Objecave The effects of PGF2α on the reactive oxygen species generation and cardiomyocyte hypertrophy were examined in experiments on the cultured neonatal rat cardiomyocytes.It is to study the role of ROS in the signaling pathway of cardiomyocyte hypertrophy induced by PGF2α.Methods The level of intracellular ROS wag measured by the ROS-specific probe 2',7'-dichlorofluorescin diacetate (DCF-DA).Cardiomyocyte hypertrophy was determined by total protein content of the cells and the cell diameter.Results In the cardiomyocytes treated with PGF2α(1nmol/l,10nmol/l,100nmol/l),the fluorescence intensity of intracellular DCF-DA increased by 38.99%,61.76% and 93.55% respectively compared with control group(F=195.69,P＜0.01).It is indicated that PGF2α can induce intracellular ROS generation on the cultured neonatal rat cardiomyocytes in a dose-dependent manner.Compared with control group,the total protein increased by 39.51%, 69.93%and 139.06%respectively(F=74.014,P＜0.01),and the cell diameter increased by 29.02%,60.79%and 127.40%respectively(F=721.02,P＜0.01).It is indicated that PGF2α induce cardiomyocyte hypertrophy on the cultured neonatal rat cardiomyocytes in a dose-dependent manner.Conclusion PGF2α can induce intracellular ROS generation and cardiomyocyte hypertrophy on the cultured neonatal rat cardiomyocytes in a dose-dependent manner.

Objective To assess the clinical diagnostic value of left atrial function index (LAFI) for heart failure with normal left ventricular ejection fraction (HFNEF).Methods One hundred and ten patients with HFNEF were divided into 3 groups by New York Heart Association (NYHA) cardiac functional grading:NYHA Ⅱ grade group (43 cases),NYHA Ⅲ grade group (40 cases) and NYHA Ⅳ grade group (27 cases),and another 33 healthy subjects were selected as control group.The 4 groups were examined by ultrasoundcardiogram,LAFI and E/E' were calculated,and the plasma brain natriuretic peptide (BNP)levels were determined.The relation between LAFI and NYHA cardiac functional grading and the correlation between LAFI and plasma BNP,E/E' were observed.Results The worse heart function,the higher plasma BNP and E/E',and the lower LAFI,and there were statistical differences [control group:(40.9 ± 26.1) ng/L,6.4 ± 2.0,0.73 ± 0.10,NYHA Ⅱ grade group:(230.1 ± 85.9) ng/L,10.0 ± 3.7,0.46 ± 0.30,NYHA Ⅲ grade group:(398.6 ±98.7) ng/L,16.9 ±4.0,0.26 ± 0.30,NYHA Ⅳ grade group:(680.3 ± 146.6) ng/L,23.8 ± 3.9,0.17 ± 0.20,P ＜ 0.05].The correlation analysis results showed that LAFI and plasma BNP,E/E' were negatively correlated in patients with HFNEF (r =-0.868,-0.873,P ＜ 0.01).Conclusions In HFNEF patients,there is significant correlation between LAFI and left ventricular diastolic dysfunction.LAFI can well diagnose diastolic heart failure early and evaluate the diastolic function of the left ventricle.

Objective To investigate the effects and mechanisms of extract of ginkgo biloba(EGb)on the cell of cardiocyte hypertrophy induced by prostaglandin F2 alpha.Methods normal cells were used as negative control,and spontaneously PGF2? and EGb were used as experimental groups.The cells were isolated and cultured on cultured neonatal rat cardiocyte by PGF2?,cardiocyte hypertrophy was determined by the cell surface and the total protein of cells;the level of intracellular ROS measured by the fluorescence microscope.Results In cardiocyte hypertrophy,the cell surface,the level of intracellular ROS and the total protein of cells increased significantly in cutured neonatal rat cardiac treated with PGF2?.Compared with cells treated with PGF2?,EGb(40?g/ml,80?g/ml,100?g/ml)inhibited cardiocyte hypertrophy by PGF2?-induced,decreased in the cell surface by 19%,27%,33% and in the total protein of cells by 21%,39%,47% respectively(all P

