To produce specific monoclonal antibody (McAb) against human thrombomodulin (hTM), the full-length hTM cDNA-expressing plasmid pThr402 was transfected into CHO cells by Lipofectamine 2000 reagent. The hTM-expressing CHO cells, which was confirmed by flow cytometry and Western blot, were obtained by G418 selection. Then the McAb against hTM was prepared with classic hybridoma technique. A cell line of CHO-TM5 with high level of hTM was used to immunize female Balb/c mice 3 times at an interval of 4 weeks. On the third day after the third immunization, mice were sacrificed and spleen cells were harvested to prepare hybridoma cells with SP2/0 cells at the ratio of 10 to 1. Hybridoma cells were then cultured at 96-well plates for screening. Cellular enzyme-linked immunoabsorbent assay (CELISA) was applied twice. The first CELISA was done with polythene ELISA plate with a monolayer of CHO-TM5 cells. The positive clones from the first screen were then selected by reacting with similar screening ELISA plate but with CHO cell monolayer instead. Only clones that were positive for the first screening and negative for the second screening were kept, and called as CHO-TM5(+)CHO(-) hybridoma cells. Balb/c mice were intraperitoneally injected with the selected hybridoma cells. Ascites were collected and monoclonal antibodies were purified using FPLC, and its Ig class, subclass, and titer were then determined respectively. The specificity of the yielded McAb was identified with CELISA, flow cytometry, ABC immunohistochemistry and immunoblotting. One line of hybridoma cells with high expression of specific McAb against hTM, NH-1, was obtained. The Ig subclass of the McAb was IgG1 and the titer of ascitic McAb was 1x10(-6). Flow cytometry, CELISA and Western blot assays demonstrated that McAb NH-1 could specifically recognize hTM expressed in CHO-TM5 cells and human umbilical vascular endothelial cells. Meanwhile, the tissue specificity of antigen recognized by McAb NH-1 was identified by immunohistochemical ABC staining. NH-1 can specifically recognize the natural hTM expressed mainly in vascular endothelial cells, which will potentially be useful for investigation of the functions and clinic values of hTM.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibody Specificity ; CHO Cells ; Cricetulus ; Female ; Humans ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Thrombomodulin ; immunology ; Transfection

OBJECTIVETo investigate the risk factors of lung cancer in Tianjin and to provide evidence for further monitor there of.

METHODSA case-control study involving interviews with 193 new cases and 259 controls aged 30 - 76 years was carried out. Structured questionnaires were used to collect information on general condition, living environment, living style, disease and family history, etc. Logistic regression model univariate and multivariate analysis were used to pick out the significant lung cancer risk factors.

RESULTSBy monovariate analysis, risk factors such as smoking, passive smoking, drinking, history of malignancy in family and occupation were found. By multivariate analysis, smoking, passive smoking, higher body mass index (BMI) and average income and living space per capita ten years earlier were ascertained, their operations research (OR) values were 3.302, 1.193, 1.003, 1.067 and 0.913.

CONCLUSIONSmoking and passive smoking are independent risk factors of lung cancer. Monthly income per person and living space per person 10 years earlier are associated with elevated risk of lung cancer. Higher body mass index has protective effects on lung cancer risk.

Adult ; Aged ; Humans ; Logistic Models ; Lung Neoplasms ; etiology ; genetics ; Middle Aged ; Occupations ; Risk Factors ; Smoking ; adverse effects

OBJECTIVELeukemia is a major cause of death of children in China, which accounts for 50 % of all cancers of children. Data from Tianjin Cancer Hospital was analyzed for mortality of leukemia in children under 20 years from 1981 to 2000 in the city of Tianjin.

METHODSAll physicians and medical staff of the hospitals and clinics in the registry area were responsible for filling out the report forms for every new case diagnosed as malignant tumors. Death certificates for malignant tumors have been registered at the local police station and the residential files were checked. All cancer cases with insufficient information were traced to his/her family and relevant persons worked in the clinic. Tianjin Cancer Registry Center periodically conducted an active re-checking program to review all patient records on cancers that was not registered in this period. Tumors diagnosed in this study were coded according to the International Classification of Diseases for Oncology (ICD-O). Mortality rates were calculated by age, sex and date of death.

