Objective To select an optimal non-specific antigen blocking method by using immuno-infiltration assay so as to suit protein chip preparation. Methods Human papillomavirus type 16 L1 protein expressed by insect-baculovirus espressin system was incubated with skimmed milk powder, calf serum, bovine serum albumin (BSA) combinations of five kinds of methods to block the non-specific antigen. PBS was used as control. The effect of eliminating non-specific stain was detected by immuno-infiltration assay. Results After repeated tests, the results showed that the stability and repeatability of blocking effects were poor for the fixing up antigen first and then blocking method, and the blank control was prone to false positive. The infiltration rate of NC membrane would be affected by using skimmed milk powder as a blocking agent because the pore of NC membrane was easily plugged by milk powder particles. The use of calf serum as a blocking agent made it very difficult to determine the result because the calf serum absorbed by NC membrane produced the background; however, when 20g/L BSA was used to blocking before fixing up antibody, the results became satisfactory. Conclusion Fixing up antibody after blocking in immuno-infiltration assay showed that the blocking effect against non-specific antigen was satisfactory, stable and repeatable, indicating this method is a novel optimal blocking method compared with others.

Objective To construct an expression vector directed by hU_(6)snRNA promoter for synthesizing small RNA and to identify its functional activity in the gastric carcinoma cells——SGC-7901.Methods Using human genomic DNA as template,U_(6) snRNA promoter was obtained by PCR method,and then cloned into PUC19 vector to produce the recombinant plasmid PUC-hU_6-extra,which was sequenced and then transfected into gastric carcinoma cell——SGC-7901 with liposome.The effect of expression directed by U_6 promoter was detected by RT-PCR method,and the cell proliferation curve analysis was performed by stained dye.Results The hU_6 snRNA promoter with the first 27 nucleotides followed were successfully cloned into PUC19 plasmid.The recombinant vector could efficiently transcribe small RNA molecules and exerted no effect on cell proliferation in SGC-7901 cells in vitro.Conclusion We have successfully constructed the recombinant PUC-hU_6-extra plasmid vector that can efficiently transcribe small RNA molecules directed by hU_6 snRNA promoter in the gastric carcinoma cells——SGC-7901.

[ ABSTRACT] AIM:To observe the effect of high glucose on the protein expression of calreticulin ( CRT) and its association with cell apoptosis and mitochondrial dysfunction in the cardiomyocytes.METHODS: AC-16 cardiomyocytes were randomly divided into normal glucose group, high glucose group, high glucose+CRT siRNA group and isotonic con-trol group.The cell apoptotic rate, reactive oxygen species (ROS), mitochondrial membrane potential level, respiratory enzyme activity, and protein expression of CRT were observed.RESULTS: Compared with the cardiomyocytes in normal glucose group, the apoptotic rate and ROS production of cardiomyocytes increased in high glucose group, accompanying with the decreases in the mitochondrial membrane potential level and enzyme activitiy of the respiratory chain.The protein expression of CRT was significantly increased in high glucose group.However, compared with high glucose group, high glucose+CRT siRNA decreased the expression of CRT and attenuated the damage of mitochondria, but CRT siRNA did not reduce the ROS level in cardiomyocytes.CONCLUSION:High glucose brings about CRT over-expression to induce mito-chondrial injury, thus increasing myocardial apoptosis.

