Objective To investigate molecular diagnostic method for hereditarymethemoblobinemia. Methods The cDNA coding sequence of NADH-cytochrome b5 reductase (b5R) from 3 patients with hereditary methemoglobinemia was analyzed by direct sequencing of RT-PCR products and the genomic DNA of b5R gene by PCR-restriction endonuclease digestion or PCR-sequencing. Results The b5R cDNA of patient A was T/C heterozygous at nucleotide 527 and G/A heterozygous at nucleotide 608. The b5R cDNA of patient B was G/A heterozygous at both nucleotide 170 and nucleotide 179. The b5R cDNA of patient C was G/A heterozygous at nucleotide 608 and C/T heterozygous at nucleotide 791. Result of genomic DNA analysis was in agreement with that of cDNA approach. Conclusion The method for molecular diagnosis of hereditary methemoglobinemia was established and 3 novel b5R gene mutations were identified in compound heterozygosity in 3 Chinese patients.

Objective To study the effect of whole-process nursing intervention on comfort of patients undergoing radial artery puncture. Methods 100 patients with radial artery puncture from July, 2007 to June, 2008 were divided into the control group and the experimental group with 50 cases in each group accord-ing to time sequence. Routine whole nursing mode was used in the control group, the whole-process nursing intervention mode based upon routine nursing mode under the instruction of evidence-based method was used in the experimental group. Pain, psychological tensity, body numbness, success rate of radial artery puncture, patients' satisfaction degree were compared between the two groups. Results Every indexes of the experi-mental group were better than those of the control group. Conclusions The whole-process nursing interven-tion mode can promote the comfort degree of patients undergoing radial artery puncture.

Objective To clone and express human autoantigen Sm B'in methylotrophie yeast Pichia Pagtoris.Methods The gene Sm B' was cloned bv PCR The PCR product wag inserted into the vector pPIC9k.The recombinant plasmid pPIC9k.Sm B' was transformed into yeast Sm D1168 by electroporation.The positive clones were screened in MD plates.The high copy number transformants were rapidly selected by using G418 and were induced by methan01.Supematants after induction were analyzed by SDS-PAGE and western blot.Sera collected from thirty patients with SLE.thirty patients with mixed connective tissue disease(MCTD)and thirty healthy volunteers were detected by immunodot and immunoblot.Results The PCR product wag about 700 bD in size which Wag in accordance with predicted 657 bp.The pPIC9k-Sm B'showed the same seqencing result with GenBank's report and restriction enzyme analysis confirmed our prediction.The pPIC9k-Sm B' positive clone produced a 32 000 protein which had natural immunogenicitv of human autoantigen Sm B'by SDS-PAGE and western blot.The positive rate of immunodot and IBT were 46.7%(42/90)and 51.1%(46/90),respectively.The agreement between immunodot and IBT was very close(Kappa value=0.911 2,P＜0.01).Conclusion Successfully cloning and expression of human autoantigen Sm B' in methylotmphic yeast Pichia Pagtoris hid a foundation for further research work.

Objective To analyze the suspicious results of serum HBV DNA by fluorescence quantitative PCR and develop appropriate countermeasures in order to improve the quality of detection of HBV DNA.Methods Blood samples of patients from the First Affiliated Hospital of Wenzhou Medical College from 2008 to 2011 were analyzed for HBV DNA by fluorescence quantitative PCR.1969 cases of suspicious results,judged by the rule of review the results of serum HBV DNA combined with the historical results,PCR amplification curve,HBV serum markers and clinical diagnosis,were analyzed and redetected by using of two different reagents,careHBV PCR Kit and careHBV PCR Kit V2,at the same time.The consistency and inconsistency ratio of the results were evaluated.Both the reasons of inconsistent and the undetected rates of careHBV PCR Kit were analyzed.The two reasons for the inconsistent results included the reagent related factors,e.g,showing no amplification curve caused by the false negative and abnormal low efficiency of amplification curve,and the non reagent related factors such as operating pollution and other sample factors.Results There were 115 154 blood samples were detected for HBV from 2008 to 2011 and 1969 samples (1.71％) with suspicious results were redetected.The consistency and inconsistency results were 1588 (80.65％) and 381 (19.35％),respectively.Every year from 2008 to 2011,the percentage of the inconsistent results caused by the reagent related factors were 18.87％,20.23％,51.33％ and 59.57％ respectively,which showed an increasing trend,and the percentage of inconsistent results caused by the nonreagent related factors were 81.13％,79.77％,48.67％ and 40.43％ respectively,which showed a declining trend year by year.The undetected rates of careHBV PCR Kit were 2.49％,4.08％,10.09％ and 14.47％ respectively,showing an increasing trend.Conclusions The redetection for the specimens with the suspicious results by using of different reagents can avoid the blind detection of HBV DNA and reduce the experimental error.All the clinical samples for quantitative HBV DNA including the mutations of HBV gene can be measured accurately and effectively,which is helpful to hepatitis B patients for antiviral therapy.