RESULTSThe types of acute lymphoid leukemia, acute myeloid leukemia and chronic myeloid leukemia were the most common types of childhood leukemia in Tianjin, comprised 69.3%, 20.9 % and 8.0%, respectively. The mortality for childhood leukemia decreased slowly during the period of 1981 to 2000 in Tianjin. Mortality and morbidity ratios were 0.51.

CONCLUSIONCombined with characteristics of individual forms of childhood leukemia mortality, further epidemiological research is needed to prevent childhood leukemia.

Adolescent ; Cause of Death ; Child ; Child, Preschool ; China ; epidemiology ; Humans ; Infant ; Leukemia ; mortality ; Young Adult

Acquired Immunodeficiency Syndrome ; epidemiology ; virology ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; China ; epidemiology ; Female ; Humans ; Male ; Middle Aged ; Young Adult

OBJECTIVEThe aim of this study is to observe the effect of combined amiodarone and antiarrhythmic peptide (AAP10) use on the incidence of induced ventricular arrhythmias in healed myocardial infarction (MI) rabbits.

METHODSTwenty Japanese rabbits underwent thoracotomy without coronary artery ligation (Sham, group A), the middle left circumflex branch were ligated to induce MI in 180 Japanese rabbits. Eight weeks after operation, 124 rabbits survived MI operation and were divided into four groups: control group (group B, n = 31), amiodarone group (group C, n = 31), AAP10 group (group D, n = 31) and amiodarone plus AAP10 group (group E, n = 31). The A and B and D groups were treated with saline 2 ml/d, the C and E groups were treated with 2 ml saline containing amiodarone 100 mg×kg(-1)×d(-1). All rabbits were examined by echocardiogram at 12 weeks after operation, then anesthetized by sodium barbital, the left wedge ventricular preparations were cannulated and artery perfused by Tyrode's solution in vitro in the absence (Group A, B and C) and presence of AAP10 (500 nmol/L, Group D and E). The volume electrocardiogram, QT Interval, QRS interval, effective refractory period (ERP), the T-peak to T-end interval (T(p-e)), and ventricular tachycardia episodes induced by programmed stimulation were recorded. The T(p-e)/QT ratio was calculated. After perfusion, gap junctions protein connexin 43 (Cx43) expression was detected by Western blot and immunofluorescence.

RESULTSThe incidence of induced ventricular tachycardia episodes of group A, B, C, D, E was 0, 62.5%, 26.9%, 40.0%, 22.2% respectively. The incidence of induced ventricular tachycardia episodes of group E was less than group B. The T(p-e)/QT ratio in group B, C, D were greater than in group A. The T(p-e)/QT ratio of group E was less than group B. The myocardial Cx43 in the group B was down-regulated and disorganized compared to group A, up-regulated in group C and E compared to group B, up-regulated in group E compared to group D. The Cx43 in the heart of group D and E were well organized than in group B and C.

CONCLUSIONSThe artery perfused rabbits wedge preparations with healed myocardial infarction with high incidence of induced ventricular tachycardia episodes are good platform for ventricular arrhythmias research. Combined amiodarone and AAP10 use could decrease the T(p-e)/QT ratio and the incidence of induced ventricular tachycardia episodes. Amiodarone and AAP10 have synergistic effects on upregulating Cx43 and preventing ventricular arrhythmias in this rabbit model of healed myocardial infarction.

Amiodarone ; pharmacology ; therapeutic use ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; therapeutic use ; Arrhythmias, Cardiac ; etiology ; Connexin 43 ; metabolism ; Male ; Myocardial Infarction ; drug therapy ; metabolism ; physiopathology ; Oligopeptides ; pharmacology ; therapeutic use ; Rabbits ; Treatment Outcome

OBJECTIVETo determine the karyotype of a patient with Prader-Willi-like syndrome features.

METHODSChromosomal high resolution banding was carried out to analyze the karyotype of the patient, and methylation-specific PCR was used to analyze the imprinting region of chromosome 15. Subtelomeric region was screened by multiplex ligation-dependent probe amplification (MLPA), and fluorescent in situ hybridization (FISH) and real-time quantitative PCR were further performed to identify the deleted region.

