OBJECTIVETo establish and evaluate a BA-ELISA method for the quantitative detection of cystic fibrosis transmembrane conductance regulator (CFTR) protein.

METHODSWe deliberately selected three tables of CFTR and made the synthetic peptide be expressed in E. coli, then used the antigen to immunize rabbits to obtain the anti-CFTR polyclonal serum. After that, 96 well plates were coated with the purified antibody against CFTR. The antigen CFTR which was extracted from human sperm was detected by anti-CFTR antibody labeled with biotin, horseradish peroxidase conjugated avidin, and the substrate. The concentrations of two kinds of antibodies and the experiment parameters were optimized. Thereby, the double antibody sandwich BA-ELISA method for the quantitative detection of CFTR protein was established. Furthermore, the reproducibility, specificity and so on were evaluated by clinical specimens of sperm.

RESULTSThe optimal concentration of coated anti-CFTR IgG was 4 µg/ml, while the biotin labeled anti-CFTR IgG was 10 µg/ml; the optimal blocking buffer was 1% BSA-PBST, the optimal time of the reaction between antigen and antibody was 60 min, the optimal chromogenic time was 15 min, the intra-assay and inter-assay coefficient were 2.16%-9.23% and 2.29%-11.71% respectively; The lowest detectable limit was 0.15 ng/ml; the standard curve had a good linear correlation of R2 = 0.962.

CONCLUSIONThe BA-ELISA method for the quantitative detection of CTFR protein is successfully established, and it is demonstrated that the method has strong specificity, high sensitivity and good reproducibility. It provides the basis and evidence of the further application of the method.

Animals ; Antibodies ; Cystic Fibrosis Transmembrane Conductance Regulator ; analysis ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; Humans ; Peptides ; Rabbits ; Reproducibility of Results ; Sensitivity and Specificity

Animals ; Cold Temperature ; adverse effects ; Heart ; physiopathology ; Inflammation ; physiopathology ; Myocardium ; metabolism ; Oxidative Stress ; Particulate Matter ; adverse effects ; Rats

OBJECTIVETo investigate whether Resolvin E1 (RvE1) could protect against ox-LDL-induced injury on human vein vascular endothelial cells and reveal related molecular mechanisms.

METHODSHuman vein vascular endothelial cells were randomly assigned to six groups, which were treated with saline, RvE1, wortmanin, ox-LDL, ox-LDL and RvE1, ox-LDL and RvE1 and wortmanin, respectively. After 48 h, survival rates were determined by MTT, apoptosis rate of cells were determined by flow cytometry, TNF-α contents were assayed by ELISA, caspase 3 and 9 activities were measured by microplate reader, and the expression of p-AKT and LOX-1 were determined by Western blot.

RESULTCompared with normal saline group, survival rate was markedly decreased and apoptosis rate, TNF-α content, caspase 3 and 9 activities, and the expression of LOX-1 were significantly increased in ox-LDL group (P < 0.01). Survival rate was significantly increased and apoptosis rate, TNF-α content, caspase 3 and 9 activities, and the expression of LOX-1 were significantly decreased in ox-LDL + RvE1 group compared to ox-LDL group (P < 0.01), these beneficial effects of RvE1 could be blocked by PI3K inhibitor wortmanin (P < 0.05).

CONCLUSIONThe present data showed that RvE1 could effectively protect against ox-LDL-induced endothelial cell injury, which might be mediated by PI3K-AKT signaling pathway.

