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Objective To study if IRF1 could regulate the polarization by IRF 1 and M1 status and affect M1 media-ted antitumor function .Methods U937 derived M1 macrophage ( U937-M1 ) model was established .The cells were devided into 4 groups:the PMA pretreated unpolarized macrophage (M0), the PMA, IFN-γand LPS induced M1 macrophage (M1), the siRNA of IRF1 knocked down M1 macrophage (siIRF1) and the negative control siR-NA treated M1 macrophage (siC).Furthermore, the expression of CD86 and CD206 was detected by flow cytome-try, the M1/M2 associated markers (IL-12p35,IL-12p40,IL-23p19,IL-6,TNF-α/IL-10) and IFNB1 were ana-lyzed by qPCR,the expression of IL-12p70 and IL-10 was examined by ELISA, the expression of IRF1 and IRF5 was detected by Western blot , the proliferration and apoptosis of HCC were analyzed by CCK 8 and flow cytometry , respectively.Results Compared with the U937-M1, the IRF1 knocked down group showed impaired CD 86 expres-sion, but enhanced CD206 expreesion ( P<0.05 ); the expression of M1 related cytokines including IL-12p35, IL-12p40,IL-23p19,IL-6,TNF-αand IFNB1 was decreased, but M2 related cytokine IL-10 level was increased (P<0.01);the expression of IFN-β, IL-12p70 and IRF5 was impaired, but IL-10 was enhanced (P<0.05).In IRF1 knocked down U937-M1, the CCK8 analysis indicated that the M1 mediated anti-proliferation effects on hepatoma carcinoma cell were turned to pro-proliferation ( P<0.05);the flow cytometry showed that the M 1 mediated pro-ap-optosis effects were reversed to anti-apoptosis ( P<0.01 ) .Interestingly , IRF5 and IFN-βwere decreased at both mRNA and protein levels in IRF1 knocked down U937-M1 compared with the U937-M1 (P<0.01).Conclusions IRF1 may partly modulate IRF5 and IFN-β, and further regulate M1 polarization and its antitumor effects .
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For the reform and development of clinical laboratory education, based on the previous course reform, Laboratory Medicine College of Chongqing Medical University has put the leading teachers'responsibility system of laboratory medicine course into practice. In the recent 3 years, the course is better organized.The students are more interested in the course and they communicate more with teachers than ever before. The effect of the course is obvious. The leading teachers' responsibility system in laboratory medicine course should be promoted.
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It is for estimating the laboratory statistics from the view of clinical medicine objectively,completely and appropriately that we establish ＜ Laboratory Sciences and Clinical Medicine ＞ which fits the requirement for the modern medical laboratory specialists. In this selective course, we use problem-based learning ( PBL ) teaching method which is different from the traditional one, stimulating students to participate in the study actively and passionately,training them to think in a scientific way and to analyse and solve the problem in a rational way. As a result, it works well which makes us believe it is worth popularizing.
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It is confirmed that the adult cells can be re-programmed to embryonic stem cells(ESCs) by presenting some certain factors in oocytes in the clone process of animals. In recent years, some transcription factors that can induce pluripotent stem cells(iPS) have been identified and which made it possible to obtain induced pluripotent stem cells similar to embryonic stem cells. iPS provides a unique platform to study the pluripotent mechanism and to establish some specific disease models. This major scientific discovery can not only avoid the use of ES which involves ethics debate, but also lead the stem cell research to a new field.
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This paper is aimed to establish and optimize proteomic research platform using isobaric tags for relative and absolute quantitation (iTRAQ) so as to facilitate further proteomic research of human peripheral blood mononuclear cells (hPBMC). We collected hPBMC, after protein extraction and trypsin digestion, we labeled the samples with iTRAQ reagents and then subjected to mass spectrometry. In triplicates, thirty precursors were randomly selected and detected; as a result, 26, 28 and 29 peptides were respectively tagged with ITRAQ reporter ions. The labeling efficiencies ranged between 86.7%-96.7%, with no significant difference among the groups (P>0.05). The coefficient of variance for the relative ratios of peptides from different proteins was ranged from 7.6% to 25.5% and there were no significant differences across the groups (P>0.05). The coefficient of variance for the relative ratios of different peptides from the same protein was varied from 9.3% to 19.1% and the differences across groups were not significant (P>0.05). The labeling of iTRAQ combined with tandem mass spectrometry in PBMC was successful with favourable reproducibility and accuracy, which could lay a foundation for further proteomic study of hPBMC in autoimmune disorders.