RESULTSNo abnormality was discovered by high resolution karyotype analysis and methylation-specific PCR studies. MLPA analysis showed that the patient had a deletion of 1p subtelomeric area, which was confirmed by FISH analysis. The deleted region was shown within a 4.2 Mb in the distal 1p by 3 BAC FISH probes of 1p36 combined with real-time PCR technique. Family pedigree investigation showed the chromosome abnormality was de novo. Therefore, partial monosomy 1p36 was likely responsible for the mental retardation of the patient.

CONCLUSIONMolecular cytogenetic techniques should be performed to those patients with Prader-Willi-like syndrome features, to determine their karyotypes.

Child ; Chromosome Deletion ; Chromosomes, Human, Pair 1 ; genetics ; Female ; Humans ; Karyotyping ; Prader-Willi Syndrome ; genetics

OBJECTIVETo establish a single-cell whole genome amplification (WGA) technique, in combination with comparative genomic hybridization (CGH), for analyzing chromosomal copy number changes, and to explore its clinical application in preimplantation genetic diagnosis (PGD).

METHODSTwelve single-cell samples with known karyotypes, including 5 chorionic villus samples, 4 human embryonic stem cell (hESC) samples and 3 peripheral lymphocyte samples, and 4 single blastomere samples carrying chromosomal abnormalities detected by PGD, were collected for whole genome amplification by combining primer extension preamplification (PEP) with degenerate oligonucleotide primed-PCR (DOP-PCR) amplification. The amplified products labeled by red fluorescence were mixed with control DNA labeled by green fluorescence, and then the mixture was analyzed by CGH. As a comparison, 10 single cell samples were amplified by DOP-PCR only and then CGH analysis was performed.

RESULTSThe amplification using PEP-DOP-PCR was more stable than traditional DOP-PCR. The products of PEP-DOP-PCR range from 100 bp to 1000 bp, with the mean size being about 400 bp. The CGH results were consistent with analyses by other methods. However, only 6 out of 10 single cell samples were successfully amplified by DOP-PCR, and CGH analysis showed a high background and 2 samples showed inconsistent results from other methods.

CONCLUSIONPEP-DOP-PCR can effectively amplify the whole genome DNA of single cell. Combined with CGH, this WGA method can successfully detect single-cell chromosomal copy number changes, while DOP-PCR was easy to fail to amplify and amplify inhomogeneously, and CGH analysis using this PCR product usually showed high background. These results suggest that PEP-DOP-CGH is a promising method for preimplantation genetic diagnosis.

Comparative Genomic Hybridization ; methods ; DNA Primers ; Genetic Testing ; methods ; Humans ; Karyotyping ; methods ; Nucleic Acid Amplification Techniques ; methods ; Nucleic Acid Hybridization ; methods ; Oligonucleotides ; chemistry ; Preimplantation Diagnosis ; methods

