evaluate the efficiency of using nested PCR in restriction fragment length polymorphism analysis (P1-RFLP) for genotyping Mycoplasma pneumonia (M.pneumonia) in clinical specimens.Methods Based on the gene sequence of RepMp4 and RepMp2/3 in P1 gene of reference strains M129 (type 1) and FH (type 2),two sets of inner primers were designed with a HaeⅢ restriction enzyme site (GGCC).The nested PCR was set up to detect the target DNA in clinical specimens.The amplification products were mixed and digested with Hae Ⅲ enzyme.The genotypes were analyzed by comparing with various restriction maps and the results were verified by sequencing analysis.The concentration of DNA extracted from standard and clinical strains were detected by ten-fold dilution to evaluate the sensitivity of nested PCR-P1-RFLP and P1-RFLP.M.pneumonia-positive specimens isolated from Beijing in 2012 were analyzed by the nested PCR-P1-RFLP and the results were compared with those by P1-RFLP analysis.Results The nested PCR-P1-RFLP could effectively genotype M.pneumonia in clinical specimens and the results were consistent with those by sequencing analysis.The sensitivity of new assay was 103 times higher than that of the original P1-RFLP.Of the 115 M.pneumoniae positive clinical specimens,97.4％ (112/115) were type 1 and the rest were type 2.Conclusion The nested PCR-P1-RFLP shows high efficiency for genotyping of M.pneumonia in clinical specimens.It might be useful for the surveillance of M.pneumoniae infection.

Objective To study the application of P1 adhesin protein epitopes in diagnosis of Mycoplasma pneumoniae(Mp) infected patient. Methods The major epitope(P1-534) of P1 adhesin protein were predicted by ProPred and ANTIGENIC according to its primary structure. The high value fragment was cloned into a constructed recombinant vector. The gene was induced to express fusion protein in E. coli host strain BL21(DE3) and the fusion protein was identified by Western blot. BALB/c mice were immunized with purified protein to test its immunogenicity. Then the purified protein was used as antigen to test the serum of Mp infected patient by ELISA, and compared with the Mp whole cell antigen. Results The P1-534 protein was successfully expressed and purified. ELISA data showed that P1-534 protein could elicit high levels of IgG in immunized mice, the sensitivity and specificity of P1-534 were determined to be 85.00% and 97.67%, while the Mp whole cell antigen were 72.50% and 74.42%. Conclusion The results conformed that the recombinant epitope has certain immunogenicity,and its sensitivity and specificity are better than Mp whole cell antigen. P1-534 protein can be used as an antigen for immunodiagnosis of Mp infection.

Objective To inspect the source of an outbreak with Mycoplasma pneumoniae (Mp).Methods We carried out real-time PCR to analyze specimens collected from pediatric patients in Beijing during January 2010 to May 2012,diagnosed as pneumonia or a respiratory infection according to clinical symptoms.These positive samples were analyzed by the M-P typing system(M:multiple-locus variable-number tandem-repeat analysis,MLVA; P:P1-restriction fragment length polymorphism analysis,P1-RFLP).Results Sixty-nine specimens were tested positive to Mp by the real-time PCR in 446 specimens from pediatric patients.The infection rate was 11.69％,15.56％ and 20.00％ respectively in 2010,2011 and the first half of 2012.According to the M-P system,11 distinct genotypes were identified from 69 positive specimens,M43562P1 and M53562P1 were the two main genotypes that showed an increasing trend from 2010 to 2011,and M33562P1 and M63562P1 showed an increasing trend from 2011 to 2012 in China.Conclusion During this international Mp epidemic,the infection rate of Mp was also increase in Beijing in 2011,and M43562P1 and M53562P1 were the two main genotypes.Among them,M43562 were consistent with pop genotypes in Europe,and M53562 were consistent with pop genotype in Israel.The M-P system would be valuable to monitor the epidemic of Mp in different countries in the world.

