AIM:To investigate the effect of RWDD3 gene silencing on the biological characteristics of human glioma U251 cells.METHODS: A lentiviral vector expressing RWDD3 shRNA was constructed and transfeeted into the U251 cells.The expression of RWDD3 at mRNA and protein levels was detected by real-time PCR and Western blot , re-spectively .The cell activity was determined by MTT assay .The colony formation ability was detected by the colony forma-tion assay .The cell proliferation ability was detected by BrdU incorporation assay .The cell invasion and migration were evaluated by Transwell assay .Flow cytometry was used to monitor the changes of cell cycle distribution and apoptosis .RE-SULTS:Recombinant lentivirus was successfully transfected into U 251 cells.Compared with the cells transfected with the scrambled shRNA and control cells, the cell activity, colony formation ability, and the invasive and migratory activities were inhibited, the cell cycle was arrested in G 0/G1 phase, and the apoptosis was increased in the U 251 cells transfected with RWDD3 shRNA ( P <0.05 ) .CONCLUSION: RWDD3 plays a vital role in proliferation and invasion of glioma cells.It may serve as a potential target of gene therapy for glioma .

OBJECTIVETo examine the effects of the whey basic protein on bone metabolism of Sprague-Dawley (SD) rats and healthy mid-aged women.

METHODSForty-four female SD rats were randomized by weight into four groups of eleven rats each and fed 10 mg x kg BW(-1) x d(-1), 20 mg x kg BW(-1) x d(-1), 30 mg x kg BW(-1) x d(-1) of whey basic protein and control diet was given respectively by intragastrically injection for 90 days. Bone mineral density of femur was measured by dual-energy X-ray absorptiometry in vitro. Sixty-three health women [(37.9 +/- 4.3) years old] were randomly assigned to treatment with placebo, 30 mg whey basic protein per day or 60 mg whey basic protein per day for 24 weeks. The bone mineral density (BMD) of the lumbar vertebrae L2-LA, femoral neck and right calcaneus of each subject were measured by dual-energy X-ray absorptiometry (DXA) at 0 and the 24th week of treatment. Serum bone specific alkaline phosphatase and N-telopeptide (NTX) were measured at 0 and the 14th week.

RESULTSThe mean BMD value of the distal end of the femur in 10 mg x kg BW(-1) x d(-1) whey basic protein group was significantly higher than that of the control group at the end of the trail. But after treatment by doses of whey basic protein used in the study, there were no differences between the control group and others groups on bone mineral density in the human trail.

CONCLUSIONWhey basic protein should enhance the bone mineral density of the rats' femur and no obvious effect was detected in the human trail.

Adult ; Animals ; Bone Density ; drug effects ; Female ; Food, Formulated ; Humans ; Lactalbumin ; pharmacology ; Milk ; Milk Proteins ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Whey Proteins

Ten compounds were isolated from the barks of Jasminum giraldii by means of various of chromatographic techniques such as silica gel, Sephadex LH-20 and Rp-HPLC. Their structures were identified by spectroscopic data analysis as (+)-medioresinol (1), (+) -syringaresinol (2), syringaresinol-4'-O-beta-D-glucopyranoside (3), oleanic acid (4), 3-methoxy-4-hydroxy-trans-cinnamaldehyde (5), trans-sinapaldehyde (6), syringaldehyde (7), 1-(4-methoxy -phenyl) -ethanol (8), trans-cinnamic acid (9), and 4-(1-methoxyethyl) -phenol (10). Among them, compounds 1-3, 5-8 and 10 were isolated from the J. genus for the first time and compounds 4 and 9 were obtained from J. giraldii for the first time. In the DPPH free radical scavenging assay, compound 1 exhibited significant activity (IC50 55.1 micromol x L(-1)), compared with vitamin C(IC50 59.9 micromol x L(-1)); and compound 2 showed moderate activity (IC50 79.0 micromol x L(-1)), compared with 2, 6-di-tert-butyl4-methylphenol (IC50 236 micromol x L(-1)).
Antioxidants ; chemistry ; isolation & purification ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Jasminum ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization

