Clinical diagnostic value of serum β-hCG and AFP in mediastinal germ cell tumor / 中华检验医学杂志

Objective To explore the clinical diagnostic value of serum human chorionic gonadotropin beta subunit (β-HCG) and alpha fetoprotein ( AFP) in mediastinal germ cell tumors .Methods A retrospective analysis was conducted on the patients who were definitely diagnosed as mediastinal tumors or mediastinal neoplastic lesions .A total of 133 patients were included for analysis between January 2008 and May 2014, divided into two groups.42 cases of mediastinal germ cell tumor patients were marked as case group while 91 cases of other mediastinal tumor or mediastinal neoplastic lesion patients were marked as control group ( including 31 cases of thymoma , 10 cases of mediastinal neurogenic tumor , 2 cases of intrathoracic goiter , 25 cases of mediastinal cyst , 2 cases of mediastinal lipoma , 11 cases of mediastinal lymphoma and 10 cases of thymic carcinoma ) .AFP was detected by chemiluminescence detection , and -HCG was detected by electrochemical luminescence .K-S test was performed to investigate normality of data , non-normally distributed data were described as Median ( interquartile range ) .Mann-Whitney U test was done for measurement of data between two groups .Logistic regression analysis was performed as multivariate analysis.Receiver operating characteristic curve ( ROC) was used to determine the cut-off values.Results The levels of serum AFP and β-HCG in case group were 13.26 (2.39-48.09) ng/ml and 1.99 (0.10-15.7) IU/L, respectively, significantly higher than those in control group [AFP:2.47 (1.78-3.16) ng/ml,β-HCG:0.10 (0.10-0.55) IU/L].The difference of levels of AFP and β-HCG between the case group and the control group were statistically significant ( P=0.000 ) .There were no significant difference when it comes to β-HCG between the case group and intrathoracic goiter patients in control group .Apart from it, the difference of levels of AFP and β-HCG between the case group and every single control group were statistically significant .Cut-off values of AFP and β-HCG for distinguishing mediastinal germ cell tumors from mediastinal tumors were 5.07 ng/ml and 2.32 IU/L.In this scenario, for AFP and β-HCG, sensitivity were 57.1%and 50%, specificity were 97.8%and 96.7%, accuracy were 54.9%and 46.7%, area under the curve ( AUC ) were 0.773 and 0.755, positive likelihood ratios were 26.00 and 15.17respectively.Parallel experiments contributed to increase the sensitivity to 71.4%. Predictive probability (P) =1/[1+exp ( -0.319AFP-0.253HCG+2.850)] was obtained by logistic regression model.When cut-off value of predictive probability ( P ) was 0.30, specificity, AUC, and positive likelihood ratio were increased to 98.9%, 0.835 and 65.00respectively, negative likelihood ratio was decreased to 0.29, positive predictive value and negative predictive value were increased also (96.8%and 88.2%respectively).Conclusion Serum β-HCG, AFP and predictive probability ( P ) is valuable in the diagnosis of mediastinal germ cell tumor .

The preliminary establishment and application of a new method for evaluating K-ras mutations based fluid chip / 中华检验医学杂志

Objective A new method for detecting K-ras mutations based liquid chip was used to evaluate K-ras mutations associated with non-small cell lung cancer (NSCLC) patients,to direct the personalized treatment and prognosis evaluation.Methods Take the diagnosis technology research methods,the sensitivity and repeatability of the liquid chip K-ras gene mutation detection method were assessed.A total of 100 NSCLC patients from Nov 2011 to Feb 2012 in Shanghai Chest hospital were included in this study,the fresh tumor tissues were collected for DNA extraction.The 2nd exon 12 and 13 codons,containing 8 K-ras mutations occuring in high frequency were amplified by polymerase chain reaction (PCR),followed by ligation of the PCR products to a series of special probes using ligase detection reaction (LDR),then the PCR-LDR products were analyzed by liquid chip platform.Direct sequencing was applied to compare with the detection results.Results The sensitivity of liquid chip technology detection was 10％-20％,higher than the traditional sequencing method by 1％.Average CV value was 4％-15％ and showed good repeatability.5 K-ras mutations in 100 patients (5％) were detected using multiplex PCR-LDR combined fluid chip methods,including 3 Glyl2Val and 2 Gly12Asp mutations in exon 2.The 5 K-ras mutations were verified accurately by direct sequencing.Conclusions The novel detection method of K-ras mutations based PCRLDR and fluid chip shows high throughput,high sensitivity,good repeatability and the results are reliable and accurate.This method can be used to accurately identified K-ras mutations for NSCLC patients prior to their targeted therapy with TKIs.

The establishment of a new high-throughput method for evaluating the methylation status of lung cancer gene / 中华检验医学杂志

Objective To explore a new high-throughput method with internal standards for analyzing the methylation profiles of lung cancer related genes. Methods The promoter sequences of 7 lung cancer related genes were cloned into plasmids and the target segments were amplified by their special primers respectively. The products were treated with M. Sss Ⅰ methylase and bisulfite. The multiplex ligation PCR method was established by designing probes containing CpGpCpG(for methylatedsequence) at the 3' ends and choosing the optimal ligation enzyme, annealing and ligation temperatures. The standard calibrators and clinic samples were tested by fluid chip platform. The results were validated by methylationspecific PCR. Results We successfully set up the standard calibrators for methylation and unmethylaiton of 7 lung cancer related genes and established a multiplex ligation PCR combined with fluid chip method, which was used to detect methylation status of 7 genes simultaneously. The fluorescence value of p16INK4A, APC,DAPK, RARIβ, RASSF1 A, MGMT and GSTP1 methylation standard calibrators were 863,909,703,701,901,1 060 and 885, much higher than that of unmethylation standard calibrators. The results were consistent with the results of methylation-specific PCP. ConclusionThe new high-throughput method can be used to evaluate the methylation status of 7 lung cancer related genes simultaneously and might be useful for clinical practice.