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Humans , Leukocytes, Mononuclear , Chemistry , Proteins , Chemistry , Proteome , Proteomics , Methods , Staining and Labeling , Tandem Mass Spectrometry , MethodsABSTRACT
Objective To establish a novel real-time PCR method to detect HCV RNA using Selfreporting duplex mutation primers.Methods The recombinant vector pMD18-T-HCV 5′-NCR was used as the calibrator.The Self-reporting duplex mutation primers were designed according to the gene sequence.And then the PCR reaction system was optimized and evaluated.The specificity,sensitivity and reproducibility of real-time PCR were estimated,The serum specimens from 90 cases(30 cases of HCV,30 cases of other viral hepatitis and 30 healthy volunteers) were tested with this real-time PCR; Results were compared with those obtained using a commercial TaqMan kit.Results The assay was established.It showed linearity over a wide range from 20 - 109 IU/ml.Intra-experimental coefficients of variation(CVs) were 1.37% -4.59%,and inter-experimental CVs were 1.58% -4.81%,respectively.There was no significant difference of HCV genome number tested by the two methods(R2 = 0.95) in 30 hepatitis C patients; HCV DNA was not detected in any serum samples of 30 healthy volunteers by the two methods.The specificity was 100%(60/60).All the samples in patients with clinically confirmed HCV infections showed HCV RNA positive.There wass good correlation between the quantitaive results and results obtained using the commercial TaqMan kit.Conclusions It is demonstrated that real-time PCR is a reliable,accurate and feasible assay for HCV.The establishment of this assay provided alternative technology for clinical diagnosis or therapeutic drug monitoring in the field of HCV infection and epidemiologic survey.
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Objective To understand genomic copy number variations (CNVs) and ascertain karyotype for a 46,X0, + der(?) fetus, and investigate possibility and superiority of array-based comparative genomic hybridization (array-CGH ) in clinical cytogenetic diagnosis. Methods G-banded chromosome analysis was carried out. The whole genome of the fetus was scanned and analysed by array-CGH. The results of array-CGH were confirmed by RT-qPCR. Results G-banded chromosome analysis showed that the fetal karyotype was 46,X0, +der(?). Array-CGH revealed the derivative chromosome as Y chromosome without CNVs. A total number of 118 submicroscopic CNVs were identified. Comparable results between array-CGH and RT-qPCR were obtained for 9 novel CNVs. Conclusion Comparing with conventional cytogenetic analysis, array-CGH is of high resolution, high-throughput and high accuracy, which provides a technical platform for accurate detection of submicroscopic chromosomal aberrations.
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Objective To establish a continuous monitoring assay of serum argininosuccinate lyase (ASL) activity with automatic biochemistry analyzer, and perform methodology validation and preliminary clinical application.Methods According to the chemical reaction catalyzed by ASL and the working characteristics of automatic biochemistry analyzer, an enzyme coupled reaction system with high specificity was set up, and the methodology validation was performed.Three hundred and nine patients with various liver diseases, 269 non-liver disease patients and 40 healthy controls were enrolled in this study.Serum ASL, ALT, and AST level were determined in all subjects.Results A new kinetics assay of ASL activity was set up with automatic biochemistry analyzer.The methodological validation demonstratod that inter-assay and intra-assay coefficient of variation were 4.0% and 5.9% respectively and the mean recovery was 100.5%.The linear range was 0-167.7 U/L.The lowest detection limit was approximately 0 U/L.The interference test showed that there is no significant interferences while the concentration of bilirubin is less than 342 μmoL/L or commonly used anticoagulants is employed at their routine concentrations.However,interference was significant when Hb level is more than 0.06 g/L.Preliminary study of clinical application showed that there was no significant difference of serum ASL level between non-liver disease group and healthy group ( q = 0.027, P = 0.979 ), but there was significant differences for both serum ALT and AST levels (ALT:q =6.461,P =0.000;AST:q =6.481,P =0.000).Conclusions A continuous monitoring assay for the determination of serum ASL activity is successfully established. Serum ASL may be a good biomarker for liver injury.
