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OBJECTIVES@#To explore the correlation between cytosine-phosphoric-guanylic (CpG) site of Septin 9 gene and colorectal cancer, and to develop a real-time PCR detection system in plasma in patients with colorectal cancer.@*METHODS@#The methylation of training samples was detected by high-throughput sequencing technology, and the sites highly consistent with the clinical information of colorectal cancer were identified. Then the detection system of real-time PCR was designed to analyze the consistency of plasma and tissue based on methylationa sensitive enzyme digestion. Finally, 100 clinical trials were conducted to evaluate the performance of the detection system with the methylation sensitive enzyme digestion-real-time PCR.@*RESULTS@#The highly consistent sites, which were selected by high-throughput sequencing from 71 training set samples, was the 38th CpG. Based on the detection region, the screened methylation sensitive enzymes were @*CONCLUSIONS@#The 38th CpG site of Septin 9 detected by the detection system of methylation sensitive enzyme digestion-real-time PCR can highly predict the occurrence of colorectal cancer with great clinical application value.
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Humans , Colorectal Neoplasms/genetics , CpG Islands/genetics , DNA , DNA Methylation , Plasma/metabolism , Septins/metabolismABSTRACT
Objective To compare the three different methods of enzyme linked immunosorbent assay(ELISA),to select the best method for clinical diagnosis and treatment.Methods Addcare ELISA800,TECAN freedom evolyzer and manual ELISA method were used to detect hepatitis B virus Pre S1 antigen(preS1Ag) hepatitis B virus Pre S2 antigen(preS2Ag) in confrontation control product samples and serum specimens from patients with HBV,and the results were analyzed by statistical methods.Results The batch precisions of the three methods to detect pre-S1Ag were 4.73%,5.38%,11.87%,the batch precisions of the three methods to detect pre-S2Ag were 4.91%,5.04%,11.75%.The inter batch precisions of the three methods to detect pre-S1Ag were 6.63%,7.90%,13.26%,the inter batch precisions of the three methods to detect pre-S2Ag were 6.74%,7.81%,12.59%.All the sensitivities were 100.00%.Conclusion All the three methods have good consistency,which could be used in the detection of Pre-S1Ag and Pre-S2Ag.The precision of Addcare ELISA800 is the best,which could further improve the quality of clinical testing.
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Objective To construct an immunosensor for detecting CD4+ T lymphocytes without labeling .Methods The staphy-lococcus protein A(SPA) method was adopted to conduct the oriented immobilization of CD4 monoclonal antibodies on the gold in-terdigitated microelectrode surface for capturing CD4+ T lymphocytes .Then cyclic voltammetry(CV) method was used to conduct the representation of modification situation on the gold interdigitated microelectrode surface .Finally the electrochemical impedance spectroscopy(EIS) was used to detect the impedance of CD4+ T lymphocytes captured by the immunosensor .The standard curve was drawn by the impedance values change obtained by the equivalent electric circuit fitting .Results The linear range of this im-munosensor for detecting CD4+ T lymphocytes was (5 × 103 -5 .0 × 106 )/mL ,with lower detection limit of 5 .0 × 102/mL .Conclu-sion The constructed immunosensor has accurate and reliable detection results uhidn is simple to operate accurate ,convenient and cheap ,which might be expected to be used in the real-time detection system ,and offers help for realizing rapid ,accurate and inex-pensive CD4+ T lymphocyte count .
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To enhance the scientific research participation of the medical postgraduates, and pro-mote core competitiveness of the medical colleges, based on analysis of the necessity of patent application in medical colleges postgraduate, we constructed the teaching teams including the medical professors, patent engineers, graduate teaching manager, and science and technology managers. The problem-based learning (PBL) and case-based learning (CBL) as teaching methods were used in practice. The patent courses included the reference search and analysis, basic knowledge of patent law, and patent application training module were constructed; and the teaching effect were evaluated and optimized through the scores of the patent basic theory test, research output, and the training of the patent application. The patent course of the medi-cal postgraduates provides a reference for cultivating the compound talents have scientific research innova-tion and patent application capability.