Objective To investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) genes among clinical isolates of Klebsiella pneumoniae (K.pneumoniae) in pediatrics.MethodsA total of 131 non-duplicate clinical isolates of K.pneumoniae were collected in the Affiliated Children′s Hospital of Capital Institute of Pediatrics from 2010 to 2012.PMQR genes [qnrA, qnrB, qnrS, aac(6′)-Ⅰb-cr and qepA], mutations in the quinolone resistance-determining region (QRDR) and extended spectrum β-lactamases (ESBLs) genes in those strains were analyzed by PCR.Minimum inhibitory concentrations (MIC) of different antibiotics against those K.pneumoniae strains were determined by broth microdilution method and E-test according to the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI).Transferability of the PMQR genes was examined by conjugation test with the sodiumazide-resistant Escherichia coli J53.Results Among the 131 isolates, 9.92% were resistant to quinolone and 30.5% were positive for PMQR genes, including 6.87% harboring qnrB gene, 22.9% harboring qnrS gene and 4.58% harboring aac(6′)-Ⅰb-cr gene.Neither qnrA-positive nor qepA-positive strain was detected.Among these PMQR genes-positive isolates, 90% were ESBLs-producing strains and two presented mutations in gyrA and parC genes.Conjugation test showed that these PMQR genes could be transferred horizontally and the ciprofloxacin resistance increased 2 to 32 folds in transconjugants.Conclusion This study indicates that the PMQR gene-carrying rate is high in K.pneumoniae strains isolated in paediatrics in China.Most of the PMQR gene-positive strains are also ESBLs-producing strains.The PMQR genes could be transferred horizontally in bacteria.

AIM To study the effect of astragalosides (AST) on pr ol iferation and collagen production of hepatic stellate cells (HSC). METHO DS The proliferation and collagen production of rat hepatic stellate cell s, HSC-T6, stimulated with Kupffer cell-conditioned medium (KCCM), mixed sera containing new bovine serum (NBS) and rat serum (RS) were measured with 3H -TdR and 3H-proline incorporation. RESULTS Both of the p roliferation and collagen production of HSC-T6 stimulated with KCCM at the dilu tion of 1∶4 were suppressed after being treated with AST (16, 32, 64, 128 and 2 56 mg?L -1 ). When HSC-T6 cells were stimulated with mixed sera containing 10% NBS and 3% RS, their proliferation was suppressed after being treated with AST(32, 64, 128 and 256 mg?L -1 )for 48 h and so was the collagen produ ction after being treated with AST(16, 32, 64, 128 and 256 mg?L -1 )for 72 h. CONCLUSION The suppression of hepatic stellate cell prolif eration and collagen production by astragalosides may be one of the mechanisms f or the depression of hepatic fibrosis by astragalosides.

Objective This study aims to investigate the effect of Lipoxin A4 receptor on acute lung injury (ALI) induced by intestine ischemia-reperfusion (IIR). Methods Thirty-two 8-week old SD rats were randomly divided into four groups: sham, intestine ischemia-reperfusion (IIR), IIR + BML111 (BML-111), Boc-2 + IIR +BML111 (Boc-2). BML-111 (1 mg/kg) was given intraperitoneally at the onset of reperfusion in the BML-111 and the Boc-2 group. Boc-2 (50 μg/kg) was given intraperitoneally after anesthesia in the Boc-2 group. Rats were subjected to superior mesenteric artery occlusion consisting of 45-min ischemia and 6-h reperfusion, and the sham laparotomy was served as controls. The lung pathology was assayed by the H&E staining. Lung water content was detected using dry/wet ratio. Concentrations of TNF-α, IL-1β, and IL-6 in lung tissue were determined by ELISA. The protein expression of p38 MAPK and NF-κB of lung was assayed by western blot. Results IIR induced serious ALI, with poor lung pathology and increased lung water content, elevation of TNF-α, IL-1β, and IL-6 levels in lung, accompanied with activation of p38 MAPK/NF-κB pathway. However, BML-111 could inhibit the activation of p38 MAPK/NF-κB pathway, leading to the reductions of TNF-α, IL-1β, and IL-6 in lung and attenuation of IIR-induced ALI. Conclusion BML-111 treatment could attenuate inflammation in lung after IIR injury via inactivating the p38 MAPK/NF-κB signaling pathway.