Ten compounds were isolated from the leaf of Eucommia ulmoides by means of recrystallization and chromatographic techniques such as D-101 macroporous resin, MCI resin, ODS gel, Sephadex LH-20 and Rp-HPLC. Their structures were identified by NMR spectral analyses as kaempferide 3-O-beta-D-glucoside (1), quercetin-3-O-beta-D-glucoside (2), quercetin (3), quercetin-3-O-beta-D-xylosyl-(1-->2)-beta-D-galactoside (4), kaempferol-3-O-alpha-L-rhamnosyl-(1-->6)-beta-D-glucoside (5), (2S,3S)-taxifolin 3-O-beta-D-glucoside (6) ,4-hydroxy cinnamic acid (7), (+)-cycloolivil (8), pinoresinol beta-D-glucoside (9), squalene (10). Among them compounds 1,5-7,10 were isolated from the Eucommia genus for the first time. In the DPPH free radical scavenging assay, compound 2 exhibited significant activity (IC50 13.7 micromol x L(-1)), compared with vitamin C (IC50 59.9 micromol x L(-1)); compounds 1, 3 and 9 showed moderate activity (IC50 161,137, 214 micromol x L(-1)), compared with 2,6-di-tert-butyl-4-methylphenol (IC50 236 micromol x L(-1)); compound 4 and 6 showed weak activity (IC50 264, 299 micromol x L(-1)).
Antioxidants ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Eucommiaceae ; chemistry ; Magnetic Resonance Spectroscopy ; Molecular Structure ; Plant Leaves ; chemistry

Objective To discuss the clinical efficiency of endoscopic submucosal dissection (ESD) in treatment of early gastric cancer (EGC) and precancerous lesions. Method Clinical data of 106 patients with EGC or precancerous lesions who received the treatment of ESD from June 2012 to June 2015 was collected. Then analyzing the treatment effect, complications, postoperative pathology and long-term efficacy of ESD. Results The overall en bloc resection rate was 100.0％, the mean operation time was (61.8 ± 17.3) min and the mean diameter of the lesions was (2.7 ± 1.3) cm. No endoscopic massive haemorrhage occurred; The incidence of perforation and postoperative delayed bleeding was 6.6％ and 5.7％ respectively, which were cured by endoscopic treatment and there was no surgical treatment. Postoperative pathological results showed high differentiated adenocarcinoma in 23 cases, moderately differentiated adenocarcinoma in 29 cases, poorly differentiated adenocarcinoma in 19 cases, signet ring cell carcinoma in 3 cases and high grade intraepithelial neoplasia in 32 cases. Among them, 7 cases with basal tumor invasion, and there were no margin positive cases. So the R0 resection rate was 93.4％ and the R1 removal rate was 6.6％. The 7 cases with R1 resection reached R0 resection after second endoscopic treatment. 5 cases recurred within 1 years after the operation, and the recurrence rate was 4.7％. Up to December 2016, 3 patients died, the median follow-up period was 34 months and the 3 year survival rate was 97.9％. Conclusion ESD is safe and feasible in the treatment of EGC and precancerous lesions with the advantages of less trauma, faster recovery, less complications and reliable curative effect. Its clinical efficiency is similar to surgery.

OBJECTIVE: To evaluate the efficacy of autogenous bone marrow injection and elastic intramedullary injection in the treatment of bone cyst in children. METHODS: From January 2012 to December 2016, 56 children with simple bone cyst were divided into two groups: autogenous bone marrow blood injection group and elastic intramedullary needle group. There were 28 cases in the autogenous bone marrow blood injection group, 16 boys and 12 girls, aged (7.7±1.9) years old, 10 cases of proximal humerus, 8 cases of proximal femur, 6 cases of proximal tibia and 4 cases of femoral shaft. In the elastic intramedullary needle group, there were 28 cases, 18 boys and 10 girls, aged(7.5±2.2) years old, 11 cases of proximal humerus, 7 cases of proximal femur, 5 cases of proximal tibia, 4 cases of femoral shaft and 1 case of distal femur. The treatment effect was evaluated by Capanna standard. RESULTS: All the patients were followed up, including 17 to 35(25.6±4.2) months in the elastic intramedullary needle group and 19 to 35(27.4±4.8) months in the autogenous marrow blood injection group. According to Capanna's evaluation standard of bone cyst, 27 patients in the elastic intramedullary needle group were treated effectively(25 patients cured, 2 patients healed but some remained lesions), 1 patients recurred, 0 patient had no response to treatment; 18 patients in the autogenous bone marrow blood injection group were treated effectively(13 patients cured, 5 patients healed but some remained lesions), 8 patients of cyst recurred, 2 patients had no response to treatment; the difference between the two groups was statistically significant(<0.01). The overall cure time was calculated by the follow-up of 25 cases in the elastic intramedullary injection group and 13 cases in the autogenous marrow blood injection group. The cure time was(20.2±3.5) months in the elastic intramedullary injection group and(27.7±4.9) months in the autogenous marrow blood injection group. The difference was statistically significant(<0.05). CONCLUSIONS For the treatment of bone cyst in children, the therapeutic effect of elastic intramedullary needle is better than that of autogenous bone marrow blood injection, and the cure time is shorter.
Bone Cysts ; Bone Marrow ; Child ; Child, Preschool ; Female ; Fracture Fixation, Intramedullary ; Humans ; Male ; Neoplasm Recurrence, Local ; Treatment Outcome