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Objective To detect quantitatively hepatocyte growth factor(HGF)mRNA expressions of bone marrow mononuclear cells(MNCs)in acute leukemia(AL)and investigate its clinical significance.Methods Total mRNA of quantitated bone marrow MNCs isolated from 67 de novo AL cases was extrated and then cDNA was synthesized.Expression of HGF mRNA was quantified absolutely using real-time fluorescence quantification PCR(FQ-PCR).Results Expressions of HGF mRNA in a group of AL were higher significantiv than these in a control group(6.936 ±1.613,0.407 ±0.170,P<0.001),but there was similafitv between a group of acute myeloid leukemia(AMI,)and group of acute lymphoblastic leukemia (ALL)(7.127±1.911,6.635±0.934,P>0.05).In AL subtypes,the expression of M5(9.998 4±1.454)was higher than that of M2,M3,M4,L1,L2 and L3(P<0.001),but there ware no differences among the latters(P>0.05). Meanwhile,there was no statistical significance on the expressions of HGF mRNA between different age and sex(P>0.05).In addition,expressions of HGF mRNA in the remission group were lower than these in the non.remission group(6.393±1.165,8.041±1.848,P<0.005).Conclusions There are statistical significances of the expressions of bone marrow MNCs HGF mRNA among the AL group and control group.As to AL subtypes,there are no statistically significant differences between AML and ALL as well as between different age and sex.Besides,lower HGF mRNA level is correlated with better curative effect.It is suggested that HGF mRNA is a suitable index for AL diagnosis and treatment.
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Objective To screen the serum biomarkers by surface-enhanced laser desorption-ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and establish the decision-tree model for the silica-exposed populations.Methods In addition to 20 healthy people without silica-exposed history chosen as control group,all the subjects diagnosed as different stages (stage 0 group,silica-exposed population,n=20;stage 0+ group, doubtful silicosis,n=20;stage Ⅰ group,stage Ⅰ silicosis) of silicosis according to the national diagnostic criteria (GBZ70-2002) of pneumonoconiosis with no complications were recruited. SELDI-TOF-MS and CM10 chip assay were applied to acquiring serum protein finger printing. The bioinformatic software was adopted to perform data ananlysis and establish Wilks'lambda decision equation and decision tree.Results As compared with that of control group, differential peaks appeared in three groups of silica-exposed population, accounting for 80% of total differential peaks. Wilks'lambda decision equation was established with M/Z 3711.73, M/Z 4183.66, M/Z 5487.28 and M/Z 6124.64, which sensitivity and specificity in differentiating healthy controls and silica-exposed population was 95% and 90% respectively. The sensitivity and specificity of decision tree was 100% and 95% respectively. Neither single peak nor Wilks'lambda decision equation worked well for discrimination of silica-exposed population, while the discriminating rates of decision tree accounted for 100%, 95%, 85% and 90% for healthy control group, stage 0 group, stage 0+ group, stage Ⅰ group respectively.Conclusion M/Z 5933.63,M/Z 4183.66,M/Z 5487.28,M/Z 6124.64,M/Z 5915.14,M/Z 2745.7, M/Z 3711.73, M/Z 2954.78 and M/Z 3401.03 are potential biomarkers for discriminating healthy controls and silica-exposed population, and the decision-tree works well for discrimination of controls, silica-exposed population, doubtful silicosis patients and stage Ⅰ silicosis ones.