Objective:To evaluate the rapid nucleic acid amplification detection of Mycoplasma pneumoniae (MP)-DNA and MP-RNA in the diagnosis of MP infection and therapeutic values in children. Methods:Patients who were diagnosed with pneumonia were enrolled from the Department of Respiration, Children′s Hospital of Capital Institute of Pediatrics from January 2018 to December 2018.Specimens were detected using the MP and Macrolide-Resistant isolates Diagnostic Kit (PCR Fluorescence Probing, Jiangsu Mole Bioscience Co., Ltd.) and MP Diagnostic Kit (Isothermal RNA amplification, Shanghai Rendu Biotechnology Co., Ltd.).Results:Among them, 42.1%(840 cases) of the 1 994 cases were positive for MP-DNA, and the macrolide associated gene mutations were detected in 96.0% (806/840 cases) of them, while 33.9% (551 cases) of 1 624 cases were positive for MP-RNA.Seven hundred and fifty-eight specimens were simultaneously detected by adopting MP-DNA and MP-RNA, and the positive rate was 43.1% (327/758 cases) and 36.7% (278/758 cases), accordingly, which were inconsistent (Kappa=0.604) in 613 (80.9%, 613/758 cases) cases, with significant differences ( χ2=6.60, P=0.01). Part of the specimens were rechecked with the interval of 7 days: MP-RNA was negative in 70.1% (47/67 cases) specimens and MP-DNA was negative in 36.1% (22/91 cases) specimens ( χ2=33.20, P<0.01). Conclusions:The positive detection rate of MP was at a high level in 2018, in Beijing, China.The results of MP-DNA and MP-RNA are consistant.But RNA detection can help to diagnose MP in the early stage, and monitor the survival of MP and its efficiency.

Objective To compare the capabilities of culture method, polymerase chain reaction ( PCR) and serological test in identifying Mycoplasma pneumoniae infection in children with confirmed com-munity acquired pneumonia. Methods Bronchoalveolar lavage fluid and serum samples were collected from hospitalized children with community acquired pneumonia in Capital Institute of Pediatrics from March to May in 2016. Three methods, traditional culture method, PCR and serological test, were respectively used to de-tect Mycoplasma pneumoniae infection in those children. Statistical analysis was performed by using SPSS18. 0 software and chi-square test. Results Seventy-nine children with community acquired pneumonia were enrolled in this study. Eight (10. 13%) patients were diagnosed with Mycoplasma pneumoniae infec-tions by the traditional culture method with an average positive culture period of 21 days. Twenty-three (29. 11%) patients showed positive results by using PCR analysis, including the 8 patients identified by the culture method. Forty-one (51. 90%) patients were found to be positive for Mycoplasma pneumoniae infec-tions by the serological test. However, four negative samples identified by the serological test were confirmed to be positive by PCR analysis, including two positive samples confirmed by the culture method. Statistical analysis showed that the differences in positive rates detected by using the three methods were statistically significant. Conclusion It is recommended that both serological test and PCR analysis should be used in combination with clinical symptoms for a comprehensive assessment of Mycoplasma pneumonia infection in children.

Objective To analyze the relationship between macrolide resistance mutations in My-coplasma pneumoniae (Mp) and its genotype by multiple-locus variable-number tandem-repeat analysis (MLVA). Methods One hundred and forty-three Mp-positive specimens were collected in Beijing(54 col-lected at the Affiliated Children′s Hospital of the Capital Institute of Pediatrics),the United States(59 col-lected at four different geographical locations:Kansas City,Missouri;Seattle,Washington;New York,New York;Chicago,Illinois) and Australia(30 provided by the diagnostic laboratory at the Centre for Infectious Diseases and Microbiology Laboratory Services,Institute of Clinical Pathology and Medical Research,West-mead Hospital,Sydney). Nested PCR was used to detect mutations in 23S rRNA. A capillary electrophore-sis-based single tube multiplex PCR (mPCR-CE) was used to analyze the MLVA types of Mp in those sam-ples. Results A2063G mutation was identified in 57 specimens including 49 from Beijing,seven from the United States and one from Australia. The 143 Mp-positive specimens were typed into 10 distinct MLVA types. Fifty-four specimens collected in Beijing belonged to four MLVA types, which were M4-5-7-2 (44/54,81.5%),M3-5-6-2 (7/54,13.0%), M4-5-6-2 (2/54,3.70%) and M4-5-5-2 (1/54,1.85%). Fifty-nine specimens collected in the United States belonged to six MLVA types including M4-5-7-2(27/59, 45.8%),M3-5-6-2 (18/59,30.5%),M3-6-6-2 (11/59,18.6%),M3-5-6-1 (1/59,1.69%),M4-5-7-3 (1/59,1.69%) and M5-5-7-2 (1/59,1.69%). Thirty specimens of Mp from Australia were grouped to five types with M3-5-6-2 (12/30, 40.0%) and M4-5-7-2 (10/30, 33.3%) and M3-5-7-2 (5/30, 16.7%) being the predominant types. Macrolide resistance mutations were detected in 57 out of 143 speci-mens (49 from Beijing,seven from the United States and one from Sydney) and 50 of them were MLVA type of M4-5-7-2. Conclusion The MLVA type of M4-5-7-2 is associated with macrolide resistance in Mp.