BACKGROUNDAccurate evaluation of response following chemotherapy treatment is essential for surgical decision making in patients with breast cancer. Modalities that have been used to monitor response to neo-adjuvant chemotherapy (NAC) include physical examination (PE), ultrasound (US), and magnetic resonance imaging (MRI). The purpose of this study was to evaluate the accuracy of PE, US, and MRI in predicting the response to NAC in patients with breast cancer.

METHODSAccording to the response evaluation criteria in solid tumors guidelines, the largest unidimensional measurement of the tumor diameter evaluated by PE, US, and MRI before and after NAC was classified into four grades, including clinical complete response, clinical partial response, clinical progressive disease, clinical stable disease, and compared with the final histopathological examination.

RESULTSOf the 64 patients who received NAC, the pathologic complete response (pCR) was shown in 13 of 64 patients (20%). The sensitivity of PE, US, and MRI in predicting the major pathologic response was 73%, 75%, and 80%, respectively, and the specificity was 45%, 50%, and 50% respectively. For predicting a pCR, the sensitivity of PE, US, and MRI was 46%, 46%, and 39%, respectively, and the specificity was 65%, 98%, and 92% respectively.

CONCLUSIONSCompared with final pathologic findings, all these three clinical and imaging modalities tended to obviously underestimate the pCR rate. A more appropriate, universal, and practical standard by clinical and imaging modalities in predicting the response to neo-adjuvant chemotherapy in vivo is essential.

Adult ; Aged ; Breast Neoplasms ; diagnostic imaging ; drug therapy ; pathology ; Chemotherapy, Adjuvant ; Female ; Humans ; Magnetic Resonance Imaging ; Middle Aged ; Physical Examination ; Ultrasonography

OBJECTIVEGonadotropin releasing hormones (GnRH) regulate the expression of annexin 5 in Leydig cells, and annexin 5 is supposed to be a signal molecule in regulating testosterone secretion. This study aimed to investigate the function of annexin 5 in male reproduction by observing its effect on human sperm motility in vitro.

METHODSThe encoding sequence of rat annexin 5 was chemically synthesized and inserted into the HIS fusion expression vector pET28a. The expression of the fusion protein HIS-annexin 5 was induced by isopropyl-beta-D-thiogalactoside (IPTG) under the control of the T7 promoter, and the products were purified by affinity chromatography. The anticoagulant activity of annexin 5 was determined by the modified activated partial thromboplastin time (APTT) test. Semen samples from 15 donors were assigned to a control and an annexin 5 group, the latter treated with recombinant annexin 5 at the concentration of 10(-8) mol/L. Sperm motility and the percentage of grade a + b sperm were measured by computer-assisted semen analysis (CASA) after 20 and 60 min exposure, and the sperm ascending experiment was done after 20 min treatment.

RESULTSThe product of the synthesized target gene was 947 bp in length, and the inserted sequence corresponded to the published encoding sequence of rat annexin 5. The plasmid pET28a-annexin 5 was transformed into E. coli BL21(DE3) and IPTG induced a fusion protein with a relative molecular weight of about 36,000, a purity of 95% and a high anticoagulant activity. Compared with the control group, sperm motility and the percentage of grade a + b sperm were increased by 40% (P < 0.01) and 21% (P < 0.01), respectively, after 20 min treatment with annexin 5, but neither showed any significant improvement after 60 min. The sperm ascending altitude was remarkably elevated after annexin 5 treatment, with extremely significant difference from the control group (37.84 +/- 6.35 vs. 49.5 +/- 12.27, P < 0.01).