Objective To observe the curative effect of low molecular heparin for treating secondary high coagulation state in the patients with nephrotic syndrome(NS) .Methods Total 87 cases of NS in our hospital were divided into the conventional treat‐ment group (n=42) and the low molecular heparin treatment group (n=45) .The routine treatment group was given the prednisone treatment and the low molecular heparin treatment group was treated by low molecular heparin combined with prednisone .The re‐lated indicators of blood coagulation before and after treatment were detected and the clinical curative effects in two groups were an‐alyzed .Results The coagulation related indicators in the conventional treatment group had no statistically significant difference be‐tween before and after treatment (P>0 .05) ,the prothrombin time(PT) and activated partial thrombin time(APTT) after treat‐ment in the low molecular heparin treatment group were significantly extended compared with before treatment ,while the concen‐trations of D‐dimer and fibrinogen were significantly decreased and the concentration of antithrombin Ⅲ was markedly increased compared with before treatment ,showing statistically significant differences between the two groups (P<0 .05);the patients of the low molecular heparin group patients had no bleeding after treatment .Conclusion Low molecular heparin combined with predni‐sone can reduce the secondary high condensation state in NS without bleeding and has a significantly clinical effect .

Objective To study the effects of taking clopidogrel on relevant indicators of platelet aggregation function in 138 cases acute coronary syndrome (ACS) .Methods The platelet function analyzer and flow cytometry were adopted to detect the ADP‐induced platelet aggregation rate ,P selectin and activated GP Ⅱ b/ Ⅲ before medication and on 7 d after taking clopidogrel . Results The platelet aggregation rate after taking clopidogrel for 7 continuous d was decreased significantly (P<0 .01);the P se‐lectin level and activated GP Ⅱ b/ Ⅲ a expressed on platelet surface were significantly reduced (P<0 .01) as well .Conclusion Taking clopidogrel could reduce the platelet aggregation significantly in the patients with ACS and has the effect for inhibiting the platelet aggregation .

Objective To analyze the expression of T lymphocyte activation antigen in peripheral blood of aged patients with non-small cell lung cancer . Methods The lymphocytes from peripheral blood in 45 aged patients with non-small cell lung cancer were immunologically labeled in double fluorescence and CD3-FITC, CD25/HLA-DR and CD69-PE and determined by flow cytometry. Normal aged donors, young patiens with lung cancer and aged benign lesion group were used as controls. Results Peripheral blood CD3 +/CD25 +, CD3 +/HLA -DR + and CD3 +/CD69 + in T lymphocyte with 45 aged lung cancer(7.24?1.85,28.46?5.39 and 7.78?2.63, respectively) were significantly lower than those in normal aged controls(10.35?2.54,37.16?5.51,11.02?2.18, respectively)and aged benign lesion (9.53?3.02, 35.33?5.23, 10.67?2.45, respectively)( P 0.05). Significant differences were found among them in stages Ⅲ, Ⅳ(7.15?1.13, 25.32?5.23, 7.14?2.81, respectively) and stagesⅠ,Ⅱ(8.06?1.21, 30.27?6.05, 8.43?2.67, respectively)( P 0.05). Conclusions Detection of CD3 +/CD25 +, CD3 +/HLA -DR + and CD3 +/CD69 + levels by flow cytometry might be helpful for reflecting the human immune function and the prognosis evaluation in patients with aged non-small cell lung cancer.

