Objective　To evaluate the role of transfected angiotensinⅡ(Ang Ⅱ) receptor AT1 anti-sense nucleotide (AT1A) in the expression of subtypes of AngⅡ receptor mRNA, synthesis of protein and nucleic acid in cardiomyocytes. Methods　AT1 cDNA sequence (476 bp) was cloned with RT-PCR and reversely inserted into PcDNA3.1 (5.4 kb) to construct an intact plasmid containing AT1A (PAT1A). The plasmid was then transfected into the cultured cardiomyocytes and identified with RT-PCR and Western blot. The synthesis of protein and nucleic acid identified by 3H-Leu and 3H-TdR incorporation, and expressions of AT1 and AT2 mRNA by RT-PCR, were compared between transfected and nontransfected cardiomyocytes after being stimulated with 10-7 mol/L AngⅡ for 24 h. Results　The plasmid PAT1A were successfully constructed. The AT1 mRNA and its protein were expressed significantly less in the transfected cardiomyocytes than in the control (P<0.01). In the transfected cardiomyocytes, AT1 mRNA expression was markedly decreased, but that of AT2 mRNA obviously increased (P<0.01) when compared with the nontransfected cardiomyocytes after stimulation for 24 h with AngⅡ 10-7 mol/L; no significant difference was found in 3H-Leu and 3H-TdR incorporation between them. Conclusion　After the cardiomyocytes was tranfected with AT1A, the expression of AT1 mRNA was markedly suppressed，while AT2 mRNA up-regulated at the same time. Our results indicate that AT1A blocking can not effectively interrupt the Ang Ⅱ-induced synthesis of the protein and nucleic acid in cardiomyocytes.

Aim To investigate the effect of salvianolic acid B (Sal B)on c-Jun N-terminal kinase (JNK)ac-tivation and apoptosis of INS-1 cells induced by inter-mittent high glucose.Methods INS-1 cells were pre-incubated with Sal B for 24 h,followed by exposure to intermittent high glucose (IHG,11.1 mmol·L-1 12 h,33. 3 mmol·L-1 12 h)for 72 h.Cell viability was assessed by MTT assay and cell apoptosis was evalua-ted by flow cytometry.Glucose induced insulin secre-tion capacity and intracellular reactive oxygen species (ROS)contents were measured by enzyme linked im-munosorbent assay (ELISA)and a fluorescent probe DCFH-DA,respectively.Levels of JNK activation and PDX-1 protein expression were determined by Western blot analysis.Results Sal B significantly alleviated IHG-induced cell injury and apoptosis,with glucose induced insulin secretion capacity improved evidently (P<0.05 or P<0.01).Preincubation with Sal B no-tably decreased intracellular ROS and JNK activation in INS-1 cells,while the level of PDX-1 protein was in-creased markedly (P<0.05 or P<0.01 ).Conclu-sion Sal B is capable of ameliorating IHG-induced cell injury and apoptosis in INS-1 cells,which might be derived from suppression of JNK activation and up-regulation of PDX-1 protein expression.

AIM: To explore the relationship between proliferation, hypertrophy of vascular smooth muscle cells(VSMC) and platelet-derived growth factor-AA(PDGF-AA) and PDGFR-? expression in spontaneously hypertension rats(SHRs) and the role of tyrosine protein kinase (PTK) in it. METHODS: 1. The difference of PDGF-AA, PDGFR-? and PDGFR-? expressions in SHR/Wistar-kyoto rat(WKY)-VSMC was examined by Western blot. 2.The effect of PTK inhibitor (genistein) on proliferation and hypertrophy of SHR-VSMC induced by PDGF-AA was observed by Western blot and incorporation. RESULTS: 1.PDGF-AA and PDGFR-? expression markedly increased in SHR-VSMC compared with WKY-VSMC. There was no difference in PDGFR-? expression levels between SHR and WKY-VSMC. 2.Dose-dependent PDGF-AA-stimulated PCNA expression and incorporation were observed in SHR-VSMC. 3. Genistein inhibited PCNA expression and incorporation induced by PDGF-AA in a dose-dependent manner in SHR-VSMC. CONCLUSIONS: Spontaneous increasing expression of PDGF-AA and PDGFR-? in spontaneously hypertension rats(SHRs) may be one of the important factors in vascular reactivity and vascular modeling mediated through proliferation and hypertrophy in SHR-VSMC. Tyrosine protein kinase plays an important role in this process.

