Objective To establish a method for detecting the EGFR gene mutations by the real-time fluorescence quantification PCR combined with Sanger sequencing and to preliminarily explore its clinical application value .Methods With EGFR gene hotspot mutations region exon 19 and 21 as the research locus ,the specific amplification and the sequencing primer were designed ,the known wild-type and mutant samples were utilized to construct the corresponding plasmid as the standard substance by the TA clone technique .Then the EGFR gene mutation detection method by the real-time fluorescence quantification PCR combined with Sanger sequencing was established and the methodological and the application evaluation were performed .Results The wild-type and mutant standard plasmids of the EGFR gene exon 19 and 21 were constructed successfully .The EGFR gene mutations detection method of the real-time fluorescence quantification PCR combined with Sanger sequencing was established ,which had high sensitivi-ty(101copies/μL)andgoodrepeatability(intra-assayCVandinter-assayCVofthereal-timefluorescencequantificationPCRofex-on 19 and 21 were 1 .42% /3 .52% and 0 .97% /2 .44% ,respectively ) .20 clinical samples were simultaneously detected by this method and the traditional Sanger sequencing ,the results were completely consistent .Conclusion The EGFR gene mutations detec-tion method of the real-time fluorescence quantification PCR combined with Sanger sequencing is successfully established ,which can be used in the clinical sample detection .

Health Knowledge, Attitudes, Practice ; Humans ; Occupational Diseases ; prevention & control ; Rural Population ; Sampling Studies ; Surveys and Questionnaires ; Transients and Migrants

An electrospray/ultraviolet lamp dual-source ion trap mass spectrometer was developed for the rapid detection of gas and liquid samples.The instrument used the discontinuous atmospheric pressure sampling technique that both the electrospray ions and gaseous analytes were sampled and transferred using a pinch valve device.The two ionization sources were generally suitable for different kinds of analytes and provided complementary applications.Electrospray was used for the ionization of polar compounds in solution, while the UV ionization source was mainly applied for the analysis of gaseous organic compounds.A variety of samples such as anisole, toluene, 2,4-dimethylaniline, arginine, reserpine and aspartame were employed to test the performance in different working mode of the instrument.The results showed that the two sources were feasible for ionization of different types of samples, and different types of molecular ions could be generated when 2,4-dimethylaniline was analyzed.The two ionization sources could be used alternately or simultaneously without interference, and the working mode was also switched to fit the application requirements.The dual-source configuration was an effective way to extend the applications for miniature mass spectrometers.It did not significantly increase the size of the instrument, but provided more versatile analysis to meet the need for the measurement of different types of samples.

Objective Bladder cancer , which has a high rate of recurrence and invasion , is the most common genitourinary cancer.The article was to study the effect of specific chemokine receptor CXCR 4 on invasion capacity and intraluminal implantation of human bladder cancer cells . Methods A CXCR4 specific recombinant plasmid vector (short hairpin, shRNA) was constructed to select those cells which could inhibit the expression of CXCR 4, and these cells were divided into blank control group , negative control plasmid group and recombinant plasmid group (pshRNA-CXCR4-1, pshRNA-CXCR4-2).RT-PCR and immunofluorescence technique were used to detect the mRNA and protein expression of CXCR 4 respectively .Invasion capability in vitro of the cells was evaluated by Boyden chamber .20 nude mice were randomly divided into experimental group and control group ( n=10 ) .The experimental group was established by injection of 100μL shRNA-EJ-M3 into the bladder , while the control group was established by injection of 100μL EJ-M3, aiming to detect the effect of shRNA-CXCR4 on intraluminal implantation of human bladder cancer cells . Results The CXCR4 mRNA expression of the pshRNA-CXCR4-1 group (62.05 ± 1.35) was significantly lower than that of blank control group (174.38 ±1.96, P <0.05 ) and negative control plasmid group (166.27 ±1.82, P <0.05).There was no significant difference among the CXCR4 mRNA expression of the pshRNA-CXCR4-2 group, blank control group and negative control plasmid group ( 182.58 ± 4.2, 174.38 ±1.96, 166.27 ±1.82, P>0.05).In immunofluores-cence experiment, the red cell amount of the pshRNA-CXCR4-1 group(32.24 ±2.23) was lower than that of the blank control group (89.61 ±4.47,P<0.05) and negative control plasmid group (92.45 ±3.68, P<0.05).There was no significant difference among the red cell amount of the pshRNA-CXCR4-2 group, the blank control group and the negative control plasmid group (76.87 ±5.11, 89.61 ±4.47, 92.45 ±3.68, P>0.05).The Boyden chamber experiment showed that the number of penetrating cells of the pshRNA -CXCR4-1 group (39.67 ±8.45) was significantly lower than that of the blank control group (135.33 ±9.28, P<0.05) and that of the negative control plasmid group(123.63 ±6.36, P<0.05).As to the intraluminal implanting capability, the difference between the ex-perimental group and the control group of statistical significance (10%vs 70%,P<0.01). Conclusion CXCR4 shRNA can inhibit the expression of CXCR4 and significantly decrease the invasion capacity and intraluminal implantation of human bladder cancer cells .