Apoptosis ; drug effects ; Cells, Cultured ; Eicosapentaenoic Acid ; analogs & derivatives ; pharmacology ; Endothelial Cells ; drug effects ; metabolism ; Humans ; Lipoproteins, LDL ; adverse effects ; Signal Transduction ; Umbilical Veins ; cytology

To investigate the mechanisms underlying the cholinergic agonist carbachol-induced cardiovascular responses, changes of renin-angiotensin system were examined in fetal hormonal systems. In the ovine fetal model under stressless condition, the cardiovascular function was recorded. Blood samples were collected before (during baseline period) and after the intravenous administration of carbachol. Simultaneously, the levels of angiotensin I (Ang I), angiotensin II (Ang II) and vasopressin in the fetal plasma were detected by immunoradiological method. Also, blood gas, plasma osmolality and electrolyte concentrations were analyzed in blood samples. Results showed that in chronically prepared ovine fetus, intravenous infusion of carbachol led to a significant decrease of heart rate (P < 0.05), and a transient decrease followed by an increase of blood pressure (P < 0.05) within 30 min. After the intravenous infusion of carbachol, blood concentrations of Ang I and Ang II in near-term ovine fetus were both significantly increased (P < 0.05); however, blood concentration of vasopressin, values of blood gas, electrolytes and plasma osmolality in near-term ovine fetus were not significantly changed (P > 0.05). Blood levels of Ang I and Ang II in the atropine (M receptor antagonist) + carbachol intravenous administration group was lower than those in the carbachol group without atropine administration (P < 0.05). In conclusion, this study indicates that the near-term changes of cardiovascular system induced by intravenous administration of carbachol in ovine fetus, such as blood pressure and heart rate, are associated with the changes of hormones of circulatory renin-angiotensin system.
Angiotensin I ; blood ; Angiotensin II ; blood ; Animals ; Blood Pressure ; Carbachol ; pharmacology ; Cholinergic Agonists ; pharmacology ; Fetus ; Heart Rate ; Renin-Angiotensin System ; Sheep ; Vasopressins ; blood

The rhizome of Panax japonicus var. major have been used as the natural medicinal agent by Chinese traditional doctors for more than thousand years. Most of the therapeutic effects of P. japonicus var. major had been reported due to the presence of tetracyclic or pentacyclic triterpene saponins. In this study, Illumina pair-end RNA-sequencing and de novo splicing were done in order to understand the pathway of triterpenoid saponins in this species. The valid reads data of 15. 6 Gb were obtained. The 62 240 unigenes were finally obtained by de novo splicing. After annotation, we discovered 19 unigenes involved in ginsenoside backbone biosynthesis. Additionally, 69 unigenes and 18 unigenes were predicted to have potential function of cytochrome P450 and UDP-glycosyltransferase based on the annotation results, which may encode enzymes responsible for ginsenoside backbone modification. This study provides global expressed datas for P. japonicus var. major, which will contribute significantly to further genome-wide research and analysis for this species.
Gene Expression Profiling ; Panax ; genetics ; Saponins ; biosynthesis ; Sequence Analysis, RNA

OBJECTIVETo evaluate the prognostic factors in treating primary liver cancer (PLC) patients by Pi-strengthening and qi-regulating method (PSQRM), thus providing evidence and optimizing Pi-strengthening and qi-regulating program.

METHODSClinical data of 151 PLC patients treated by PSQRM at Oncology Department, Guangdong Provincial Hospital of Traditional Chinese Medicine from May 2007 to March 2009 were retrospectively analyzed. The univariate analysis was determined to analyze possible prognostic factors. Selected key factors were introduced into the COX proportional hazard model, and multivariate analysis was carried out.

RESULTSThe 1-year survival rate was 21.85%, the median survival time was 6.80 months, and the mean survival time was 8.98 months. The univariate analysis showed that Chinese medicine (CM) syndrome types, clinical symptoms at the initial diagnosis, ascites, tumor types, ratios of foci, portal vein tumor thrombus, intrahepatic metastasis, a-fetoprotein (AFP) levels, total bilirubin classification, albumin classification, Child-Pugh classification, and domestic staging of liver cancer were significant prognostic factors (P < 0.05). The statistic data of multivariate analysis indicated that CM syndrome types, ascites, tumor types, portal vein tumor thrombus, AFP levels, Child-Pugh classification, and domestic staging of liver cancer were independent factors influencing prognosis (P < 0.05).

CONCLUSIONThe prognosis of PLC treated with PSQRM is determined by multiple factors including CM syndrome types, ascites, tumor types, portal vein tumor thrombus, AFP levels, Child-Pugh classification, and domestic staging of liver cancer.