Objective To investigate the clinical diagnostic value and pathogenesis of serum protein identification in Keshan disease (KD).Methods A total of 65 chronic KD patients were selected as the patient group in KD endemic areas,while 29 cases of dilated cardiomyopathy (the DCM group),62 healthy cases from KD endemic areas (control 1 group) and 28 healthy cases from non-endemic areas (control 2 group) were selected as controls.Liquid chip time of flight mass spectrometry (ClinProtTM MALDI-TOF-MS) was used to determine the expression of proteins/peptide peaks.ClinProTools 2.2 software was used to analyze the protein profiles to determine differentially expressed proteins/peptide peaks.The Genetic Algorithm (GA),QuickClassifer Algorithm (QC) and Supervised Neural Network Algorithm (SNN) methods were used to screen marker proteins.Matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometry technique (MALDI-TOF/TOF) was also used as a secondary mass spectrometry to identify differentially expressed peptides.Results Between the KD and control 1 groups,34 differentially expressed proteins/peptides and 5 marker proteins were identified,while 52 differentially expressed proteins/peptides and 5 marker proteins were identified between the KD and control 2 groups,and there were 67 differentially expressed proteins/peptides and 5 marker proteins between the KD and DCM groups.During secondary mass spectrometry,two peptides for mass-to-charge ratio (m/z) 2 079 and 1 465 were obtained,peptide of matching β-globin showed low expression while peptide of matching fibrinogen showed high expression in the KD patients.Conclusions Serum marker proteins can be used as biomarkers for diagnosis and differentiation of KD.β-globin and fibrinogen play an important role in the development of KD myocardial injury.

Objective In order to investigate the relationship between erythrocyte immune function and seleni- um(Se) level. Method Red blood cell immune adherence(RCIA) function,serum RCIA regulatory factor, blood Se content and activities of glutathione peroxidase(GPX) of residents in Keshan disease(KD) endemic and non-endemic areas were comparatively studied. Forty-eight residents in KD endemic area aged 13～ 16 years were divided into 2 groups. The residents in the experimental group were orally given 200μg Se daily as Se yeast for 12 weeks, and their erythrocyte Se content and activity of glutathione peroxidase (GPX),RCIA function,serum RCIA regulatory function and circulating immune complexes(CIC) content were determined. ResultsThe results showed that the rosette for- mation rates of erythrocyte and blood Se levels of the residents in KD area were significantly lower and the rosette formation inhibitory rate of serum RCIA of the residents in KD area was significantly higher than those in the non-endemic area. Erythrocyte Se contents, GPX activities and rosette formation rates of erythrocyte were sig- nificantly increased and the rosette formation inhibitory rates of serum RICA were significantly decreased after sup- plementing Se,but the difference in the contents of serum CIC was not significant. ConclusionThe increase of ery- throcyte immune function by Se-supplement might be one of the effective mechanisms in the prevention of KD by Se- supplement.

Objective:To investigate the relationship between single nucleotide polymorphisms of transforming growth factor-β2 (TGFβ2) gene and Keshan disease (KD) in Han population of Shaanxi Province.Methods:KD region in Huangling County, Yan'an City, Shaanxi Province was selected as the investigation site in this study. Using the method of cluster random sampling, 52 families with KD in 6 administrative villages in Huangling County (Duanjiawan Village, Taoqu Village, Yaoping Village, Jianzhuang Village, Anjiao Village in Yaoping Town, and Houziping Village in Diantou Town) were selected for epidemiological investigation. According to the "Diagnosis of Keshan Disease" (WS/T 210-2011), 285 subjects were identified, including 79 patients with KD (case group) and 206 healthy controls (control group). Genomic DNA was extracted from the peripheral venous blood. The polymorphism of genetic variation of TGFβ2 gene rs6658835 was genotyped by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Chi-square (χ 2) test and t-test were used to analyze the baseline data, and binary logistic regression model was used to analyze the influencing factors of KD, all samples were tested for Hardy-Weinberg equilibrium using goodness-of-fit χ 2 test, differences in genotype and allele frequencies between case and control groups were compared by χ 2 test, and logistic regression analysis was used to compare the genotype frequencies between two groups after adjusting for confounding factors. Results:Epidemiological investigation showed that there were significant differences in age and heart murmur between case group and control group ( t = 7.03, χ 2 = 9.66, P < 0.05). The analysis of binary logistic regression model showed that the influence of age on KD was statistically significant (χ 2 = 20.72, P < 0.001). The gene frequency distribution of TGFβ2 gene rs6658835 in case group and control group conformed to the Hardy-Weinberg equilibrium (χ 2 = 0.02, P = 0.900). Correlation analysis results: the difference of genotype frequency of TGFβ2 gene rs6658835 in case group (GG, GA, AA: 6.3%, 38.0%, 55.7%) and control group (GG, GA, AA: 10.7%, 43.7%, 45.6%) was not statistically significant (χ 2 = 2.78, P = 0.249). After adjustment by age, the difference of genotype frequency and dominant model of TGFβ2 gene rs6658835 in case group and control group was statistically significant (χ 2adj = 5.43, 4.86, P < 0.05), the difference of recessive model of TGFβ2 gene rs6658835 in case group and control group was not statistically significant (χ 2adj = 2.12, P = 0.145). Conclusion:TGFβ2 gene rs6658835 is associated with KD in Han population of Shaanxi Province.