Objective To built the reference range of peripheral blood T-lymphocyte subsets including CD3+ ,CD4+ ,CD8+ and CD4+ /CD8+ ratio in healthy adults of Ugyur and Han nationalities and to provide basis for the diagnosis ,therapy and prognosis of disease .Methods A total of 181 blood samples were collected from healthy adults .The cell chip quantitative detection technology was used to detect CD3+ ,CD4+ and CD8+ ,CD3+ ,CD4+ absolute value ,CD8+ and CD4+ /CD8+ ratio were compared between Ugyur and Han nationalities .Results CD8+ absolute counting and CD4+ /CD8+ ratio had no significant difference between Ugyur and Han nationalities(P>0 .05) ,while the CD3+ ,CD4+ absolute counting had significant difference(P<0 .05) .Conclusion The discrepancy of territory and living environment should be taken into account for building a reference values of CD4+ .

Objective To study the rule of Apaf-1 in wnt/β-catenin signaling pathway and its regulatory expression mechanism in hepatocellular carcinoma cells.Methods The regulatory mechanism of Apaf-1 gene in Wnt/β-catenin signaling pathway was verified by the TOPflash experiment;real-time PCR was used to detect the expression amounts of Apaf-1 in various hepatoma cell lines (HepG2,H HCC,HB611) and normal liver cell line(LO2);the RNAi interference plasmid of Apaf-1 was constructed and transfected into the HepG2 cell line.Then the transfection efficiency and expression of related genes and proteins were detected.Results The TOPFlash experiment found that Apaf-1 gene could inhibit the wnt/β-catenin signal pathway in a dose dependent manner;the Apaf-1 expression level in hepatoma cells was decreased compared with the normal liver cells,moreover its expression level was lowest in HepG2 cell line;RNA interfering Apaf-1 gene in HepG2 cell line,the expression level of Apaf-1 gene was significantly decreased,and the expression levels of downstream genes and protein(β-catenin,Cyclin A,CDK2,wnt5a,STAT3,EGFR,APC) in wnt signal pathway were significantly increased or decreased,the difference was statistically significant(P＜0.05).Conclusion Apaf-1 gene plays an important role in the formation process of hepatocarcinoma cells by wnt/β-catenin signaling pathway.

OBJECTIVETo develop a method of quantitative analysis of multi-components by single marker (QAMS) for simultaneously determining paeoniflorin and albiflorin in Paeoniae Radix Alba.

METHODUsing paeoniflorin as the internal reference substance, the relative correction factor (RCF) of albiflorin was determined by HPLC and UPLC with good reproducibility. The contents of paeoniflorin in 16 samples of Paeoniae Radix Alba were authentically determined by the external standard method, and the content of albiflorin was calculated according to the RCF. The contents of these two components in the samples were determined with the external standard method.

RESULTNo siginificant differences between the quantitative results of QAMS method and external standard method were observe.

CONCLUSIONIt is a convenient and accurate method to determine multi-components when some authentic standard substances were unavailable. It can be used to control the quality of Paeoniae Radix Alba

Benzoates ; analysis ; Bridged-Ring Compounds ; analysis ; Chromatography, High Pressure Liquid ; methods ; Glucosides ; analysis ; Monoterpenes ; Paeonia ; chemistry

Objective To investigate the diagnostic value of detection of protein SP70 in differentiating benign and malignant pleural effusion.Methods A case-control study was conducted from July 2011 to February 2012.108 cases of pleural effusion from patients with clinically proven lung cancers and 122 cases of benign pleural effusion were collected.SP70 was detected by Sandwich ELISA,while CEA,CYFRA21-1,NSE were measured by electrochemiluminescence immunoassay for comparison.Meanwhile,protein SP70 on exfoliated cells in pleural effusion was detected by direct immunofluorescence,and was compared with the results of HE staining.The differences between the groups were evaluated by the chisquare test Fisher' s exact test.Results Positive rates of SP70,CEA,CYFRA21-1,NSE were 72.2％,58.3％,52.8％ and 30.6％ in malignant pleural effusion,obviously higher than benign pleural effusio (9.8％,13.1％,23.0％ and 19.7％).The specificity of SP70,CEA,CYFRA21-1,NSE were 90.2％,86.9％,77.0％ and 80.3％,NSCLC had significantly higher positive rate than SCLC(74.3％ ＞0.0％,P =0.02 ＜ 0.05),detection of protein SP70 in malignant pleural effusion had significantly higher coincidence rate than HE staining(72.2％ vs 47.2％,x2 =14.03,P ＜ 0.05).Conclusion Determination of the protein SP70 in pleural effusion and in exfoliated cells,can improve the sensitivity and specificity of the diagnosis of malignant pleural effusion.

