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.

Insolinoma is a rare disease,but with an increased incidence in recent years.From March 2003 to October 2010,16 patients with insulinoma had been admitted to the Nanfang Hospital,and the results of imaging investigation were compared with histopathological examination after operation.All cases had typical Whipple's triad,low plasma glucose and high plasma insulin concentrations. Sixteen tumors were found in total.The detection rate of ultrasonography was 44％ (7/16),and the detection rate of enhanced computed tomography (CT)and magnetic resonance imaging (MRI) were 67％ (8/16) and 75％ (6/8) respectively.The detection rate reached 88％ by combination of CT and MRI.Two patients had no abnormal findings in ultrasonography,CT and MRI,while positive results were found in the arterial stimulation procedure with venous sampiing (ASVS) in both patients.CT or MRI is the preferred approach for localization diagnosis of insulinoma. The ASVS achieves a distinct advantage in localization of insulinoma.

Objective To discuss the clinical features and prognostic factors of primary testicular lymphoma (PTL). Methods A retrospective review was performed based on the clinical records of 33 PTL cases treated at Tianjin Medical University Cancer Hospital from June 1977 to May 2009.Drawing survival curves by Kaplan-Meier method, using Log-rank test to the univariate analysis, and multivariate analysis by COX regression model to evaluate independent prognostic factors. Results The median age of patients was 62 years at presentation(range 33-81 years). Painless testicular swelling was the initial symptom. The majority of histological subtype was B cell lymphoma, 48% of which was diffuse large B-cell non-Hodgkin's lymphoma. By postoperative chemotherapy and/or radiotherapy, 23 patients achieved complete remission and 7 achieved partial remission, with a median follow-up of 23 months (4-231 months). The 5 years and 10 years of overall survival was 39.1% and 19.5%. The effect of patients who received chemotherapy≥4 cycles and B cell lymphoma patients combination with Rituximab were better. In multivariate analysis, Ann Arbor stage, B symptoms, age of patient and number of extranode site were independent prognostic factors of survival. Conclusions Primary non-Hodgkin lymphoma should be treated with multi-modality strategies. Treatment with doxorubicin-based chemotherapy after orchiectomy should be recommended. Ann Arbor stage, B symptoms, age of patient and number of extranode site were independent prognostic factors of survival in PTL.

Objective To investigate the predictive factors of progressive motor deficits (PMD) after isolated pontine infarction. Methods Consecutive patients with isolated pontine infarction admitted to hospital within 48 hours after onset were enroled. They were divided into either a PMD group (increase ≥1 within 7 days) or a non-PMD group according to the clinical course and the changes of motor scores of the National Institutes of Health Stroke Scale (NIHSS). The pontine infarction patterns were classified as basal surface infarction and deep infarction, the sides were divided into left and right, the infarct levels were divided into upper, middle, and lower according to diffusion-weighted imaging. The demographics, baseline clinical data, and imaging features were compared between the two groups. Multivariable logistic regression models were used to analyze the predictive factors of PMD after isolated pontine infarction. Results A total of 101 patients with isolated pontine infarction admitted to hospital within 48 h of onset were enroled, including 16 in the PMD group and 85 in the non-PMD group. The proportions of pontine infarction involving the basal surface (87. 5% vs. 47. 1% , χ2 = 8. 851, P = 0. 003), the infarcts on the middle levels (56. 2% vs. 24. 7% , χ2 = 4. 851, P = 0. 028), and basilar artery stenosis or occlusion (62. 5% vs. 27. 1% ,χ2 = 7. 689, P = 0. 006) of the PMD group were significantly higher than those of the non-PMD group, while the proportions of the infarcts on the left sides (18. 8% vs. 56. 5% , χ2 = 7. 664, P = 0. 006) and the infarcts on the upper levels (37. 5% vs. 72. 9% , χ2 = 7. 689, P = 0. 006) of the PMD group was significantly lower than those of the non-PMD group. Multivariate logistic regression analysis identified that pontine infarction involving the basal surface (odds ratio 5. 650, 95% confidence interval 1. 011 - 31. 580, P = 0. 049) and basilar artery stenosis or occlusion (odds ratio 4. 075, 95% confidence interval 1. 127 - 14. 741, P = 0. 032) were the independent risk factors for PMD after isolated pontine infarction. Conclusions Infarction involving the basal surface and basilar artery stenosis or occlusion may be the predictors for PMD after isolated pontine infarction.

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 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.