Objective To investigate the serum coxsackie virus B(CVB) infection and nitric oxide (NO)level of the patients suffer from latent or chronic Keshan disease and their characteristics in the etiopathology of Keshan disease. Methods Sera were isolated from 30 patients with latent or chronic Keshan disease in Huangling county.Shaanxi Province, and the CVB-specific IgM antibody and NO were tested. Control groups were health subjects in Huangling county or Xi'an city, Shaanxi Province. Results The percentage of CVB-specific IgM positive in patients in Huangling county was significantly higher than that of both control groups in Huangling county and Xi'an city (P＜0. 05). The serum level of NO in patients was significantly higher than that of the control group in Huangling county (P＜0.05) ,however,compared with control group in Xi'an city, there was no difference (P＞0.05). In CVB-specific IgM positive patients,the serum level of NO was significantly higher than that of CVB-specific IgM negative group(P＜0.05).Conclusion CVB infection and serum NO level might be related to the etiopathology and the development of Keshan disease.

Objective To investigate the protective effects of selenium on nitric oxide(NO)-mediated myocardial apoptosis.Methods The AC16 cardiomyocyte cultured in vitro were divided into control group,selenium treatmentgroup,sodium nitroprusside(SNP) treatment group and selenium + SNP treatment group,SNP was the exogenous NO donor.There was no intervention in the control group,and an equal volume of the culture solution was added to the treatment groups.The selenium treatment group added a dose of 100 μg/L of selenium,the SNP treatment group added a dose of 1.0 mmol/L of SNP,and the selenium + SNP treatment group was pretreated by 100 μg/L selenium for 4 h followed by 1.0 mmol/L SNP;the cells or supernatants were collected after 24 h of culture.The content of NO was detected by Griess method in supernatants.The level of cell reactive oxygen species was detected by flow cytometry.The changes of cell mitochondrial membrane potential and apoptosis were observed under fluorescence microscope.The real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression levels of apoptosis-related genes B-cell lymphoma-2 (Bcl-2) associated X protein (Bax) and Bcl-2,respectively.Results The NO content in the control group,selenium treatment group,SNP treatment group and selenium + SNP treatment group were (10.3 ± 1.8),(9.2 ± 2.1),(15.2 ± 3.5),(14.3 ± 2.6) μmmol/L,respectively;SNP had a main effect on NO content (F =23.33,P ＜ 0.05).The cell reactive oxygen species were 31.63 ± 1.40,29.52 ± 2.86,60.62 ± 4.83,50.08 ± 2.41,respectively;selenium and SNP had main effects on reactive oxygen species (F =12.19,187.20,P ＜ 0.05),selenium combined with SNP had an interactive effect on reactive oxygen species (F =5.42,P ＜ 0.05).The cell mitochondrial membrane potential levels were 0.42 ± 0.11,0.37 ± 0.07,7.25 ± 1.91,and 5.21 ± 1.59,respectively;selenium and SNP had main effects on cell mitochondrial membrane potential levels (F =14.21,440.01,P ＜ 0.05),selenium combined with SNP had an interactive effect on cell mitochondrial membrane potential levels (F =12.89,P ＜ 0.05).Selenium had main effects on nuclear pyknosis ratio,Bcl-2 mRNA and Bax protein expressions (F =9.52,10.84,22.17,P ＜ 0.05);SNP had main effects on nuclear pyknosis ratio,Bax and Bcl-2 mRNA expressions,and Bcl-2 protein expression (F =192.86,21.90,16.09,18.39,P ＜ 0.05);selenium combined with SNP had an interactive effect on Bax,Bcl-2 mRNA and protein expressions (F =20.51,7.59,15.38,11.97,P ＜ 0.05).Conclusion The SNP can induce apoptosis of AC16 cardiomyocyte;selenium combined with SNP has an interactive effect on AC16 cardiomyocyte,indicating that selenium has protective effect on NO modiated myocardial apoptosis.