[ ABSTRACT] AIM: To observe the effects of TNF-α/nuclear factor-κB( NF-κB) /matrix metalloproteinase-2 (MMP-2) pathway on the expression of MMP-2 in the mice with viral myocarditis.METHODS: Six-week-old inbred male mice were randomly assigned to control and myocarditis group.The mice in myocarditis group and control group were intra-peritoneally inoculated with 0.1 mL 10-5.69 TCID50 /mL coxsackievirus B3 and vehicle (PBS), respectively.Ten mice were sacrificed at the 4th and 10th days after injection.The blood and heart specimens were harvested.The serum content of TNF-αwas measured by ELISA.The myocardial levels of MMP-2, NF-κB p65 and IκBαwere determined by Western blot.RESULTS: Compared with control group, the protein expression of MMP-2 and NF-κB p65 in the myocardium and the serum content of TNF-αwere significantly increased in myocarditis group (P <0.05).The protein expression of IκBαwas lower in myocarditis group than that in control group (P <0.05).CONCLUSION: TNF-α, NF-κB p65 and MMP-2 were higher in the mice with acute viral myocarditis.The increased expression of them might be involved in the pathogene-sis of viral myocarditis.

Objective To develop a rapid quantitative detecting assay for point-of-care testing ( POCT ) of N-terminal pro-brain natriuretic peptide ( NT-proBNP ) in serum by the fluorescence immunochromatographic technology.Methods Applying double-antibody sandwich assay to establish the quantitative NT-proBNP kit.The performance of quantitative NT-proBNP kit was evaluated by the sensitivity , specificity, accuracy, precision, stability and clinical effectiveness.It compared the research kit and conference kit by the parallel experience in the 1 056(605 males, 451 females)serum specimen collected from Guangdong Provincial People′s Hospital, Sun Yat-sen Memorial Hospital and Children′s Hospital of Zhengzhou between February 2013 to April 2014.Statistical significance of the results was assessed by correlation analysis , linear regression , receive operating characteristic ( ROC) curve analysis , negative and positive consistent.Results The report range of the NT-proBNP kit was 18-35 000 ng/L.The coefficient of variation ( CV) values for low , median and high concentration calibrators respectively were all less than 15%.Common interfering substances in human serum specimens such as bilirubin , triglyceride and cholesterol were found no significant affect on NT-proBNP antigen detection and the CV were no more than 15%.According to the results of detection for calibrators , the shelf time of the NT-proBNP diagnostic kit should be longer than 12 months.The NT-proBNP kit and reference kit had good correlation ( Y=1.048 9X developed reference +121.54, R2 =0.956 6, n=1 056) to detect the target protein through the parallel experiments and the deviation of the quantitative results of clinical serum samples showed no statistical significance (Z=0.88, P=0.379>0.05).The clinical assays of two different diagnostic kits showed good consistency based on the ROC curve evaluation which is compared by two cut-off values (300 and 450 ng/L).The areas under ROC curve were 0.981 and 0.978 respectively.Conclusions A novel NT-proBNP chromatographic quantitative immunofluorescence detection method was developed in this study .The performance evaluation data indicated that the kit is suitable for rapid detection of serum NT -proBNP.

Objective To evaluate the olfactory functions in patients with Parkinson's disease (PD) with a smell identification test developed for the Chinese population and to examine possible factors contributing to PD patients' olfactory performance.Methods Fifty-five patients with idiopathic and nondementia PD and 55 demographically matched healthy controls (HC) were recruited and their olfactory identification tested using the Chinese Smell Identification Test developed by the Institute of Psychology at Chinese Academy of Sciences.Results Olfactory identification score was significantly lower (t =-6.827,P＜0.01) in the PD group (19.3 ±5.4) as compared with the HC group (26.5 ±5.7).Of all PD patients,63.6％ scored over 1 SD below the age norm.In particular,of those with Hoehn-Yahr ratings between 2 to 3,77.8％ scored over 1 SD below the age norm.Receiver operating characteristic (ROC) curve analysis indicated that a cut-off score of 22.5 on the Chinese Smell Identification Test distinguished between PD patients and HC with a sensitivity of 74.6％ and specificity of 70.9％.Within the PD group,age,education,Hoehn-Yahr rating and Mini-Mental State Examination score were not found to correlate with olfactory performance (all P ＞ 0.05).Conclusion PD patients in China show significant impairment of olfactory identification,which is effectively captured by the Chinese Smell Identification Test.