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Objective To investigate the gestation-specific reference intervals (GRIs) and dynamic changes of thyroid function at different gestational ages in Chongqing .Methods Combining self-sequential longtitudinal with cross-sectional study, the serum samples from 640 pregnant women with different gestational age were collected from June 2014 to September 2015 in the Third Military Medical University. The free triiodothyronine (FT3), free thyroxine (FT4), thyroglobulin (TG), thyroid-stimulating hormone (TSH), thyroid peroxidase antibody (TPOAb), and antithyroglobulin antibody (TGAb) were detected by the direct chemiluminescence method.According to China Guideline for the diagnosis and treatment of thyroid disease in pregnancy and postpartum in 2012, the reference interval of the thyroid function was calculated.The data were analyzed by Chi square test .Results Established GRIs of thyroid function during pregnancy in Chongqing:The GRIs was 3.68-5.59 pmol/L for FT3, 9.34-17.02 pmol /L for FT4, 0.18-5.26 mIU/L for TSH in 6-9+6 weeks of pregnancy; the GRIs was 3.69-6.03 pmol /L for FT3, 8.42-15.75 pmol/L for FT4、0.09-4.85 mIU/L for TSH in 10-13+6 weeks of pregnancy; the GRIs was 3.24-5.46 pmol /L for FT3, 6.50-14.24 pmol/L for FT4, 0.11-5.13 mIU/L for TSH in 14-27+6 weeks of pregnancy;the GRIs was 3.06-5.05 pmol /L for FT3, 6.12-11.69 pmol/L for FT4, 0.75-3.67 mIU/L for TSH in 30-34 weeks of pregnancy; the GRIs was 2.96-5.00 pmol/L for FT3, 6.26-11.36 pmol /L for FT4, 0.84-5.54 mIU/L for TSH in 36-40 weeks of pregnancy.Screening by GRIs, the prevalence of thyroid dysfunction was 8.75% (46), however, the prevalence was 37.07% (195) in according with the guidelines,χ2 =120.5,P =0.000.The overdiagnosis rate was 28.32%(149 /526).Using the guidelines of thyroid disease and our GRIs, the thyroid disease was found 116 (22.05%) and 30 (5.70%) in the first screening. Moreover, the thyroid disease was found 79(19.27%) and 10(3.23%) during the repeat screening in the normal population.Conclusions Using the GRIs for thyroid function tests in normal singleton pregnant women could reduce the risk of over diagnosis .The detection rate of repeat screening of TPOAb negative patients was close to the first screening detection rate , and repeated screening could reduce the risk of missed diagnosis for thyroid dysfunction in pregnancy women .(Chin J Lab Med, 2016, 39:511-515 )
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Objective To study the efficacy and safety of transverse compression suture in the lower uterine segment for the control of postpartum haemorrhage in cesarean section caused by placenta previa. Methods From Jan 2011 to Jan 2013, 21 patients with postpartum haemorrhage in cesarean section due to placenta previa were given transverse compression suture in the lower uterine segment after routine medical treatment. And the hemostatic efficacy and safety were observed. Results 20 cases of the vaginal bleeding were controlled efficient-ly, with an efficiency of 95. 2%. There was no complication occurred, and menstruation were back to normal during the follow-up, and there was nothing abnormal in the uterine double accessories through B ultrasound reexamination. Conclusion Transverse compression suture in the lower uterine segment is an easy, safe and highly effective surgical technique, it is especially suitable for the control of haemorrhage in the lower uterine segment caused by placenta previa.
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Objective To investigate the similarities and differences of 3 methods for detecting hepatitis B virus S 1(PreS1) ,and select the appropriate method for detecting clinical samples .Methods The PreS1 of the serum samples from chronic hepatitis B pa-tients were tested with enzyme immunoassay analyzer ,time-resolved method and the manual method .To compare the repetition rate ,select PreS1 antigen strongly positive serum and weak positive serum were detected 15 times by three methods ;To compare the explicit rate ,the reaction temperature was raised or lowered by 3 ℃ .Results The positive rate of three methods was 93 .53% , 92 .81% ,92 .81% .Automated ELISA reproducibility CV strong positive CV3 .62% ,weakly positive CV was 13 .42% ,CV of time-resolved method for the 2 kinds of samples were 5 .10% ,7 .92% ,manual methods CV 11 .10% 29 .88% ;changing the reaction incu-bation temperature 3 ℃ ,automatic detection ELISA all specimens S/CO value decreased ,increasing the chance of a false negative . The manual methods and time-resolved detection method for all specimens S/CO values increased ,increasing the chance of a false positive .Conclusion The detection rate and repeatability of automated ELISA were better .The time-resolved method followed and the manual methods were poor .
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ObjectiveTo investigate the effects and problems of problem-based learning ( PBL ) teaching methods that are adopted in the laboratory medicine practice teaching. MethodsOne hundred and four undergraduate students of 5-year system of laboratory medicine were selected to use the PBL teaching methods during the laboratory medicine practice. ResultsIt showed that the PBL teaching methods obtained good probation effect. But teachers and students are required to make greater efforts in PBL teaching mode than in traditional mode, and the teaching methods was also needed to be consummated.ConclusionsUsing the problem-based teaching methods the comprehensive ability of the student is enhanced. Therefore it deserves to be generalized during laboratory medicine practice teaching.