Shaanxi Province located at Midwest inland of China was a typical iodine deficient disorders region. To investigate iodine and selenium levels of neonates in the Shaanxi sub-clinical cretinism region of China after supplement of iodine salt for nearly twenty years. We collected 56 umbilical cord blood samples from cretinous regions of Yijun County (a selenium deficient region) north of Shaanxi Province and Ziyang County (a selenium-enriched region) south of the province and from Lintong in Xi'an (a non-cretinous region for control). Among these samples 17 were collected from Ziyang, 20 from Lintong and 19 from Yijun. Seven trace elements of iodine, selenium, zinc, copper, iron, calcium and magnesium in the umbilical cord blood samples were measured and the results were processed statistically. There were no significant differences in the levels of iodine among all three counties. However, the level of selenium in Ziyang was the highest and in Yijun it was the lowest. The other trace elements such as Cu Zn Fe and Mg showed no significant difference among the three counties except for the Ca level which was lower in Yijun.The regression equation was established with the backward method of multiple regression was: Se=0.180+0.00006654 Fe-0.006 Cu-0.005956 Mg+0.1.

Objective To investigate the relationship between single nucleotide polymorphisms of Bcl-2 related anti apoptotic protein 3 (BAG3) gene and Keshan disease (KD) in north Chinese Han population.Methods In 2002 a total of 285 Chinese Han subjects,including 79 KD patients and 206 control subjects were involved in this study.Genomic DNA was extracted from the peripheral venous blood sample.Blood samples were provided by the Institute of Endemic Disease Prevention,Xi'an Jiaotong University,and stored at 80 ℃.The polymorphism of genetic variation was genotyped by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF).The data was analyzed using TYPER 4.0 or SPSS16.0 software.All sample groups were tested for Hardy-Weinberg equilibrium using goodness-of-fit x2 test.Differences in genotype distribution and allele frequencies between case and control were compared by x2 test.Logistic regression analysis was applied to detect association using age as a confounding factor.Results All sample group passed the Hardy-Weinberg equilibrium test (P ＞ 0.05).Significant differences were not observed in genotype distribution between cases (rs2234962:CC,CT,TT were 0.0％,0.0％ and 100.0％,respectively;rs196295:GG,GA,AA were 22.8％,54.4％ and 22.8％,respectively;rs3858339:GG,GT,TT were 5.1％,38.0％ and 56.9％,respectively;rs3858340:TT,TC,CC were 5.1％,38.0％ and 56.9％,respectively) and controls (rs2234962:CC,CT,TT were 0.0％,1.0％ and 99.0％,respectively;rs196295:GG,GA,AA were 21.4％,51.5％ and 26.2％,respectively;rs3858339:GG,GT,TT were 5.8％,34.5％ and 59.7％,respectively;rs3858340:TT,TC,CC were 5.8％,34.5％ and 59.7％,respectively) for rs2234962,rs3858339,rs196295 and rs3858340 on BAG3 gene (x2 =0.685,0.408,0.330,0.330,P ＞ 0.05).Significant differences were not observed in genotype after agecorrecting between cases and controls for 4 SNPs on BAG3 gene (x2 =0.001,0.019,1.009,0.019,P ＞ 0.05).Conclusion The results suggest that the BAG3 gene might not be a susceptibility gene of KD in north Chinese Han population.