AIM:To study the protective effect of anti-aging Klotho protein on human umbilical vein endothe-lial cells (HUVECs) treated with high glucose (HG).METHODS:HUVECs were cultured in vitro, and divided into PBS control group, 5.5 mmol/L glucose group, 33.3 mmol/L glucose group, 0.1 μmol/L Klotho +33.3 mmol/L glucose group, 1 μmol/L Klotho+33.3 mmol/L glucose group , and 10μmol/L Klotho+33.3 mmol/L glucose group .The viabili-ty of the HUVECs was measured by MTT assay .The content of malondialdehyde ( MDA) , and the activities of lactate de-hydrogenase (LDH), superoxide dismutase (SOD) and glutathione (GSH) in cell culture supernatants were observed . The production of reactive oxygen species ( ROS) in HUVECs was analyzed by flow cytometry .The levels of nitric oxide ( NO) , endothelin ( ET-1 ) , intercellular adhesion molecule-1 ( ICAM-1 ) in HUVEC culture medium were detected by ELISA.The protein expression of nuclear factor-kappa B (NF-κB) in the HUVECs was determined by Western blot .RE-SULTS:Compared with PBS control group , 33.3 mmol/L glucose significantly decreased the HUVEC viability , increased ROS, LDH and MDA levels , reduced the activities of SOD and GSH , decreased the NO secretion , and induced the ET-1 and ICAM-1 secretion and the protein expression of NF-κB in HUVECs.When HUVECs were treated with Klotho protein at different concentrations combined with 33.3 mmol/L glucose, the cell viability was increased significantly , the ROS, LDH and MDA levels were decreased significantly , the antioxidant SOD and GSH activities were significantly increased , the se-cretion of NO was increased , but ET-1 and ICAM-1 releases and protein expression of NF-κB were significantly reduced . CONCLUSION:Anti-aging Klotho protein promotes the viability of HUVECs treated with HG , reduces the oxidative dam-age and ROS production , and restores the normal secretory function of HUVECs , thus playing a protective role in vascular endothelial cells through reducing the protein expression of NF-κB.

AIM:To explore the effects of transforming growth factor-α( TGF-α) in the monoclonal formation , proliferation, migration and adhesiveness of human endothelial progenitor cells ( EPCs).METHODS: The isolated and cultured EPCs were treated with various concentrations of TGF-α(final concentrations of 1, 5, 10μg/L, respectively).At the same time, the PBS control and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) group (10μg/L TGF-αplus 1∶1 000 EGFR-TKI) were set.The effects of TGF-αon monoclonal formation , proliferation, migration and adhesiveness of EPCs were determined by clone formation experiment , thiazolyl blue tetrazolium bromide (MTT), EdU, Transwell and adhesion assays , respectively.The expression of epithelial growth receptor (EGFR) and vascular endothelial growth factor ( VEGF) were measured by Western blotting .RESULTS:Different concentrations of TGF-αall significantly induced the monoclonal formation , proliferation, migration and adhesiveness of EPCs (P<0.01), which were inhibited by EGFR-TKI.The results of Western blotting showed that TGF-αalso induced the expression of EGFR and VEGF with a cer-tain concentration effect ( P<0.01) .CONCLUSION:By combining with EGFR induced the expression of VEGF , TGF-αsignificantly promotes the related cell function of monoclonal formation , proliferation, migration, adhesiveness in EPCs.