Objective To investigate the lipid levels of Han ethnicity Chinese children at school-age,to provide objective data for the formulation of prevention and management strategy regarding dyslipidemia among children and adolescents.Methods 20 191 children (with 10 669 boys and 9522 girls) aged 7 to 16 years old from 6 representative geographical areas,including Beijing,Tianjin,Hangzhou,Shanghai,Chongqing and Nanning,were surveyed in a randomly selected clustered sample in China.Data on fasting blood triglyceride (TG),total cholesterol (TC),lowdensity lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) levels were measured.Non-high-density lipoprotein cholesterol (non-HDL-C) levels were calculated with data collection,entry,and collation were under the same criteria.Results (1) In the 7-16 year-old group,TG (P95) fluctuated between 1.26 mmol/L and 1.88 mmol/L,while TC (P95) was between 4.80 mmol/L and 5.46 mmol/L.LDL-C(P95) was between 2.67 mmol/L and 3.27 mmol/L while non-HDL-C (P95) was between 3.36 mmol/L and 3.91mmol/L,sugesting that age did not seem to be an affecting factor for the lipid level (P＞0.05).The level of HDL-C (P5) fluctuated bctwcen 1.08 mmol/L and 0.83 mmol/L,and the dependability analysis on HDL-C and age showed statistically significant difference (P＜0.01,r=-0.274).(2) In the 7-9 year-old group,the levels ofTG,TC,LDL-C and non-HDL-C of boys were lower but the HDL-C level was higher than in girls.However,in the 10-16 year-old group,the levels of five lipids of boys were all lower than in girls,with all the differences statistically significant (P＜0.05).(3) The levels of TG,TC,LDL-C and non-HDL-C in the obese group were significantly higher than those in non-obesity group,as HDL-C was significantly lower than in non-obese group(P＜0.01).Incidence rates of single and multiple dyslipidemia in obese group were significantly higher than in non-obese group (P＜0.01).(4) Grouped by region,the abnormal rates of TG were descending,with the ranking as North (10.4％),Midwest (9.7％) and East (8.3％),while the abnormal rates of TC were descending with the ranking as Midwest (6.0％),North (5.2％)and East (4.8％).The abnormal rates of LDL-C were descending as the ranking of North (3.1％),East (2.6％) and Midwest (0.9％),with the abnormal rates of non-HDL-C were descending as Midwest (6.5％),North(4.2％)and East (3.6％).The abnormal rates of HDL-C were descending as Midwess (14.2％),North(5.7％) and East(5.5％).All the differences in the above-said items were statistically significant (P＜0.05).(5) According to the standards of hyperlipidemia formulated by the American Academy of Pediatrics,the incidence rates of abnormal TG,TC,LDL-C,non-HDL-C,HDL-C were 9.4％,5.4％,2.2％,4.8％,8.6％ respectively.Conclusion (1) Levels of lipids were affected by many factors,but age was not one of them in children and adolescents.However,HDL-C was declining along with the increase of age,to some extent.(2)Girls had a relatively protective tendency through the increasing HDL-C level when they entered the puberty years.(3)Lipids levels in non-obese group were significantly better than the obese group.(4)The lipids levels of children and adolescents in the Eastern region of the country were better than that in the northern and mid-western areas.

Objective To analyze transmission factors of norovirus outbreaks in Guangdong province during 2008-2015 and provide evidence for the prevention and control of norovirus infection.Methods Epidemiological analysis was performed on the data of norovirus outbreaks reported in Guangdong from January 1,2008 to December 31,2015,which were obtained from the Public Health Emergency Management Information System of Guangdong province.The samples collected from the norovirus outbreaks were detected for norovirus by RT-PCR and the gene sequencing of the positive PCR products were performed.Results A total of 96 norovirus outbreaks were reported in Guangdong during 2008-2015.Sixteen outbreaks were reported during 2008-2012and 80 outbreaks were reported during 2013-2015 (83.3％).Eighty-two outbreaks (85.4％) occurred in schools.The infection routes included foodborne transmission in 39 outbreaks (40.6％),person to person transmission in 23 outbreaks (24.0％) and waterborne transmission in 8 outbreaks (7.3％).The gene sequencing results showed that variant G Ⅱ.4/Sydney2012 was the predominant pathogen for 6 of the 20 outbreaks (30.0％) during 2012-2013.Variant G lⅡ.17 was the predominant pathogens for 33 of the 53 outbreaks (62.3％) during 2014-2015.Conclusion The norovirus outbreaks in Guangdong during 2008-2015 were caused by foodborne and person to person transmissions of two emerging variant:G Ⅱ.4/Sydney2012 and G Ⅱ.17.

Objective To understand the antibody levels against pandemic influenza A (H1N1) virus (2009 H1N1 ) among aged ≥3 years population in 2009, from Jiangsu province, and to describe the distribution of 2009 H1N1. Methods Serum specimens were collected from natural populations at four different periods in Jiangsu, and tested with hernagglutination-inhibition (HI)assays. Rates of protective antibody against 2009 H1N1 and Geometric mean titers (GMTs)were estimated. Results The rates of protective antibody against 2009 H1N1 rose with the and November, 2009. There were no significant differences on the rates of protective antibody between males and females at four different cross-sectional periods (P＞0.05), and no significant differences on GMTs were observed at different periods except for November 2009. Significant differences on rates of protective antibody and GMTs among various age groups were observed at four different periods (P＜0.05), and similar results were observed among different periods in various age groups (P＜0.05). There were significant differences on rates of protective antibody and GMTs among different areas (P＜0.05). Conclusion The 2009 H1N1 strain had been widely spread out in Jiangsu province since July 2009. People aged 12-17 years became the major victims after August. As of November 2009, the rate of protective antibody against 2009 H1N1 was still low, predicting that the epidemic might continue to exist for a certain period of time.