Objective:To construct a chemiluminescense immune quantification assay based one paired mAbs against complexed prostate specific antigen ( c-PSA).Methods:Six week-old female BALB/c mice were immunized with the commercial c-PSA antigen.After serum titer reaching a platform stage ,the spleen was immunized and fused with mouse myeloma cell lines ( Sp2/0 ) .The hybridoma were screened by indirect ELISA ,and eight generated antibodies were paired to obtain a quantitative analysis of the chemical luminescence.Results:7D6 specifically recognized c-PSA,while 1A10 recognized total PSA(t-PSA).And the paired antibody 1A10/7D6 were determined to successfully construct a chemiluminescense immune response quantitative detection method through the detection of c-PSA standard and clinical serum samples .had,positive samples have statistically significant difference ( P<0.000 1 ) with negative samples.And the correlation coefficient R 2 was 0.97 between our c-PSA quantitative results and that of the Siemens c-PSA chemiluminescense immunoassay kit .The detection linear range was 0.1-100 ng/ml,and the sensitivity was 0.005 ng/ml.Conclusion:The paired monoclonal antibodies specifically detecting c-PSA were generated and a c-PSA chemiluminescense immunoassay were developed in this study .The detection capability of our method was comparable with that of the international commercial kit .

Objective To construct a CaME141G fusion protein-expressing plasmid,and to express,purify,and identify the activity of the recombinant protein. Methods The 141st site of the wild type CaM,E (GAG),was mutated to G (GGG),using site-specific mutagenesis technology. Escherichia coli BL-21 was transformed with the mutant plasmid. The GST-CaME141G fusion protein was mass-cultured and induced for expression. Subsequently,the GST-CaME141G fusion protein was purified using GS-4B beads. PreScission protease was applied to remove the GST,the Bradford method used to determine the concentration of purified protein,and SDS-PAGE used to detect its relative molecular weight and purity. The GST pull-down assay was used to study the protein's biological activity. Results The CaME141G protein was successfully purified at a high concentration and purity. The protein could interact with PreIQ protein fragments from the myocardial CaV1. 2 calcium channel C terminal,in a CaME141G concentration-dependent manner. Therefore,CaME141G has the ability to bind with the CaV1. 2 calcium channel. Conclusion This study successfully constructed a CaME141G fusion protein-expressing plasmid and purified the CaME141G protein. This lays a foundation for regulating the function of CaM mutations in the myocardial CaV1. 2 calcium channel,and for the study of its relationship with diseases of the cardiovascular system.

Objective To study the characteristics of resting energy expenditure (REE) and evaluate the association between REE and obesity-related comorbidities in obese children and adolescents.Methods Recruited obese children and adolescents were recruited from the Department of Child Health Care in Nanjing Children's Hospital from July,2015 to September,2016.Height,weight(WT),waist circumference,hip circumference,blood pressure were measured.Puberty status was determined by Tanner staging.REE was measured by indirect calorimetry.Body composition,including fat mass (FM),fat free mass (FFM) were assessed in terms of bioelectrical impedance.Serum biochemical parameters were assessed,including fasting blood glucose (FBG),fasting blood insulin (FBI),total cholesterol (TC),triglyceride (TG),high-density lipoprotein cholesterol (HDL-C) and uric acid (UA).B-model ultrasonography of the liver was conducted.Results One hundred and ninety-six obese children (143 boys and 53 girls) aged 7-15 years were recruited.The measured REE was (1 497.2 ± 312.1) kcal/d.Pearson correlation analysis indicated that REE was positively related to age (r =0.386,P ＜ 0.001),WT(r =0.676,P ＜ 0.001),FM (r =0.629,P ＜0.001),FFM(r=0.635,P＜0.001) and FM％ (r =0.335,P＜0.001),but negatively related to FFM％ (r=-0.335,P ＜0.001).By studying stepwise linear regression,it was found that the factors which influenced REE were age and weight[REE(kcal/d) =899.469-32.098 × age (year) + 16.143 × weight (kg),P ＜ 0.001].REE was expressed per kg of body weight (REE/WT),and there was a significant difference in REE/WT during pubertal development (P ＜0.001) in both males and females and the pubertal males had lower REE/WT than females.Moreover,REE/WT was lower in insulin resistant group compared to the normal insulin group.Meanwhile,REE/WT in hypertension group was lower than that in the normal blood pressure group.The above differences remained after adjusted for age adjustment(P ＜0.O1).Conclusion The decrease in REE/WT level in obese children and adolescents is correlated with puberty development and obesity complicated with insulin resistance and hypertension.