OBJECTIVETo establish a HPLC method of characteristic chromatographic profile for the quality control of Paeoniae Radix.

METHODThe 67 batches of samples were analyzed on a Polaris C18-A column with a gradient elution of acetonitrile and phosphate solution (pH 3.0) at a flow rate of 1.0 mL x min(-1) and detected at 230 nm.

RESULTNine main marker peaks were identified and semi-quantificated. By the similarity evaluation software for chromatographic fingerprint of traditional Chinese medicine (Version 2004A) and hierarchical clustering analysis, 67 batches of Paeoniae Radix were classified.

CONCLUSIONThe method can be applied for quality assessment of Paeoniae Radix.

Chromatography, High Pressure Liquid ; methods ; Paeonia ; chemistry ; Quality Control

Objective To investigate the expression level of circular RNA circ-TNRC6A in bladder cancer tissues and its mechanism of regulating the proliferation and migration of bladder cancer cells.Methods The expression level of circ-TNRC6A in bladder cancer tissues and its relationship with clinical stage of patients with bladder cancer were analyzed using the Cancer Genome Atlas database.The expression levels of circ-TNRC6A in human normal bladder epithelial cell SV-HUC-1 and bladder cancer cell lines(MGH-U3,5637,RT-4,T24,J82)were analyzed by real-time fluorescence quantitative PCR(qPCR).The circ-TNRC6A plas-mid(circ-TNRC6A group)and the control plasmid(NC group)were transfected into 5637 bladder cancer cells,respectively.The effects of circ-TNRC6A on the proliferation and migration of bladder cancer cells were detected by colony formation assay and cell scratch assay,respectively.The targeting relationship between circ-TNRC6A and microRNA(miR)-494-3p was predicted by bioinformatics technology and confirmed by lu-ciferase reporter gene assay.qPCR was used to detect the effect of circ-TNRC6A on miR-494-3p expression.Western blot was used to detect the effect of circ-TNRC6A on the expression of key proteins in Wnt/β-catenin signaling pathway.Results circ-TNRC6A was down-regulated in bladder cancer tissues compared with adja-cent tissues(P＜0.01).The expression level of circ-TNRC6A was correlated with the clinical stage of bladder cancer(P＜0.05).Compared with SV-HUC-1 cells,the expression of circ-TNRC6A was lower in bladder cancer cell lines(all P＜0.05),and the expression level of circ-TNRC6A was the lowest in 5637 cells(P＜0.01).Compared with the NC group,overexpression of circ-TNRC6A inhibited the proliferation of 5637 cells(P＜0.01)and reduced the migration ability of 5637 cells(P＜0.01).circ-TNRC6A could target miR-494-3p(P＜0.01).Compared with NC group,overexpression of circ-TNRC6A significantly reduced the expression level of miR-494-3p(P＜0.01)and inhibited the activation of Wnt/β-catenin signaling pathway(P＜0.01).Conclusion circ-TNRC6A inhibits the proliferation and migration of bladder cancer cells by down-regulating miR-494-3p.circ-TNRC6A may be a new therapeutic target for bladder cancer.

OBJECTIVETo investigate the effect of environmental risk factors on the development of stroke.

METHODSWith the use of cold-stimuli plus high-salt intake as environmental risk factors, a hypertension model with the complication of stroke was established in rats, then, a new technique, suppression subtractive hybridization (SSH), was used to identify the differential genes which specifically expressed in total cerebrum tissue of rat in each group. Comparison was made between control group and stroke group.

RESULTSBy the application of SSH, a total of 576 clones were generated in this study from two subtractive libraries, among them 456 clones were usable and were analyzed. Genes for cell/organs defense were down-regulated in stroke group and metabolism transcripts were shown to be up-regulated (P<0.01).

CONCLUSIONCell/organs defense genes may play important roles in the development of stroke. The above findings suggested that environmental risk factors could genetically alter individual sensitivity to stroke.

Animals ; Brain ; metabolism ; pathology ; Cell Division ; genetics ; Cold Temperature ; DNA, Complementary ; chemistry ; genetics ; Expressed Sequence Tags ; Gene Expression Regulation ; drug effects ; Immunity, Innate ; genetics ; Male ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Rats ; Rats, Wistar ; Sequence Analysis, DNA ; Signal Transduction ; genetics ; Sodium Chloride, Dietary ; administration & dosage ; Stroke ; genetics