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Objective To analyze the drug-resistance and distribution of nonferments bacteria infection in our hospital and provide the diagnosis and treatment evidence to doctor.Methods 241 nonferments bacteria strains had been separated from patients of Xinqiao Hospital from July 2006 to Jan 2008. Bacteria identify and drug-resistance test were performed by VITEK-Ⅱanalysis system. The drug-resistance result was determined by MIC with the standard of NCCLS.Results The separate rate in patients of neurosurgery, respiration, orthopaedics were 24.94、23.27 and 10.79 respectively. The main source of specimen are sputum. The drug resistance rate were Imipenem (16.9), Amikacin(29.3), Cefepime (33.9) and Cefazidime(39.1) in 241 nonferments bacteria strains. Conclusion The infection of nonferments bacteria must be recognized by doctors, and it would prompt us to use antibiotic properly.
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Objective To establish a new double-antibody sandwich ELISA to detect the antigen AP33(a 33 kDa adhesive protein) of Trichomonas vaginalis based on the quantum dots and magnetic beads.Methods After BALB/c mice were immunized by AP33,the multiclonal antibodies in the antiserum was conjugated with the quantum dots and magnetic beads by carbon diimine crosslinking method respectively.Then the antibodies combined with magnetic beads were coated to microwell of plate as capturing antibody,and the antibodies bound to quantum dots were regarded as the marked antibody which can be directly observed under fluorescence microscope and quatitated by spectrofluorometry.The specificity and sensitivity of our established system were investigated.Results AP33 was successfully detected at the concentration as low as 50 ng/ml by this with-filling method.No crossreaction was observed when this system was used to detect Trichomonas vaginalis and other common bacteria in the vagina.The accuracy was 88% and the specificity was 90%.Conclusion This new double-antibody sandwich ELISA to detect Trichomonas vaginalis is successfully prepared and of sound specificity and sensitivity.
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Objective To establish an immunoassay based on microfluidic chip for detection of TSH and total T4.Methods The direct-write laser micromachining technology was used to fabricate microfluidic chip,luminol-H2O2 as luminator.This method and Roche E2010 electrochemiluminescence immunoassay were used to examine TSH by double-antibody sandwich method and total T4 by competitive principle in 50 serum samples collected at our outpatient clinic.Results The detection was completed within 25 min.Ten microliter sample and reagent was required.The detection range of TSH was 0-50 ?IU/ml and its average intraassay and interassay precision was 4.7% and 4.6% respectively and its average recovery rate was 95.3%.The detection range of T4 was 9.375-300 ng/ml,and its average intraassay and interassay precision was 4.9% and 4.7% respectively and its average recovery rate was 91.27%.Conclusion Rapid detection of TSH and T4 by microfluidic chip-based immunoassays is convenient,stable and accurate and of less reagent and time consuming.
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Objective To study the diagnostic value of multiplex protein microarray and ELISA for human serum pathogens.Methods Totally 230 national standard samples were tested by multiplex protein microarray and ELISA respectively for the pathogens of HBV,HIV,HCV and treponema pallidum(TP).Results The matched pair test for numeration data showed ?2=0.5,P=0.125,indicating that no statistical significance between the two methods.Conclusion Multiplex protein microarray shows high specificity and sensitivity to the four pathogens so that it is worth extensive application.
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Objective To explore how the antibody of glucocorticoid-induced tumor necrosis factor receptor (GITR) exerts inhibitory effect on the L615 leukemia cells by strengthening the activation of the NK cells. Methods The 24 established L615 leukemia mice were equally and randomly divided into 4 experimental groups according to different drugs given intraperitoneally, groupⅠ (normal saline), Ⅱ (GITR), Ⅲ (cyclophosphamide), and Ⅳ (GITR +cyclophosphamide).Then the NK cells were extracted from the spleen of mice as effective cells, and L615 leukemia cells served as the target cells. The changes of the NK cells’killing activation was observed in vivo. The mRNA levels of 3 proteins tightly related to the NK cells’activation Perforin, IFN-? and Fas mRNAs were detected with RT-PCR. Results The GITR-antibody enhanced the killing activity of the NK cells obviously, with the expressions of the 3 proteins increasing obviously. Conclusion By regulation of the Treg cells, the GITR-antibody can inhibit the L615 leukemia cells through enhancing the NK cells' killing activity.
