Background The proliferation and migration of vascular endothelial cells is a primary link during angiogenesis.Studies showed that heat shock protein B6 (HspB6) promotes the secretion of multiple angiogenesis-related factors and therefore leads to neovascularization.Understanding the effects of neutralizing HspB6 antibody on the biological behavior of human choroidal vascular endothelial cells has an important significance in the target treatment of choroidal neovacularization diseases.Objective This study was to address the role and mechanism of neutralizing HspB6 antibody in tube formation of human choroidal vascular endothelial cells.Methods Human choroidal vascular endothelial cell line was normally cultured and harvested for total RNA extraction.Expressions of HspB6 mRNA and protein in human choroidal vascular endothelial cells were detected by reverse transcription PCR (RT-PCR) and flow cytometry (FCM).The cells were seeded on 96-well plate covered with matrigel at the density of 2×104/hole.Then the neutralizing HspB6 antibody at the concentration of 100 μg/Land 500 μg/L was added into the medium respectively,and the control cells were set without the addition of HspB6 antibody.The number of capillary tubes was calculated 12 hours after culture by three-dimensional matrigel assay.In addition,0,50,100,500 μg/L of neutralizing HspB6 antibody were added into the cell medium separately for 24hours,cell counting kit-8 (CCK-8) method was employed to assay the inhibitory rate(IR) of the cells.Transwell test was used to count the cell number across chamber membrane for the evaluation of migration ability of the cells.The apoptosis of the cells was assayed by FCM.Results Both HspB6 mRNA and protein were expressed on human choroidal vascular endothelial cells.The number of capillary tube formation of human choroidal vascular endothelial cells was (67.25±5.75),(60.39±6.41) and (39.76±10.73) /field in the 0,100 and 500 μg/L neutralizing HspB6 antibody groups,with significant difference among them (F =10.210,P =0.012),and the tube number was significantly less in the 500 μg/L neutralizing HspB6 antibody group compared with 0 μg/L neutralizing HspB6 group (P =0.005).The IR of neutralizing HspB6 antibody to the cellular proliferation and migration was enhanced with the increases of concentration and time lapse(Fconcentration =7.485,P =0.002 ; Ftime =16.684,P =0.001).The number of the cells through Transwell chamber membrane was 14.0 ± 2.5,11.1 ± 0.8,6.6 ± 0.1,6.7 ± 0.2 in the 0,50,100,500 μg/L neutralizing HspB6 antibody group respectively,and that in the 100 μg/L and 500 μg/L neutralizing HspB6 antibody group was lessened in comparison with the 0 μg/L neutralizing HspB6 antibody group(both at P=0.000).The apoptosis rate of the cells was (22.73 ± 2.53)％ in the neutralizing HspB6 antibody group,which was significantly lower than (13.33±2.08) ％ of the control group (t=4.967,P=0.008).Conclusions Neutralizing HspB6 antibody inhibits capillary tube formation of human choroidal endothelial cells in vitro in dose-and timedependent manner,probably through suppressing the proliferation and migration and promoting the apoptosis of choroidal endothelial cells.

AIMTo identify the factors influencing diagnosis and treatment of chronic prostatitis (CP) among Chinese urologists.

METHODSA sample of 656 urologists from 29 provinces of China were asked to complete a questionnaire that explored attitudes towards CP as well as diagnosis and treatment patterns in the management of CP. Both univariate and multivariate logistic regression analysis schemes were used to determine the factors that influence the diagnosis and treatment of CP.

RESULTSA total of 656 questionnaires were given out. All were returned and 410 of those were included in the final univariate and multivariate analysis. Multivariate logistic regression analysis indicated that belief of bacterial infection in the etiology of CP (odds ratio [OR], 2.544; 95% confidence interval [CI], 1.650-3.923; P < 0.001) was the most significant factor influencing the routine performance of bacterial culture test. Using the same model, the type of hospital (OR, 2.799; 95% CI, 1.719-4.559; P < 0.001) and the routine use of the 4- or the 2-glass test (OR, 3.194; 95% CI, 2.069-4.931; P < 0.001) were determined to be significant factors influencing the use of the National Institutes of Health (NIH) new classification system. According to the same model, belief of bacterial infection in the etiology of CP (OR, 3.415; 95% CI, 2.024-5.762; P < 0.001) and the routine use of bacterial culture test (OR, 2.261; 95% CI, 1.364-3.749; P < 0.01) were important factors influencing the routine prescription of antibiotics.