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Objective To construct quantitative standard for quantification of hepatocyte growth factor (HGF) mRNA and establish its real-time fluorescence quantitative(FQ)-PCR assay to estimate its clinical relevance in lymphoma.Methods Recombinant plasmid Was constructed with target cDNA obtained from isolated total RNA by RT-PCR After PCR products were identified and purified,recombined plasmids were quantitated and then acted as quantitative standard.A new real time FQ-PCR analysis system Was established with the second pair of primers and the probe after amplification condition and the concentrations of components were optimized.HGF mRNA expressions in 47 lymohoma cases[11 Hodgkin disease(HD) cases,36 non-Hodgkin lyphoma(NHL)cases.among these patients,36 patients in remission while 11 patients without remission ] were analyzed quantitatively,and its specificity and sensitivity for lymphoma diagnosis were evaluated by receiptor operation character(ROC)curve method.Results HGF mRNA quantitative standard was constructed successfully.and its real time FO.PCR analysis system Was established combined with hot.start PCR and down.touch PCR technique. According to slope of standard curve (-3.513)and correlation cofficient(0.999),amplification efficiency of the system was 92.6%.Coefficient variation of intra-assay,intra-day and inter-day-assay were 2.1%,4.0% and 6.8%,respectively.Sensitivity of FQ-PCR Was 2 eopies/μl.Expressions of HGF mRNA in lymphoma group Was higher than that in control group(6.425±2.172 and 0.317±0.192,respectively,t=15.883,P<0.001),and its expressions in remission group was lower than no remission group(6.157±1.712 and 7.59l ±1.184,respectively,t=2.768,P<0.05).However,there Was not difference of HGF mRNA level between group HD and group NHL(P>0.05).According to ROC analysis,its sensitivity and specificity were 93.6% and 100% when cutoff value for lymphoma clinical diagnosis Was 3.136.Conclusion HGF mRNA'8 quantitative standard and its real time F9-PCR analysis system have been successfully constructed,and it can be used for quantitative detection of its mRNA expression in lymphoma.
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Objective Signal transducers and activators of transcription 3 (STAT3) silenced by RNA interference (RNAi) technique were used to induce the apoptosis and growth inhibition in T24 and 5637 bladder cancer cells. Methods Three recombinant plasmids pGenesil-1-shRNA-STAT3 was constructed and transfected into T24 and 5637 cells. The expression of STAT3 gene was detected by RT-PCR and Western blotting. FCM was used to observe the apoptosis in T24 and 5637 cells. Results pGenesil-1-shRNA-STAT3 was successfully constructed, and transfected into T24 and 5637 cells. RT-PCR and Western blot analysis demonstrated that pGenesil-1-shRNA-STAT3 could significantly inhibit the expression of STAT3 in T24 and 5637 cells; FCM results show that it could suppress the growth of 1'24 and 5637 cells. Conclusion pGeneSiI-1-shRNA-STAT3 could significantly inhibit STAT3 expression, suppress the growth of T24 and 5637 cells.
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To identify in vivo-induced gene in Streptococcus pneumoniae (S. pn) through a novel in vivo expression technology (IVET), a large promoter-trap library using galU and lacZ as the reporters was constructed. Based on the suicide vector pEVP3, a new vector pEVP3-galU was constructed with promoterless galU gene as an in vivo reporter. Firstly, promoterless galU gene was directly cloned into pEVP3 fusing with promoterless lacZ gene (an in vitro reporter). Then the random pieces of S. pn chromosomal DNA (200-500 bp), obtained by partial Sau3AI restriction digestion, were subcloned into the Bgl II site of pEVP3-galU. Upon introduction of the ligated plasmid library into E. coli DH5alpha by transformation, about 70,000 recombinants were recovered. Considering insert DNA orientation and insert size, this represents 5 coverage of the 2.2 Mb S. pn genome; 90% of these clones had 250- to 500-bp inserts. Thus, the library retained maximal complexity. Transformation by this plasmid library yield 450,000 S. pn transformants. The library was used to infect animals in intraperitoneal model. Those strains survived in vivo while exhibiting a white colony phenotype on TSA agar containing X-gal would indicate that the DNA fragment upstream of the galU reporter contained an in vivo-induced promoter. The promoter-trap library is suitable for screening in vivo-induced gene of S. pn.