Objective To analyze the perioperative clinical characteristics of non-small cell lung cancer ( NSCLC ) complicated with dermatomyositis ( DM) . Methods Six cases of NSCLC complicated with DM received lobectomy and lymph node dissection by video-assisted thoracoscopic surgery. The same kind of operations were carried out in another six NSCLC cases without DM in the same period. The twelve cases in two groups were 1 ∶ 1 matched study. The operation time, operative blood loss, postoperative pneumonia or atelectasis rate, postop-erative wound recovery, thoracic drainage in postoperative three days, incidence of postoperative atrial fibrillation, perioperative hospital stay were compared between the two groups. Results In operative time, blood loss and thoracic drainage volume in postoperative three days, there was no significant difference in two groups (P>0. 05). In paients with and without DM, the occurrence rate of atrial fibrillation was respectively 50. 00% (3/6) and 16. 66% (1/6), the occurrence rate of postoperative pneumonia or atelectasis was respectively 50. 00%(3/6) and 16. 66% (1/6), and postoperative hospital stay was respectively (16. 28 ± 3. 61) d and (11. 11 ± 2. 92) d. It was of significant differences (P<0. 05). Conclusion Compared with NSCLC patients without DM, postoperative atrial fibrillation and pneumonia or atelec-tasis occured more often in the DM cases. And patients with DM have longer hospital stays.

Objective To explore the causes,clinical characteristics and prognosis of children′s completely at-rioventricular block(CAVB).Methods The clinical data of 73 patients with CAVB were analyzed retrospectively from January 2004 to December 201 3 at the Cardiology Department,Nanjing Children′s Hospital Affiliated to Nanjing Medi-cal University.Within those 73 patients,34 patients were male and the others were female,from 3 months old to 1 2.5 years old,the mean age of 6 years.Results There were 21 congenital CAVB patients and 52 acquired CAVB patients with myocarditis undergoing ventricular septal defect (VSD)closure operation.All congenital CAVB patients were re-fractory to drug therapy.Electrocardiogram and echocardiogram were performed in 1 9 cases without clinical symptoms during follow -up,but 2 cases had permanent pacemaker implanted.Among 27 fulminant myocarditis,Adams -Stokes attacks were found in 1 5 cases,3 cases had Adams -Stokes attack in 1 5 cases with sequelae of myocarditis,and 2 out of 6 cases undergoing VSD closure operation had Adams -Stokes attack,and other 4 cases without clinical symptoms were followed up periodically.The acquired CAVB patients were given energy composition and intravenous megavitamin C. The cases with fulminant myocarditis were given adrenal cortical hormone and intravenous gamma globulins simulta-neously.A total of 27 acquired CAVB patients were implanted temporary pacemaker and 5 with permanent pacemaker. Among 52 acquired CAVB patients,31 cases were cured,9 cases were improved,1 1 cases were ineffective,and 1 case died.Conclusions Most congenital CAVB children without clinical symptoms need clinical follow -ups.Myocarditis is a major cause of acquired CAVB.The CAVB prognosis caused by fulminant myocarditis may be related to antimely im-planting the temporary pacemaker timely.Permanent pacemaker should be implanted in patients who have no response to drug therapy with frequent Adams -Stokes or heart failure.

[Objective] To assess the predilection age,clinical features,treatment and pognostic factors of primary cutaneous B-cell lymphoma (PCBCL).[Methods] A total of 31 patients with PCBCL were registered in Tianjin Cancer Central Registry from January 1970 to September 2010,and 26 patients had complete medical recortds.A retrospective analysis was conducted on the 26 patients.The following indices were analyzed,including gender,age,body sites of initial involvement,extent of cutaneous involvement,pathological subtypes,metastatic sites,treatment,survival and prognosis.Data were processed with SPSS16.0 software,survival analysis was carried out by using Kaplan-Meier method,univariate analysis of prognostic factors by Log-rank test,multivariate analysis of prognostic faetors by COX proportional hazards model,and variables were selected by Forward LR method.[Results] Clinical manifestafions were atypical in these patients.The ratio of man and women was 1:1.6.Of the 26 cases,12 were primary cutaneous follicle-center lymphoma (PCFCL),8 were primary cutaneous marginal zone lymaphoma (PCMZL),and 4 were PCLBCL,leg type.Follow-up revealed lymph node involvement in 8 patients (4 in cervical nodes and 4 in inguinal nodes),distant metastasis of organs in 3 patients ( 1 in lung and pleura,1 in bone marrow and 1 in central nervous system).Most patients received surgical treatment combined with chemotherapy or chemoradiation.The 5-year overall survival rate was 80.8％(21/26).Statistical analysis showed that the prognosis of PCBCL was associated with histological subtype,lactate dehydrogenase (LDH) level and globulin level,but unrelated to gender,age,site of initial involvement,extent of cutaneous involvement,involvement of lymph nodes and organs,presence of B symptoms,treatment strategies,number of relapses,level of β2-globulin and hemoglobin,or lymphocyte absolute value.[Conclusion]s PCBCL,as a kind of rare extra-nodal lymphoma,usually occurs with atypical clinical mainfestations in persons aged from 39 to 66 years.The 5-year overall survival rate was 80.8％ in these patients.The prognosis of PCBCL seems to be related to histological subtypes,LDH and globulin levels.