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Objective To investigate the effect and the mechanism of the GITR-antibody(glucocorticoid-induced tumor necrosis factor receptor-ligand antibody) on the mouse leukemia model induced by L615.Methods The mouse leukemia models induced by L615 cells were divided into 4 groups: negative controls(peritoneal injection of normal saline,0.2 ml/d),GITR group(GITR,100,infused through caudal vein 2 d before leukemic lymphocytes inoculation,again at dose of 50 ?g/each mouse after inoculation),Cyclophosphamide group(200 mg?kg~(-1)?d~(-1),intraperitoneal injection from the 3~(rd) day after inoculation for 3 d),GITR+ Cyclophosphamide group(100 mg?kg~(-1)?d~(-1) Cyclophosphamide instead).The survival time,leukocyte counting in the peripheal blood,liver and spleen index were calculated and the pathological examination of liver,spleen were performed.Results GITR-ligand could prolong the survival time of mouse leukemia model,lead the necrosis and apoptosis of leukemic cells in bone marrow,decrease the liver and spleen index,decrease and relieve the leukocyte increase of peripheal blood and the irregular swelling of liver and spleen.Conclusion Through immunoregulation,GITR-antibody can inhibit the L615 leukemic cells effectively,therefore inhibit the progress of leukemia to some extent.
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Objective To establish two PCR assays to rapidly and simultaneously detect U.parvum and U.urealyticum.Methods Two PCR assays were established through designing two sets of specific primer targeting urease gene B of U.parvum and U.urealyticum,respectively.The standard strains of U.parvum and U.urealyticum and the clinical samples were detected by these PCR assays and PCR products of the standard strains and two clinical specimens were directly sequenced and carried out specific and sensitive assays.Forty-eight clinical specimens were tested for culture and the PCR assays and tow methods were compared.Results The standard strains of U.parvum,U.urealyticum and the clinical specimens were successfully and differentially detected by the PCR assays and the sequencing results were found to have a complete similarity to the GenBank sequences.The 14 strains of other bacterial genera including 5 strains that produced urease were not amplified by these PCR assays.Each assay had a detection limit of 10 copies/?l of plasmid DNA.The positive rate in 48 clinical specimens by PCR(54.2%)was higher than that by culture(39.6%)(P
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Objective: To investigate the effects of normal and pathological bone marrow stromal cells(BMSC) on the proliferation and apoptosis of K562 cells in separated culture and contact culture.Methods: On 2,4,7 and 12 d after culture,the growth of K562 cells was observed under optical microscope;the apoptosis of K562 cells was detected by TUNEL. Results: the growth of K562 cells on CML BMSC in contact culture was faster than the else.The growth of K562 cells in contact culture was significantly faster than that in separated culture after 7 d(P
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0.05). Conclusion The sensors can eliminate the interference of Ac effectively and lay a basis for the development of practical blood sugar detector.
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Objective To investigate the significance of TGF-β1, TGFRl and TGFR2 in the pathogenesis and prognosis in patients with myelofibrosis. Methods The expression of TGF-β1 and its receptors (TGFR1 and TGFR2 ) in bone marrow tissues and the level of TGF-β1 in the blood of 23 patients with myelofibrosis were detected by SABC immunocytochemistry and ELISA repectively. Results Expression of TGF-β1 and TGFR 1 was significantly higher in primary and secondary myelofibrosis patients than that of the control. No significant difference of TGFR2 expression was found between the groups of myelofibrosis and the control (P>0.05). The level of TGF-β1 in the blood of the patients with myelofibrosis was significantly higher than that of the control (P<0.01) and more obvious in secondary cases while TGF-β1 decreased nearly to the normal level when patients were in clinical remission. Conclusion TGF-β1 and it's receptors may be involved in the pathogenesis of myelofibrosis and might be of importance for the prognosis of the patients with myelofibrosis.
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Objective To construct and identify the recombinant adenovirus vector containing the Toll-like receptor 2(TLR2) extracellular domain gene of human.Methods Human TLR2 extracellular domain cDNA was amplified by RT-PCR from peripheral blood mononuclear cells(PBMCs) and inserted into pMD18-T vector.After being confirmed by enzyme digestion and sequencing,the DNA fragment,digested with Kpn Ⅰ and Hind Ⅲ,was directionally cloned into adenovirus shuttle plasmid pAdTrack-CMV.After linearization by Pme Ⅰ digestion,the recombinant plasmid pAdTrack-CMV-TLR2 was transformed into competent AdEasier-1 germs and then homologically recombined with an adenoviral backbone plasmid pAdEasy-1 in bacteria BJ5183 to obtain the recombinant adenovirus plasmid.After confirmation,the recombinant adenovirus plasmid pAd-TLR2 was linearized with Pac Ⅰ digestion and transfected into 293 cells via liposome,and then package and adenovirus amplification were performed.The expression of green fluorescent protein(GFP) was observed,the virus titer was determined and the recombinant adenovirus was identified by PCR.ResultsThe gene fragment obtained by RT-PCR was of the same sequence as in GenBank.It was certified by restricted endonuclease digestion and PCR analysis that the recombinant adenovirus containing the TLR2 extracellular domain gene of human had been successfully constructed with a satisfactory high titer of 3?109pfu/ml.Conclusion The recombinant adenovirus containing TLR2 extracellular domain gene of human has been successfully constructed,which lays a foundation for further study on the structure and biological activity of TLR2.