Objective To establish a stable and useful method for culturing human osteoclast-like cells in vitro,and investigate the effect of 1?,25-(OH)2D3,M-CSF and PGE2 on osteoclasts differentiation,proliferation and activation so as to lay the foundation for further study of the biological mechanism for tooth movement.Methods The HCMNC were isolated and cultured in 24-well plate with coverslips and human dentine slices.The experiment group was cultured with 1?,25-(OH)2D3,M-CSF and PGE2,respectively,while the control group was not.The liquid was changed every 3 days and the whole culture process lasted for 7 days.The phase contrast microscopy and TRAP staining were adopted to identify osteoclast-like cells.Results On the 3rd day the monocytes began to fuse and on the 7th day positive multinucleated cells could be seen with TRAP staining,but absorption pit was not formed on the dentin slices.The group with 1?,25-(OH)2D3 had the largest number of osteoclast-like cells.Conclusion After the monocytes in UCB are cultured by 1?,25-(OH)2D3,M-CSF,PGE2 induction,they can turn into TRAP(+) multinucleate osteoclast-like cells,the 1?,25-(OH)2D3 10-8mol/L being the most effective.

Objective To establish a liquid chromatography-tandem mass spectrometric ( LC-MS/MS ) for determination of clopidogrel in human plasma and stability of clopidogrel under different conditions,which was used subsequently to investigate the pharmacokinetics of clopidogrel in healthy Chinese volunteers. Methods Clopidogrel-d4 hydrogen sulfate was used as an internal standard. Separation was achieved on a WATERS Xterra? RP18 column (4.6 mm× 100 mm,3.5μm) with a mobile phase consisting of acetonitrile-0.1% formic acid (66:34) at a flow rate of 1.0 mL.min-1 within 3.2 min. ESI source was applied and operated in positive ion mode and multiple reaction monitoring (MRM). Plasma samples were pretreated by acetonitrile precipitation. Results A good linearity of clopidogrel was obtained in the concentration range of ( 5-5 000 ) ng.L-1. The lower limit of quantification was 5 ng.L-1. The intra-and inter-run precisions at three quality control levels were within 1.3%–9.9%,the relative deviation of the assay was within -6.2%-14.3%. The blood samples were stable when chilled with crushed ice ( 0℃) and cold water ( 4℃) ,as well as kept at room temperature ( 20℃) for 40 min. The plasma QC samples were stable at room temperature ( 20 ℃) for 4 h,at -70℃ for 38 days and during three freeze-thaw cycles. Hemolysis in blood sample drawn didn’ t affect the plasma concentration. The determination and the result of incurred sample reanalysis met the requirements. Conclusion A specific, rapid, sensitive and stable LC-MS/MS method is developed and validated for determination of clopidogrel in human plasma. The method is proven to be suitable for study of the pharmacokinetics of clopidogrel in healthy Chinese volunteers after a single oral dose of 75 mg clopidogrel hydrogen sulphate tablet.