CONCLUSIONOur findings suggest that attitudes towards CP, and the characteristics of individual urologists' practices may influence the diagnosis and treatment of CP among Chinese urologists.

Adult ; Aged ; Anti-Bacterial Agents ; therapeutic use ; China ; Chronic Disease ; Cross-Sectional Studies ; Health Knowledge, Attitudes, Practice ; Health Surveys ; Humans ; Logistic Models ; Male ; Middle Aged ; Multivariate Analysis ; Practice Patterns, Physicians' ; Prostatitis ; diagnosis ; drug therapy ; microbiology ; Surveys and Questionnaires

OBJECTIVE: To localize the gene of autosomal dominant familial dilated cardiomyopathy with conduction defect. METHODS: A Chinese family which was diagnosed as dilated cardiomyopathy with conduction defect was studied. Venous blood (3 - 5 mL) from some family members was collected, and genomic DNA was extracted from the blood. Then whole genome wide scan was performed after excluding the known markers on the candidate loci (CMD1A, CMD1 E, CMD1F, and CMD1H) by two-point linkage analysis. RESULTS: No significant evidence for linkage was found in the two point linkage analyses to the known markers in the analyzed family. And the whole genome wide scan showed the maximum LOD score reached 2.68 at marker D3S1614 ( at recombination fraction theta = 0). CONCLUSION The related gene in this kindred is located on 3q26 other than on CMD1A, CMD1H, CMD1E, and CMD1F.
Adult ; Arrhythmias, Cardiac ; etiology ; genetics ; Cardiomyopathy, Dilated ; genetics ; Chromosomes, Human, Pair 3 ; genetics ; Female ; Genetic Linkage ; Humans ; Male ; Microsatellite Repeats ; Middle Aged ; Pedigree

OBJECTIVETo isolate and characterize human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLSs).

METHODSThe synovial membrane tissues were obtained from 4 RA patients, 1 chondroma patient and 1 healthy subject and FLS were isolated by means of tissue culture. The cell morphology was observed by phase-contrast microscope and the cell surface markers were detected by flow cytometry.

RESULTSThe FLSs were successfully cultured from the synovial membrane tissues with good cell homogeneity after the third passage. The FLSs of the 3rd to 7th passages were stable and proliferated actively, followed by slow proliferation and aging since the 8th passage. Flow cytometry showed that the 4th-passage FLSs from the RA patients contained 99.04% CD90(+) cells, 2.73% CD3(+) cells, 0.29% CD3(-)CD19(+) cells, 2.81% CD3(-)CD16(+)CD56(+) cells, 5.89% CD14(+) cells, and 54.17% CD55(+) cells. The presence of interleukin-1 receptor type I (IL-1RI, 158.63-/+20.32 pg/ml) and IL-1beta (4.67-/+0.82 pg/ml) were detected in the cell culture supernatant of the 4th-passage FLSs from the RA patients by enzyme-linked immunosorbent assay ELISA.

CONCLUSIONFLSs from RA patients can be effectively culture by means of tissue culture, and the cultured FLSs show high expressions of CD90, IL-1RI and IL-1beta.

Adult ; Aged ; Arthritis, Rheumatoid ; pathology ; Cell Proliferation ; Cell Separation ; Cells, Cultured ; Female ; Fibroblasts ; pathology ; Humans ; Interleukin-1beta ; metabolism ; Male ; Middle Aged ; Receptors, Interleukin-1 Type I ; metabolism ; Synovial Membrane ; cytology ; pathology ; Thy-1 Antigens ; metabolism

Adult ; Aged ; Aged, 80 and over ; Humans ; Male ; Middle Aged ; Mouth Mucosa ; transplantation ; Reconstructive Surgical Procedures ; methods ; Surgical Flaps ; Treatment Outcome ; Urethral Stricture ; surgery ; Young Adult