Objective To construct eukaryotic expression plasmid pIRESneo3-pigment epithelium-derived factor (PEDF) and detect its transient expression in SP2/0 cells. Methods Specific primers were designed based on the mature peptide sequence of human PEDF cDNA in the GenBank. Human PEDF gene was cloned into the eukaryotic expression vector pIRESneo3. The PEDF DNA was transfected into SP2/0 with LipofectamineTM 2000. The recombinant human PEDF protein expressed in SP2/0 cell culture supernatant was identified by Western blot and enzyme-linked immunosorbent assay. The biological activity of the recombinant human PEDF was measured by 3-(4,5-dimethylthiazol-z-y1)-2,5-diphenytetrazolium bromide(MTT) method. Results PCR amplification, restriction enzyme digestion and DNA sequencing confirmed that the mature peptide sequence of human PEDF cDNA was successfully cloned into the eukaryotic expression vector pIRESneo3. And the plasmid was transfected into SP2/0 cells, which could secret PEDF. Western blot analysis showed that there was only one obvious band at the position of relative molecular weight of 50 000, and it is equivalent to the expected value. Enzyme-linked immunosorbent assay suggested that the content of PEDF began to rise after transfection, and peaked at 36 h [(0.92±0.04) μg/ml]. The proliferation of human umbilical vein endothelial cell line was significantly inhibited by supernatant after transfection of 36 h (P<0.05). Conclusions The eukaryotic expression plasmid pIRESneo3-PEDF had been successfully constructed and active human PEDF was transiently secreted, which made a foundation for further study of stable expression and purification of PEDF. This protein could be a potential medication for preventing and managing retinopathy of prematurity.

Objective To summarize the nursing key points of one patient of external ankle foot of gout patients with purulent infection which caused by gout stone burst in order to provide reference for the similar case. Methods Using silver dressing for local treatment of gout during wound, on the basis of control the basic diseases, local wound treatment focus was mainly reflected in: infection control, fluid management and wound debridement. Results After 86 days of treatment. The wound had already healed. Conclusions Rational use of silver dressing gout can promote wound healing and relieve the pain of patients.

Objective To explore the role of pancreatic cancer-derived microvesicles (MV) and their enclosed microRNAs (miRNA) in the pathogenesis of pancreatic cancer induced diabetes mellitus (DM).Methods The supernatants of three pancreatic cancer cell lines SW1990, BxPC3 and PANC1 were collected, and MV were isolated with gradient centrifugation.The entrance of MV into pancreatic islet cell line MIN6 was proved by Western blot assay and fluorescence-label method.The miRNA-19a levels were measured in MV and MV-free supernatants of three pancreatic cancer cells lines.The three experimental groups were MIN6 cells separately treated by MV derive from SW1990, BxPC3 and PANC1, and untreated MIN6 cells were assigned to the control group.The miRNA-19a levels as well as changes of glucose stimulated insulin secretion (GSIS) were measured.Afterward, pre-miRNA-19a and anti-miRNA-19a were transfected into MIN6 cells by liposome, and the effects of them on GSIS were observed.Results CD63 and AGO2 as the protein markers of MV and the entrance of MV from pancreatic cancer into pancreatic islet cell line MIN6 were detected by Western blotting.The miRNA-19a levels in MV and MV-free supernatants of SW1990, BxPC3 and PANC1 were (132.7±16.0), (32.8±4.3), (78.4±8.9),(22.6±3.3), (63.3±12.0) and (23.3±3.3) pmol/L, respectively, and the differences were statistically significant (t=10.44, 10.12 and 5.56,all P＜0.01).Compared to the MIN6 control group, the miRNA-19a levels of MIN6 treated by MV from SW1990, BxPC3 and PANC1 significantly increased, and the 2-△△Ctvalue was 2.02 ± 0.50, 1.80 ± 0.41 and 2.11 ± 0.59, respectively, and the differences were statistically significant (t=2.97, 2.77, 2.84;all P＜0.05).Stimulated with high glucose, the GSIS of pancreatic islet cells treated by SW1990, BxPC3 and PANC1 in three groups decreased, which were (103.73±16.49), (141.17±11.26), and (138.24±13.97) ng · mg protein-1 · h-1 MV, respectively, and that of control group was (256.24 ± 33.05) ng · mg protein-1 · h-1.The differences were statistically significant (t=4.13, 3.30 and 3.29, all P＜0.05).Compared with control group, GSIS of pre-miRNA-19a treated MIN6 remarkably decreased, which was (126.17± 62.87) ng · mg protein-1 ·h-1 and (316.72±91.87) ng · mg protein-1 · h-1 , and the difference was statistically significant (t=2.97, P＜0.05).GSIS of MIN6 cells transfected with anti-miRNA-19a was higher than that of control group, which was (697.47±77.62) ng · mg protein 1 · h-1 and (355.33 ±84.77) ng · mg protein-1 ·h-1 , and the difference was statistically significant (t=-2.97,P＜0.05).Conclusion The entrance of MV derived from pancreatic cancer into pancreatic islet cell line MIN6 may cause the dysfunction of insulin secretion an important signaling molecules, miRNA-19a.