Adult ; Aged ; Aged, 80 and over ; Carcinoma, Hepatocellular ; epidemiology ; therapy ; Female ; Humans ; Liver Neoplasms ; epidemiology ; therapy ; Male ; Medicine, Chinese Traditional ; methods ; Middle Aged ; Multivariate Analysis ; Prognosis ; Retrospective Studies ; Survival Rate ; Treatment Outcome

OBJECTIVETo study the effects of the extensive resection of the tumor-loading segment and artificial humerus head replacement combined with perioperative rehabilitation for the treatment of stage II to III giant cell tumor of bone in the proximal humerus.

METHODSFrom March 2007 to March 2010, 7 patients with stage II to III giant cell tumor of bone in the proximal humerus were treated. Among the patients, 3 patients were male and 4 patients were female with a mean age of 34.6 years (ranged, 18 to 49 years). The mean course of disease was 19 months (ranged, 6 to 35 months). All the patients were confirmed to suffer stage II to III giant cell tumor of bone in the proximal humerus by pathology and X-ray examinations. Clinical manifestations of the patients included persistence aggravated pain of the shoulder, swelling in the proximate arm with obviously tenderness, activity limited of the joint. All the patients were treated with extensive resection of the tumor-loading segment and artificial humerus head replacement combined with perioperative rehabilitation. CMS and OSIS score system were used to evaluate shoulder function and shoulder stability.

RESULTSAll the patients were followed up, and the duration ranged from 14 to 35 months, with an average of 17 months. There were no serious complications or recurrence in all cases. One year after the surgery CMS and OSIS score system were 70.7 scores (ranged,63 to 82 scores) and 25.1 scores (ranged, 18 to 29 scores) respectively. According to evaluation for shoulder function, 2 patients got an excellent result and 5 good. According to evaluation of shoulder stability, 1 patient got an excellent result and 6 good.

CONCLUSIONExtensive resection of the tumor-loading segment and artificial humerus head replacement combined with perioperative rehabilitation for the treatment of stage II to III giant cell tumor of bone in the proximal humerus would not only preserve the upper extremity but also preserve the function of upper extremity.

Adolescent ; Adult ; Bone Neoplasms ; pathology ; rehabilitation ; surgery ; Female ; Giant Cell Tumor of Bone ; pathology ; rehabilitation ; surgery ; Humans ; Humerus ; surgery ; Limb Salvage ; Male ; Middle Aged ; Neoplasm Staging

OBJECTIVETo investigate the expression of cyclin E in rectal carcinoma and its prognostic significance.

METHODSCyclin E expression was examined by Western blotting in tumor tissue samples from 130 potentially resected rectal cancer patients with pathological stages I- III. Blood vessel invasion (BVI) was detected by immunohistochemistry. Multivariate analysis using the COX proportional hazards models was applied to evaluate the independent prognostic tumor markers of rectal cancer.

RESULTSThe high expression rate of cyclin E in rectal carcinoma tissue was 23.1%(30/130). Except for a positive correlation with BVI and the gross configuration of tumor, the expression of cyclin E showed no significant relation to other clinicopathological factors. The 5-year disease-specific survival rate of cyclin E high expression group was 29.2%, which was significantly lower as compared to that of cyclin low expression group 70.5% (P<0.05). Multivariate analysis revealed that histology and cyclin E expression were independent prognostic indicators for rectal cancer patients at stages I- III. Compared to those with low expression levels, patients with high cyclin E levels had the hazard ratio (95%CI) for death from rectal cancer for 3.544 (1.528-8.215). In stage I- II, multivariate analysis showed that stronger predictive values of cyclin E expression even were detected. Patients with low cyclin E expression and negative BVI had a significantly better prognosis than those with high cyclin E expression and positive BVI.

CONCLUSIONSThe expression of cyclin E is independent prognostic factor in rectal carcinoma at stages I- III. Detecting the expression of cyclin E and/or combined with BVI may help to predict clinical outcome and design further individualized intensive adjuvant treatment.