Although 5-aminolevulinic acid (5-ALA)-mediated photodynamic therapy (PDT) has been demonstrated to be a novel and effective therapeutic modality for some human malignancies, its effect and mechanism on glioma are still controversial. Previous studies have reported that 5-ALA-PDT induced necrosis of C6 rat glioma cells in vitro. The aim of this study was to further investigate the effect and mechanism of 5-ALA-PDT on C6 gliomas implanted in rats in vivo. Twenty-four rats bearing similar size of subcutaneously implanted C6 rat glioma were randomly divided into 3 groups: receiving 5-ALA-PDT (group A), laser irradiation (group B), and mock procedures but without any treatment (group C), respectively. The growth, histology, microvessel density (MVD), and apoptosis of the grafts in each group were determined after the treatments. As compared with groups B and C, the volume of tumor grafts was significantly reduced (P<0.05), MVD was significantly decreased (P<0.001), and the cellular necrosis was obviously increased in group A. There was no significant difference in apoptosis among the three groups. The in vivo studies confirmed that 5-ALA-PDT may be an effective treatment for gliomas by inhibiting the tumor growth. The mechanism underlying may involve increasing the cellular necrosis but not inducing the cellular apoptosis, which may result from the destruction of the tumor microvessels.
Aminolevulinic Acid ; pharmacology ; therapeutic use ; Animals ; Brain Neoplasms ; blood supply ; drug therapy ; pathology ; Cell Line, Tumor ; Glioma ; blood supply ; drug therapy ; pathology ; Microvessels ; drug effects ; Photochemotherapy ; Photosensitizing Agents ; pharmacology ; therapeutic use ; Rats ; Rats, Wistar ; Xenograft Model Antitumor Assays

OBJECTIVETo study the predominant calcium-antagonist components of Danshen injection.

METHODThe effects of danshensu, protocatechualdehyde and Danshen injection on calcium concentration in cytoplasm of erythrocytes were examined in vitro by the fluorescent Ca+ -chelator fura-2.

RESULTEither DS182 or PCAD can decrease in dose-dependent cytosolic free calcium concentration in human erythrocytes. They had additive effect when mixed, which was similar to Danshen injection.

CONCLUSIONDS182 and PCAD may be predominant calcium-antagonist components of Danshen injection.

Adult ; Benzaldehydes ; isolation & purification ; pharmacology ; Calcium ; metabolism ; Catechols ; isolation & purification ; pharmacology ; Cytoplasm ; metabolism ; Drug Synergism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Erythrocytes ; metabolism ; Female ; Humans ; Injections ; Lactates ; isolation & purification ; pharmacology ; Male ; Middle Aged ; Plants, Medicinal ; chemistry ; Salvia miltiorrhiza ; chemistry

OBJECTIVE: To construct the recombinant lentivirus RNAi vector, and to determine whether the lentivirus mediated short hairpin RNA (shRNA) can inhibit the tissue factor (TF) expression in endothelial cells. METHODS: Two short hairpin RNAs targeting to human TF were cloned into pENTRTM/U6 plasmid to obtain an entry clone, and the positive clones were verified by sequencing. A recombination reaction was performed between the pENTR/U6 entry construction and pLenti6/BLOCKiTTM-DEST vector, and then the positive clones were confirmed by sequencing. The 293FT cell line was transfected by the above recombined plasmid and lentivirus packing materials, the culture supernatant was harvested, and the virus titer was determined. RT-PCR and ELISA were used to observe the inhibition of TF gene expression after the lentivirus transduction in human umbilical vein endothelial cells. RESULTS: The shRNA sequences targeting to human TF were cloned into the vectors, and an entry clone and an expression clone were constructed successfully, which were proved by sequence determination. Viral particles were packaged in the 293FT cell line, all virus stocks were collected, and the transfection titer was 5*10(5)/transduced unit. RT-PCR and enzyme linked immunosorbent assay demonstrated that the lentivirus stocks could suppress the TF expression in endothelial cells remarkably. CONCLUSION Lentivirus RNAi vectors containing human TF gene are successfully constructed, and lentivirus mediated shRNA can inhibit the TF expression in endothelial cells, which may provide a highly effective method for the prevention and treatment of thrombo-embolic diseases.
Base Sequence ; Down-Regulation ; Endothelial Cells ; cytology ; metabolism ; Genetic Vectors ; genetics ; Humans ; Lentivirus ; genetics ; Molecular Sequence Data ; RNA Interference ; RNA, Messenger ; biosynthesis ; genetics ; RNA, Small Interfering ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Thromboplastin ; biosynthesis ; genetics ; Umbilical Veins ; cytology

OBJECTIVETo investigate the incidences of urea cycle defects (UCDs) in the patients with hyperammonemia and study their etiology, clinical and laboratory features.