Objective:To study the clinical characteristics of growth development and metabolic disorders in obese children and adolescents with insulin resistance (IR).Methods:Normal weight or obese children and adolescents who hospitalized at the Department of Children′s Health Care of Children′s Hospital Affiliated to Nanjing Medical University from September 2015 to April 2018 were recruited.Children′s height, body weight and waist circumference were measured, and waist-to-height ratio (WHtR) and body mass index (BMI) were calculated.Puberty process was determined by Tanner stage.Blood glucose, blood lipid and insulin were measured in fasting state, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated based on fasting blood glucose and insulin levels.IR was considered when HOMA-IR was over 2.69.Non-alcoholic fatty liver (NAFLD) was diagnosed by abdominal ultrasound.Results:(1) A total of 691 subjects were included, including 183 cases with the age of (9.73±2.38) years in the normal weight group/normal group, and 508 cases with the age of (10.24±2.05) years old in the obese group.The rate of IR was higher in obese group than that in normal group (55.71% vs. 10.38%), and the difference was statistically significant ( P<0.001). (2)HOMA-IR was positively correlated with age ( r=0.256, P<0.001), BMI ( r=0.426, P<0.001), waist circumference ( r=0.454, P<0.001), and WHtR ( r=0.321, P<0.001). After the adjustment for age, sex, and puberty stage, HOMA-IR was also positively correlated with BMI ( r=0.418, P<0.001), waist circumference ( r=0.419, P<0.001) and WHtR ( r=0.375, P<0.001). (3) During puberty, HOMA-IR in both of obese group and normal group was increased, and HOMA-IR in obese group was more particularly serious compared to normal group[TannerⅠ: 2.60(1.49, 3.94) vs.1.28(0.80, 1.90); Tanner Ⅱ: 3.07(1.75, 5.17) vs.1.80(1.16, 2.96); Tanner Ⅲ: 4.33(2.80, 6.57) vs.2.47(1.41, 3.68); Tanner Ⅳ-Ⅴ: 3.49(1.04, 5.78) vs.1.91(0.54, 2.60)], and the differences were all statistically significant(all P<0.05). (4)Compared with the obese objects without IR, obese children and adolescents with IR had higher systolic blood pressure[112(104, 124) mmHg vs.109(98, 121) mmHg, 1 mmHg=0.133 kPa], triglyceride level [1.27(0.95, 1.81) mmol/L vs.1.09(0.79, 1.61) mmol/L], fas-ting blood glucose level [4.80(4.46, 5.01) mmol/L vs.4.48(4.16, 4.76) mmol/L] and fasting insulin level [21.27(16.21, 28.56) mmol/L vs.7.62(4.43, 10.83) mmol/L], and the differences were all statistically significant(all P<0.05). IR was a risk factor for NAFLD in obese children( OR=1.536, 95% CI: 1.049-2.247, P<0.05). Conclusions:Serious and abdominal obesity in children and adolescents is a major risk factor for the development of IR.HOMA-IR of obese children and adolescents is particularly serious during puberty.The obese children with IR are more likely to have metabolic disorders in blood glucose, serum lipid and blood pressure, and have the risk of NAFLD development.

Objective:To observe the effects of lifestyle intervention on diet, physical activities and health outcomes in obese children and adolescents during one year of follow up.Methods:A total of 153 obese children and adolescents with body mass index more than 95th percentage of the same age and sex were recruited consecutively from June 2015 to June 2017 in our hospital whose parents had signed the informed consent forms. The children were followed-up once every three months and their diet and physical activities, anthropometric measurement and metabolic indicators were assessed on research scheme, which lasted for one year.Results:A total of 52 obese children completed four visits plan in 12 months (34.0%, one year group), 101 children (66.0%) dropped from 3 to 9 months (66.0%, less than one year group). The intake of total energy [(8 524.5±2 068.6)kJ vs (6 464.0±1 586.9)kJ, P<0.05], dietary protein [(75.5±20.7)g vs (64.2±16.8)g, P<0.05], fat [(79.0±18.8)g vs (60.3±14.2)g, P<0.05], carbohydrates [(257.1±83.6)g vs (188.9±63.8)g, P<0.05] decreased after one year intervention in one year group. Moreover, the numbers of physical activity of medium to high intensity increased in obese children (0 vs 32.7%, P<0.05). BMI-SDS [(3.15±0.85) vs (2.46±0.81), P<0.05], WHtR [(0.60±0.04) vs (0.56±0.06), P<0.05] and FM% [(39.9±5.4) vs (33.0±7.4), P<0.05] were reduced, while SMM% [(32.1±3.2) vs (36.0±4.3), P<0.05] increased significantly in one year group. The detection rate of insulin resistance, dyslipidemia and nonalcoholic fatty liver disease (NAFLD) were reduced in one year group at the end of follow up ( P<0.05). The decrease of body fat and the increase of skeletal muscle were more obvious in one-year follow up group( P<0.05). Conclusion:Children and adolescent have less energy intake, more physical activities, good clinical outcomes and less complications of obesity through lifestyle intervention and follow up for one year, so it is worthy of being promoted.