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.

Female ; Humans ; Infant, Newborn ; Maple Syrup Urine Disease ; diagnosis ; therapy

The study of viruses traditionally focused on their roles as infectious agents and as tools for understanding cell biology. Recently, however, with the development of structural biology, viruses have now been receiving particular attention in nanotechnology. By chemical methods or by gene modification, viruses have been functionalized as potential building blocks for several applications, such as drug/gene delivery vehicles, advanced vaccine vehicles, and special inorganic or organic nanomaterials. Here we highlight some of the recent progresses in the medical applications of viruses.
Biomedical Research ; trends ; Gene Transfer Techniques ; Genetic Therapy ; Nanostructures ; Nanotechnology ; trends ; Vaccines ; Viruses

BACKGROUND:Whether lumbar spondylolisthesis needs to be treated with reduction remains controversial.OBJECTIVE:To investigate the clinical effectiveness of pedicle screw fixation combined with posterior lumbar interbody fusion for degenerative lumbar spondylolisthesis in the patients aged more than 70 years old.METHODS:The clinical data of 30 patients with degenerative spondylolisthesis aged over 70 years old undergoing pedicle screw fixation combined with posterior lumbar interbody fusion were analyzed retrospectively.The Visual Analogue Scale,Oswestry Dysfunction Index and the Japan Orthopedics Association scores were compared and analyzed before and after surgery.RESULTS AND CONCLUSION:(1) Among 30 patients,12 were male and 18 female,the average age was (75.1±6.7) years old,and all were followed up for more than 12 months.(2) The postoperative Visual Analogue Scale,Oswestry Dysfunction Index and the Japan Orthopedics Association scores were significantly improved compared with baseline (P ＜ 0.05).According to the Japan Orthopedics Association scores,3 cases were cured,24 were significantly effective,and 3 effective.(3) Graft fusion was achieved at all patients,the average fusion time was (5.13±0.65) months.(4) The average operation time was (225.6±23.4) minutes,the average intraoperative blood loss was (470±45.5) mL,and the average follow-up time was (25.8±3.5) months.(5) Six patients presented complications (20％),including one case of dural tear,two cases of paralytic intestinal obstruction,and three transient lower extremity nerve symptoms.(6) These results indicate that if the associated disorders are strictly controlled,pedicle screw fixation combined with posterior lumbar interbody fusion can achieve good surgical results in the treatment of degenerative spondylolisthesis in the elderly.

Objective To evaluate the clinical outcomes and report the second-look arthroscopic findings of reconstructed anterior cruciate ligament (ACL) using deep-frozen allograft.Methods Sixty-six patients undergoing ACL reconstruction using deep-frozen allograft in our institute with at least twelve months follow-up from January 2012 to March 2016 were included and received second-look arthroscopy.The patients consisted of 51 males and 15 females with an average age of 30.6 years (range,18 to 55 years) at the time of ACL reconstruction.Knee functions were evaluated by Lysholm score and International Knee Documentation Committee (IKDC) score.Knee stability was evaluated by drawer test,Lachman test and KT-1000 arthrometer.Second-look arthroscopic evaluation was performed in all patients,focused on continuity of the reconstructed ACL,the synovial coverage and subjective tension of the graft,and the prevalence of cyclops-like lesion and other changes after reconstruction procedures.Resuits All patients were followed up for average 18.3 months (range,12 to 36 months).Thirty-one patients underwent second-look arthroscopy from 12 to 18 months,and the other 35 patients underwent second-look arthroscopy from 18 to 36 months after ACL reconstruction.No infection,rejection reaction and other serious complication were reported after operation.The knee range of motion was normal in all cases,except that 10° extension limitation in one case.The Lysholm score significantly improved from preoperative 54.95±9.01 to 12 months postoperatively 86.14±5.86,and the IKDC improved from 54.79±9.12 to 85.11±5.77.Lachman test was positive in 8 cases,but negative in 58 cases postoperatively.KT-1000 arthrometer measurement showed that the side-toside difference significantly improved from preoperative 6.70± 1.24 mm to postoperative 1.52± 1.02 mm.Complete discontinuity occurred in 2 cases of the reconstructed grafts,graft tear in 4 cases.Cyclops-like mass was identified in 2 cases.The overall synovial coverage was slightly better in the patients who were followed up more than 18 months than those less than 18 months.However,there was no significant difference among the groups in the field of graft tension.The patients with taut grafts showed statistically better KT-1000 values (1.14±0.35 mm) than those with lax grafts (2.95±1.38 mm).Conclusion Frozen allograft could be a reasonable choice for ACL reconstruction.However,the graft integration and remodeling could tend to be slow.

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.