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Animals , Female , Mice , Cloning, Molecular , Genes, Bacterial , Genetics , Genes, Reporter , Genetics , Genomic Library , Mice, Inbred BALB C , Promoter Regions, Genetic , Genetics , Streptococcus pneumoniae , GeneticsABSTRACT
Objective To study the functions of GPⅠa/Ⅱa and GPⅣduring the course of platelet adhesion to collagen. Methods Reaction system which contains anti-GPⅠa/Ⅱa antibody ,anti-GPⅣantibody or no blocking antibody was analyzed by flow cytomety. PE labeled anti-CD42b antibody was used to select platelet and fluorescence intension of fluorescein isothiocyanate ( FITC) was detected during the course of platelet adhesion to FITC-labeled collagen. Results The antibody against GPⅠa/Ⅱa inhibited platelet adhesion to collagen, especially to activated platelet(P 0. 05 ). Conclusion GPⅠa/Ⅱa plays an important roles in platelet adhesion to collagen. Blocking GPⅠa/Ⅱa may decrease the adhesion. GPⅣmay be helpful for acceleration of platelet adhesion to collagen at early stage.
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Objective To investigate the loss of heterozygosity (LOH) of 3p D3S1300 and D3S1289 loci of plasma DNA of lung cancer (LC) patients and evaluate its value for a tumor marker of lung cancer.Methods Two microsatellite markers,D3S1300 and D3S1289,were analyzed by PCR and silver staining to investigate LOH of the plasma DNA and cancer tissue DNA from 69 cases of primary lung cancer,and the plasma DNA from 40 control subjects.Results The percentages of D3S1300 LOH detected in the tumor tissue and plasma DNA of LC patients were 40.6% and 29.0% respectively,while the percentages of D3S1289 LOH were 31.9% and 24.6% respectively.In contrast,only 2 cases of D3S1300 LOH and 3 cases of D3S1289 LOH were found in plasma DNA of control group (P
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This paper summarizes that to strengthen the construction of national key disciplines Clinical Laboratory Diagnostic and build professional post-graduate education platform for innovation,the exploration and practice of reform have been done in Chongqing Medical University,in the teaching ranks and exercise disciplinary research direction to enhance the level of scientific research and laboratory construction.
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As required to the standards of the model curricula and with the years of continuous teaching research and reforming,the clinical biochemistry curriculum has kept developing from the teaching staff,content of course,teaching method and means and the teaching condition etc.,thus forming model curricula with special features and notable results.
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Objective To investigate the clinical application of Cystatin C as a biological marker for monitoring hepatic pathological change in patients with virus hepatitis.Methods Two hundred and seven patients infected with hepatitis B or C virus(HBV, HCV)were divided into cirrhosis group(group A),chronic HBV group(group B),chronic HCV group(group C),and liver cancer group(group D). 32 healthy controls(group H) were recruited . The serum TIMP-1,TIMP-2,and Cystatin C as well as some traditional markers for monitoring liver function and renal function including creatinine, creatinine clearance rate, alanine transaminase, and aspartate transaminase were determined.Results In these groups, serum Cystatin C(F=28.334, P
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According to the characteristics of the Laboratory Medicine and our twenty years' medical educational experience ,we explore to improve the quality of education and cultivate the students' practical ability and creative ability. And we construct an educational mode for clinical practice, laboratory medical practice and research project.
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Objective To investigate the effects of quercetin on the growth of lung cancer cell line A549 and the expression of hTERT gene. Methods The number of viable cells was ascertained by trypan blue dye exclusion test. Morphological changes of apoptotic cells were observed by electronic microscopy and DNA ladder assay. The telomerase activity was analyzed by PCR-TRAP assay and hTERT mRNA expression was detected by quantitative RT-PCR. Results Quercetin had a significant inhibition on the proliferation of A549 cells in a dose-dependent manner. The IC50 was 22.5 ?mol/L after exposure to quercetin for 48 h. The results from electron microscopy and DNA ladder showed that apoptosis occurred in the A549 cells of treatment groups. The results of quantitative RT-PCR and PCR-TRAP revealed that the expression of hTERT mRNA was significantly inhibited by quercetin and telomerase activity was decreased. Conclusion Quercetin inhibits the growth of lung cancer cell line A549 in a dose-dependent manner,and induce their apoptosis. The down-regulated expression of hTERT,suppression of telomerase activity and destruction of telomere stability may all contribute to the mechanism of apoptosis induction.