[ ABSTRACT] AIM:To study the effects of anti-aging Klotho protein on neonatal rat myocardial cells with hypo-xia/reoxygenation ( H/R) injury.METHODS:The cardiomyocytes of neonatal SD rats were cultured to establish hypoxia/reoxygenation model.The myocardial cells were divided into normal control group, H/R model group, different concentra-tions of Klotho protein (0.1μmol/L, 1μmol/L and 10μmol/L) pretreatment groups.The myocardial cells pulse frequen-cy was observed before and after H/R.The cell viability was measured by MTT assay.The leakages of LDH, CK and AST, the content of MDA and the activity of SOD were detected.The apoptotic rate of the myocardial cells was analyzed by flow cytometry.The mRNA expression of endoplasmic reticulum stress markers and apoptosis-related molecules GRP78, CRT, CHOP and caspase-12 was measured by real-time PCR.The protein levels of CHOP, caspase-12 and phosphorylated Akt in the myocardial cells were determined by Western blot.RESULTS: Compared with normal control group, the pulse fre-quency, cell viability rate and SOD activity of myocardial cells were significantly decreased, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were increased in H/R model group.The mRNA expressions of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and caspase-12 were increased, whereas p-Akt level was decreased obviously.Compared with H/R model group, the pulse frequency, cell viability rate and SOD activity were in-creased significantly, the cell apoptotic rate as well as the contents of LDH, CK, AST and MDA were decreased in Klotho pretreated group.The mRNA expression of GRP78, CRT, CHOP and caspase-12 as well as the protein levels of CHOP and
caspase-12 were decreased, while p-Akt level increased significantly.CONCLUSION:Anti-aging Klotho protein improves the myocardial cell survival and inhibits the apoptosis by increasing the resistance of the cells to oxidative stress and exces-sive endoplasmic reticulum stress response, which is related with the activation of Akt phosphorylation in H/R-injured my-cardial cells.

ObjectivesTo examine the validity of high-frequency Doppler ultrasound in identifying knees synovitis in patients with rheumatoid arthritis(RA).MethodsNinety-five consecutive patients with active RA were examined withhigh-frequency Doppler ultrasound to examine synovitis signals in knees.Synovial tissue samples of 51 patients were obtained by closed needle biopsy from knees after ultrasound examination.Serial synovial tissue sections were stained with H&E and immunohistochemical staining,and the histopathological synovitis scores were evaluated.The relationship among clinical, histopathological and ultrasound synovitis indexes was analyzed by Spearman's rank order correlation test and receiver operating characteristic curve analysis.ResultsAmong 95 RA patients,the median thickness of synovial membrane in ultrasound was 2.8 mm,the median depth of effusion was 2.7 mm; Doppler signals of synovial blood flow were detected in 82％(78/95 ) of patients and the median semiquantitive grading of synovial blood flow was 1.0.The thickness of synovial membrane and synovial blood flow at Doppler ultrasound correlated positively with histological synovitis score,hyperplasia of the lining layer,and inflammatory infiltration in sublining area (the thickness of synovial membrane:r=0.438,0.424,0.368,respectively; synovial blood flow:r=0.357,0.377,0.347,respectively; all P＜0.05).Although there was no significant difference in clinical synovitis indexes between patients with histologically low-grade and high-grade synovitis,the thickness of synovial membrane and synovial blood flow in ultrasound in patients with histologically high-grade synovitis was significantly higher than those with low-grade synovitis(P=0.001,0.036,respectively).When the thickness of synovial membrane in ultrasound was ≥ 3.9 mm,the specificity of diagnosing the high-grade synovitis was 96.7％ and the sensitivity was 61.9％.ConclusionSynovitis signals at high-frequency Doppler ultrasound correlate with histopathological synovitis,and it might be helpful in evaluating the severity of histopathological synovitis.