METHODSIn the past 7 years, 26 cases (10.2%) of UCDs were detected from 254 patients with hyperammonemia. The etiological diagnoses were made by blood amino acids analysis, urinary organic acid analysis and blood acylcarnitine profile analysis. Three patients with citrullinemia type II were further confirmed by liver pathological analysis and gene diagnosis.

RESULTSAmong 26 cases with UCDs, 15 had ornithine transcarbamylase (OTC) deficiency, 5 had citrullinemia type I, 3 had citrullinemia type II and 3 patients had arginemia. The age of onset of the patients ranged from 3 days to 13 years. Three cases (11.5%) developed hyperammonemic encephalopathy during neonatal period. Thirteen (50.0%), 7 (26.9%) and 3 (11.5%) cases developed clinical symptoms at the age of 1 to 12 months, 1 to 3 years and 6 to 13 years, respectively. Positive family history was found in 11 cases (42.3%). Among 26 patients with UCDs, 9 (34.6%) were hospitalized with the complains of seizures, psychomotor retardation, vomiting and unconsciousness, 8 (30.8%) with recurrent vomiting, headache and coma, 6 due to liver dysfunction. Intrahepatic cholestatic jaundice was found in 3 patients with citrullinemia type II. Blood ammonia ranged from 58 to 259 micromol/L on their first visit to our hospital. Twenty cases (76.9%) had liver dysfunction, 4 patients (15.4%) were diagnosed postmortem. Twenty-one patients got treatment and were followed up. Among them, 7 cases died of hyperammonemic encephalopathy or upper alimentary tract bleeding. Clinical improvement was observed in 14 cases. A boy with OTC deficiency who received a partial liver transplant from his mother showed normal general condition for two years.

CONCLUSIONSUCDs are the most frequent causes of congenital hyperammonemia. In this study, 26 patients (10.2%) with UCDs were identified from 254 patients with hyperammonemia resulting in encephalopathy and liver dysfunction. Early diagnosis and treatment can contribute a lot to improve the prognosis of the patients. Blood ammonia assay and further etiological analysis should be considered in the differential diagnosis of neurological and hepatic abnormality.

Adolescent ; Ammonia ; blood ; Child ; Child, Preschool ; Female ; Follow-Up Studies ; Humans ; Hyperammonemia ; congenital ; diagnosis ; genetics ; Infant ; Infant, Newborn ; Male ; Urea ; metabolism

OBJECTIVETo discuss the effectiveness of individualized strategy of surgical management on the great arteries (TGA).

METHODSFrom March 1998 to October 2009, 127 cases (97 males and 30 females) with TGA were treated. There were 97 male and 30 female, aged from 4 hours old to 17 years old with a mean of (25 ± 37) months, weighted from 2.7 to 47.5 kg with a mean of (8 ± 8) kg. The palliative operations included Glenn operation in 14 cases (3 cases double Glenn procedure), Balalock-Taussing shunt in 14 cases, Banding operation in 8 cases, and atrial septal defect enlarge/Banding/Balalock-Taussing shunt in 15 cases. The end-stage operation included Senning procedure in 5 cases, Switch procedure in 32 cases, 2(nd)-stage Switch procedure in 11 cases, Switch procedure with VSD repairing in 20 cases, Switch procedure with Hybrid in 1 case, Nikaidoh procedure in 3 cases, Rastelli procedure in 13 cases, Fonton procedure in 18 cases, other procedure in 4 cases. Twenty-one cases underwent 2 operations, and 5 cases underwent 3 or more operations. Sixty-six cases underwent delayed sternal closure.

RESULTSThere were 12 cases of death operatively in 127 cases. The total operative mortality was 9.4%. There were 5 cases dying of low cardiac output during the operation, 2 of pulmonary hypertension crisis, 2 of hemorrhage, 1 of grafting problem of coronary artery deformation, 1 of renal failure after Fonton procedure and 1 case of newborn dying of spontaneous rupture of liver post-operatively. The patients were followed up for 1 month to 12 years. There were 10 patients with vary degrees complications such as pulmonary stenosis, residual shunt and narrow channel. Three cases underwent reoperation. The rest of survived cases had normal heart function, good growth and development state.

CONCLUSIONSIndividualized strategy of surgical management based on anatomical conditions of TGA can significantly improve the success rate of surgery and long-term survival.

Adolescent ; Child ; Child, Preschool ; Female ; Follow-Up Studies ; Humans ; Infant ; Infant, Newborn ; Male ; Transposition of Great Vessels ; surgery ; Treatment Outcome