Adult ; Aged ; Aged, 80 and over ; Cyclin E ; metabolism ; Female ; Humans ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Staging ; Neovascularization, Pathologic ; Prognosis ; Rectal Neoplasms ; blood supply ; metabolism ; pathology ; Survival Rate

OBJECTIVETo explore the role of SHP-1 promoter methylation on the pathogenesis and progression in myelodysplastic syndromes (MDS) and its related mechanism.

METHODS63 MDS patients were divided into low-grade (LG) group and high-grade (HG) group according to IPSS score system. Bone marrow samples were collected. Methylation specific-PCR (MSP) were used to detect the status of SHP-1 promoter methylation in bone marrow (BM) samples from different risk MDS patients and MDS cell line, SKK-1. Western blot was used to detect signal transduction and activator of transcription (STAT3) activation in SKK-1 cell line and MDS patients.

RESULTSNo SHP-1 promoter methylation could be detected in healthy controls BM. Partially methylation was found in SKK-1 cell line. Methylation rate of SHP-1 gene promoter was found in BM of 24.2% of low-grade MDS patients and 63.3% of high-grade MDS patients, the difference between these two groups was statistically significant (P < 0.05); Patients were divided into different groups according to WHO subtype, chromosomal karyotype and blast cells in bone marrow, methylation rates of SHP-1 were significantly higher in RAEB-II, poor karyotype group and samples with 0.11-0.19 blast cells (P < 0.05); The phosphorylation protein of STAT3 was detected in SKK-1 cell line. The expression of phosphorylation STAT3 was significantly higher in HG group than in LG group (66.7% vs 18.2%) (P < 0.05). There was a significant correlation between SHP-1 promoter methylation and STAT3 phosphorylation.

CONCLUSIONAbnormal methylation of SHP-1 gene promoter might have tentative role in the pathogenesis and progression of MDS, which may be involved in STAT3 activation. Detection of SHP-1 promoter methylation may be helpful to evaluate the prognosis of MDS.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Child ; DNA Methylation ; Female ; Humans ; Male ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; metabolism ; Prognosis ; Protein Tyrosine Phosphatase, Non-Receptor Type 6 ; genetics ; metabolism ; STAT3 Transcription Factor ; metabolism ; Young Adult

OBJECTIVEA randomized trial of breast self-examination (BSE) Program was carried out to evaluate whether the intensive BSE could reduce the number of deaths among women from breast cancer.

METHODSThis study was a randomized controlled trial (RCT). A total of 266 064 women (age of 30 to 64 years) associated with 519 textile factories in Shanghai had been randomly assigned to a BSE group (132 979 women) or a control group (133 085 women) since 1989. Initial instruction in BSE group would include demonstration of proper palpation techniques and was followed by 2 reinforcement sessions during the subsequent 4 years including video shows, BSE instruction sessions and BSE practice under medical supervision. These activities were continued for 5 years. Attendance at all events was recorded. The cohort was followed through July 2000 for development of breast diseases, and the breast cancer cases were followed through 2001 for vital status. Data analysis methods used would include Kaplan-Meier plots, log-rank test and Cox modeling.

RESULTSAmong women under instruction, 864 breast cancers detected and 133 breast cancer deaths occurred while 896 breast cancers were detected and 130 deaths recorded in the control group. The tumor size (P = 0.07), TNM stage (P = 0.39) and cumulative breast cancer mortality rate (P = 0.72) were not significantly different between the 2 groups. However, more and smaller fibroadenomas were detected in the instruction group than in the control group (P< 0.01).

CONCLUSIONIntensive instruction in BSE did not seem to have reduced the mortality rate of breast cancer, but more and smaller benign breast lumps could be detected.

Adult ; Breast Diseases ; diagnosis ; Breast Neoplasms ; diagnosis ; mortality ; Breast Self-Examination ; China ; Female ; Humans ; Middle Aged ; Patient Education as Topic