CONCLUSIONAn annexin 5 recombinant expression vector was successfully constructed. The protein annexin 5 can be efficiently expressed in E. coli and effectively improve human sperm motility in vitro.

Animals ; Annexin A5 ; genetics ; pharmacology ; Genetic Vectors ; Humans ; Male ; Plasmids ; Rats ; Recombinant Fusion Proteins ; genetics ; pharmacology ; Sperm Motility

OBJECTIVETo investigate the effects of GnRH analogues GnRHa and GnRHant on the MAPK pathway in rat Leydig cells.

METHODSRat Leydig cells were primarily cultured for 24 hours in vitro and serum-starved for 2 hours, followed by treatment with GnRHa (10(-7) mol/L) or GnRHant (10(-6) mol/L) for 0, 5, 15, 30, 60 and 90 minutes, with the 0 min group as the control. Then the protein levels of phosphorylated ERK (p-ERK) and phosphorylated p38 (p-p38) were detected by Western blot, and that of p-ERK determined by the same means after co-incubation of GnRHa or GnRHant with the PKC inhibitor GF109203X at 1, 5, 10 and 20 micromol/L.

RESULTSAfter stimulation of the Leydig cells with GnRHa or GnRHant for different times, the protein level of p-p38 showed no significant difference from that of the control group (P > 0.05). Then the Leydig cells were treated with GF109203X at different concentrations for 20 minutes and with addition of GnRHa for another 10 minutes. The level of p-ERK was significantly decreased (P < 0.05) by GF109203X at 10 and 20 micromol/L. Compared with the control, the p-ERK expression was increased by 65% at 15 minutes (P < 0.05) in the GnRHant stimulation group, by 81% (to the peak) at 30 minutes (P < 0.05), began to fall at 60 minutes, and returned to the base level at 90 minutes. The p-ERK level exhibited no significant difference from that of the control (P > 0.05) after treatment of the Leydig cells with different concentrations of GF109203X for 20 minutes and then with GnRHant for 30 minutes.

CONCLUSIONThe ERK MAPK activation induced by GnRHa depends on the PKC pathway, but not that induced by GnRHant. The p-38 MAPK pathway may not be involved in the effect of GnRH analogues on rat Leydig cells.

Animals ; Cells, Cultured ; Gonadotropin-Releasing Hormone ; analogs & derivatives ; pharmacology ; Leydig Cells ; drug effects ; metabolism ; MAP Kinase Signaling System ; drug effects ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the role of Annexin 5 in protecting human sperm membrane and DNA integrity.

METHODSWe collected 53 semen samples based on the criteria of sperm density > 20 x 10(6)/ml and motility > 60%, and divided them into an experimental group (2.5 microl 10(-6) mol/L Annexin 5 added to 47.5 microl semen), a negative control group (2.5 microl 1 mol/L Tris-HCl [pH 8.0, 25 degrees C] added to 47.5 microl semen), and a blank control group (2.5 microl 0.01 mol/L PBS [pH 7.4] added to 47.5 microl semen). After 20 minutes of incubation, we evaluated the sperm membrane integrity using the hypoosmotic swelling test and, after another 60 minutes of treatment with H2O2 at 2.5 microl 10.02 mol/L, measured the sperm nuclear DNA integrity by acridine orange fluorescent staining.

RESULTSAfter 20 minutes of treatment with Annexin 5, the experimental group showed extremely significant difference in the percentage of hypoosmotic swelling sperm ([66.17 +/- 12.02] %) from the blank control ([58.13 +/- 13.08]%, P < 0.01) and the negative control group ([59.94 +/- 11.91]%, P < 0.01), but there was no significant difference between the latter two. Treatment with H2O2 remarkably increased DFI in the experimental group (6.39 +/- 1.07) as compared with the blank control (11.16 +/- 1.16) and the negative control group (10.86 +/- 1.05, P < 0.01), but no significant difference was observed between the latter two.

CONCLUSIONAnnexin 5 can increase the percentage of hypoosmotic swelling sperm in vitro and protect sperm membrane integrity, and it can also protect sperm DNA from H2O2 damage.

Annexin A5 ; pharmacology ; Cell Membrane ; drug effects ; DNA ; DNA Fragmentation ; Humans ; Male ; Sperm Count ; Sperm Motility ; Spermatozoa ; drug effects