Objective To study the genotypes of OXA-51-like carbepenemases in Aeinetobacter beumannii and its association with drug resistance. Methods The susceptibility of 174 Acinetobacter baumannii against ceftazidime, cefotriaxon, amikacin and ciprofloxacin were detected with disc diffusion method. The minimum inhibitory concentration (MIC) values for meropenem and imipenem were determined with an agar dilution method. VIM, IMP, OXA-23, OXA-24, OXA-51 and OXA-58 β-lactamase genes were determined by PCR. DNA sequencing and genotyping were performed against OXA-51 positivestrains. Results All 174 isolates were negative by PCR for genes OXA-24, OXA-58, IMP and VIM. OXA-23 and OXA-51were amplified in 15.5% (27/174) and 72.4% (126/174) isolates, respectively. Therewere 15.5% (27/174) isolates producing OXA-51-like and OXA-23 carbapenemase simultaneously. Among126 OXA-51-like carbapenemase producing strains, 82.5% (104/126)were OXA-66 genotype, whereas theremaining 17.5% (22/126) strains belong to other genotype. Eight novel OXA-51-like Genotype were foundin this study. Conclusions OXA-66 were the primary genotype of OXA-51-like carbapenemases in A.baumannii. OXA-66 were related to low-level carbapenems resistance and may be associated with resistanceof other drugs. We found new OXA-51-like genotype in clinic isolates of A. baumannii in this study.

Objective To explore the effect of fluoxetine on the brain function of bulimia nervosa (BN) patients.Methods Seven female BN patients,who met criteria of the 3rd version Chinese Criteria of Mental Diseases (CCMD-3),accepted functional magnetic resonance imaging (fMRI) examinations before and after the antidepressant treatment (fluoxetine (20 mg/day)) for three months.Seven normal controls accepted the same fMRI examination only at baseline.fMRI imaging was block-design.Blocks of food or non-food stimulus containing pictures selected from International Affective Picture System (IAPS) which were shown by computer automatically.All subjects were evaluated by Hamilton anxiety scale (HAMA),Hamilton depression scale (HAMD17) and Likert Scale-likelihood evaluation to the same pictures in the fMRI imaging blocks.Results The average intensity and volume activated in BN before treatment were both significantly lower than that in the control (P＜0.05).But under stimulus of food pictures,bilateral prefrontal cortex and left amygdala of BN patients were significantly activated.After fluoxetine treatment,the intensity and volume activated both increased significantly (P＜0.01) and the main areas being activated were right temporal,cerebellum and bilateral prefrontal cortex.Conclusion Fluoxetine improves the bulimic symptoms of BN patients and decreases abnormal activation of prefrontal and limbic in these areas.The underline mechanism may be related to functions of serotonin system in prefrontal-limbic path.

OBJECTIVETo investigate the polymorphism in the promoter region of PCA3 gene and its relationship with risk of prostate cancer (PCa).

METHODSThe promoter region of PCA3 gene of the DNA of peripheral blood mononuclear cells was detected by sequence analysis in the 186 PCa and 141 BPH patients and 135 healthy control individuals. If the samples were detected with polymorphism of insection/deletion, clone sequence analysis was used with pBS-T carrier to verify it.

RESULTSThere were 5 polymorphisms. TAAA repeat times: 4, 5, 6, 7, 8, and 8 genotypes (TAAA 4/5, TAAA 4/6, TAAA 5/5, TAAA 5/6, TAAA 5/7, TAAA 5/8, TAAA 6/6, and TAAA 6/7) were detected in the promoter region of PCA3 gene. The eight genotypes were divided into three groups: ≤10TAAA, 11TAAA, ≥12TAAA. Unconditional logistic regression analysis models were used to analyze the relationship between different genotypes and cancer risks adjusted by sex and age. The type 11TAAA and ≥12TAAA was associated with higher relative risk for prostate cancer than the group ≤10TAAA [OR=1.74, 95% CI=1.06-2.87 (for type 11TAAA); OR=5.63, 95% CI=1.85-17.19 (for type ≥12TAAA)]. In the 186 PCa patients, there was 62.4% allele of PCA3 gene with AG/CA mutation found in the promoter 18-19 bp region of PCA3 gene and it had a close relation with the development of prostate cancer.

CONCLUSIONSShort tandem repeats are found in the promoter region of the PCA3 gene in PCa patients, and the increase of TAAA repeat sequences highly enhance the relative risk of prostate cancer development. The occurrence of such STR might be related to the mutations in their upstream loci.

Antigens, Neoplasm ; genetics ; metabolism ; Base Sequence ; Genes, Neoplasm ; physiology ; Genotype ; Humans ; Leukocytes, Mononuclear ; Male ; Microsatellite Repeats ; Mutation ; Polymorphism, Genetic ; Promoter Regions, Genetic ; Prostatic Neoplasms ; epidemiology ; genetics ; Risk