Analysis of NKT cells and related cytokines of peripheral blood in primary biliary cirrhosis animal model / 中华检验医学杂志

Objective To investigate the cell phenotype for T cells in polyI: C induced primary biliary cirrhosis (PBC)animal model.Methods 20 female C57BI/6 mice,8 weeks old,were divided into model group and control group randomly. Mice in model group and control group were injected with polyI:C at a dose of 5 ms/ks and PBS,respectively.All mice were acrificed after 16 weeks after injection, and the sections of liver specimen were subjected to hematoxylin and eosin(H.E) staining.Serum AMA and ALP were detected.CD4+,CDs8+ and NKT cells in peripheral blood were determined by flow cytometry.The level of serum IL-4 and IFN-γ were assayed by EUSA.Results PBC mouse model was developed 16 weeks fter polyI: C injection. Infiltration of lymphocytes in portal area,positive serum AMA and high level of serum ALP were observed.The ratios of CD4+ T cells in model group and control group were(25.45±11.12)% and (26.72±0.63)%,respectively(t=0.314,P>0.05).The ratios of CDs+T cells in two groups were (18.3±0.91)% and (17.8±0.58)%,espectively(t=0.226,P>0.05).No significant change Was found for CD;and CDs+T cells in mice of both groups.However,NKT cells in peripheral blood of two groups were(11.56±5.09)% and (1.26±0.53)%,respectively(t=9.504,P<0.01).The number of NKT cells in model group was more than that of control group significantly.Simultaneously,serum L-4 and IFN·γ in mice of model group were also higher than that of control group.IL4 in senlm of two groups were (22.19±2.31)pg/ml and(8.72±0.87)pg/ml,respectively(t=58.06,P<0.01).IFN-γ in serum of two groups were(3.34±0.76)ng/ml and(1.14±0.21)ng/ml,respectively(t=23.31,P<0.01).Conclusions NKT cells increase greatly in eripheral blood of polyI:C induced PBC mouse model.NKT cells may play a critical role in the pathogenesis of PBC.

Defective activation induced cell death of CD4+ T cells in primary biliary cirrhosis murine model / 中华微生物学和免疫学杂志

Objective To study the activation induced cell death (AICD) of CD4+ T cells in primary biliary cirrhosis(PBC)murine model induced by poly I∶C. Methods Thirty female C57BL/6 mice were divided into model and control group randomly, and the former were injected with 5 mg/kg of poly I∶C, the later with PBS. PBC mice were detected 16 weeks after injection. CD4+ T cells isolated from spleen were stimulated in vitro by Con A and anti-CD3, and the apoptosis were determined by Annexin-V and PI staining. The expression of Fas, FasL and TRAIL were assayed by relative quantitative real-time PCR. Bcl-2 was detected by Western blot. Results Compared with control group, the portal areas of mice in model group were infiltrated with mononuclear cells obviously. The positive rate of serum antimitochondrial antibody(AMA) and the level of alkali phosphatase (ALP) were higher than that in control group (P<0.001). AICD of splenic CD4+ T cells in model group was lower than that of control group (P<0.001). The mRNA of FasL and TRAIL in model mice was down-regulated. Simultaneously, the anti-apoptosis protein Bcl-2 was up-regulated in model group. Conclusion These observations suggest that a defect in AICD of auto-reactive TH1 cells may contribute to the pathogenesis of PBC model. Furthermore, this defect in AICD may results from the change of Fas/FasL, TRAIL pathway and the up-regulation of Bcl-2.

Expression and clinical significance of chemokine receptor in peripheral blood mononuclear cells in patients with primary biliary cirrhosis / 第二军医大学学报

0.05),but was correlated with ?-GT(r=-0.295,P

Prediction and identification of HLA-A * 0201 restricted CD8~+ CTL epitope in Mycobacterium tuberculosis Ag85C / 第二军医大学学报

Objective:To predict and identify HLA-A * 0201 restricted CD8+ CTL epitopes in Mycobacterium tuberculosis (Mtb) antigen Ag85C, so as to provide evidence for epitope-based study for tuberculosis (TB) vaccine. Methods: The online database SYFPEITHI was applied to predict the potential HLA-A * 0201 restricted epitopes from Ag85C, an antigen of Mycobacterium tuberculosis. T2 cell line was used to assay the affinity between the predicted peptides and HLA-A * 0201 molecules. The specific CTL lines were induced from peripheral blood mononuclear cells (PBMCs) of HLA-A * 0201 positive TB patients and PPD+ healthy donors by peptides with high binding affinity to HLA-A * 0201 molecules. IFN-?production, in vitro proliferation and cytotoxicity of peptide-induced CTL were determined to screen HLA-A * 0201 restricted CD8+ CTL epitopes from those candidates. Results: Fourteen potential epitopes were identified from the SYFPEITHI database. After binding affinity assay, 3 of the 14 peptides (170-178 aa, 317-325 aa, and 144-153 aa) were found to have high binding affinity to HLA-A* 0201 molecules. However, only one peptide (144-153 aa) stimulated its specific CTL to release IFN-y, proliferate in vitro and produce specific cytotoxicity. Conclusion: We have successfully identified a HLA-A * 0201 restricted CD8+ CTL epitope of Mtb Ag85C-FLTREMPAWL( 144-153 aa) , which might be a candidate epitope for TB vaccine designing. Our findings provides a basis for developing novel and effective anti-TB vaccine.