BACKGROUND: Orthodontic tooth movement is dependent on reconstruction of periodontium. Osteoclastic bone resorption is the first step of tooth movement. The present study hotspots focus on signal transduction pathway regarding osteoclast differentiation and functional development under stress and on the relationship between periodontal ligament cells and osteoclasts. OBJECTIVE: To set up a simple method to in vitro culture human osteoclast-like cells and to observe the effects of bone resorption-stimulating factors on differentiation, proliferation, and function of osteoclast-like cells, DESIGN, TIME AND STTING: A cytological in vitro controUod observation was performed at the Central Laboratory,Stomatology Hospital, Xi'an Jiaotong University between October 2007 and May 2008. MATERIALS: Umbilical cord blood was sourced from the healthy puerperae who had not suffered from high-risk pregnancy. Freshly prepared fetal femur provided by Laboratory Animal Center, Xi'an Jiaotong University and were used for preparation of bone flaps at 100-200 μm thickness. 1α ,25-(OH)2D3, macrophage colony-stimulating factor (M-CSF), prostaglandin E2 were purchased from Sigma Company, USA.METHODS: Under aseptic condition, umbilical cord blood was collected. Following Ficoll solution separation and centrifugation, supematant was discarded. Umbilical cord blood-derived mononuclear cells were suspended with o -modified minimal essential medium (α-MEM) solution and then inoculated into a 24-well culture plate, in which, coverslips and femoral slices were pre-placed, at a density of 1×109/L, 1.0 mL per well. Five groups were set, blank control, 108 mol/L 1α ,25-(OH)2D3, 10-7 mol/L 1α ,25-(OH)2D3, macrophage colony stimulating factor (M-CSF), and 1α ,25-(OH)2D3+prostaglandin E2 (PGE2). Each group was cultured for 7 days. MAIN OUTCOME MEASURES: Cellular growth morphology was observed under an inverted microscope; osteoclast-like celt formation was examined by tartrate-resistant acid phosphatase (TRAP) staining; and osteoclastic Howship's lacuna was detected by toluidine blue staining. Any cell with TRAP-positive staining and more than two nuclei was considered osteoclast-like cell and counted. RESULTS: After 3 days of culture, cells from the blank control group did not exhibit apparent changes in morphology and quantity. In the remaining groups, mononuclear cells appeared with confluent tendency. After 7 days of culture, a small number of osteoclast- like cells with 2-3 nuclei were found in the blank control group; a great many of multinucleated osteoclast- like cells with 3-20 nuclei were present in the remaining groups. Through the use of optical microscope, osteoclast-like cells could be found for the presence of red cytoplasm, bright yellow nuclei, and TRAP-positive staining in each inducing factor-treated group, in particular in the 108 mol/L 1α ,25-(OH)2D3 group, which displayed osteoclast- like cells exhibiting 14 nuclei, strong TRAP-positive staining, and a relatively big cell body. But no osteoclastic Howship's lacuna was found in any group. Compared to the blank control group, the numbers of osteoclast-like cells were greater in each inducing factor-treated group (F = 9.78, P < 0.01). There was no significant difference in the numbers of osteoclast-like cells between the 108 mol/L 1α ,25-(OH)2D3 and the 10-7 mol/L 1 a ,25-(OH)2D3 groups (P0.05). The M-CSF group and the 1α ,25-(OH)2D3+PGE2 group exhibited significantly less numbers of osteoclast-like cells than the 108 mol/L 1α ,25-(OH)2D3 group (F= 7.46, P< 0.01). CONCLUSION: After in vitro culture of 1α ,25-(OH)2D3, M-CSF, and PGE2, umbilical cord blood-derived mononuclear cells can differentiate into TRAP-positive multinucleated osteoclast-like cells, the 10-8 mol/L 1α ,25-(OH)2D3 being the most effective.

Objective By culturing the osteoclasts together with the osteoblasts directly to investigate the effect of osteoblasts on the formation of mature osteoclasts.Methods The bone marrow mononuclear cells of rats were treated with 30?g/L M-CSF and 50?g/L RANKL and cultured for 6 days.Subsequently,the primary osteoblasts which were of the same quantity as the osteoclasts were co-cultured directly.In the co-culture system,we added the liquid containing 1,25-(OH)2D3 1?10-8mol/L and PGE2 1?10-6mol/L.The morphological observation,TRAP staining and pit staining were adopted to identify osteoclasts.Results When the osteoclasts were co-cultured with primary osteoblasts,the growth of osteoblasts had more preponderances.After staining,we could see more osteoblasts than osteoclasts.Conclusion The relationship between osteoblasts and osteoclasts is related to the relative quantities of the two cells.When osteoblasts outnumber osteoclasts,osteoblasts would inhibit the formation and differentiation of osteoclasts.

Objective To investigate the prognostic value of lymph node status evaluated by computed tomography (CT) in cervical carcinoma treated with radical chemoradiation therapy.Methods 209 patients with FIGO stage Ⅰ B-ⅣA uterine cervical carcinoma treated with radical chemo radiation therapy were enrolled.Lymph node status was evaluated based on site,number and size respectively by CT.Kaplan-Meier method was used to analyze the impact of lymph node status as well as other pathoclinical factors on overall survival (OS).Cox regression model was used to explore the relationship between independent prognostic factors and OS.Results Lymph node metastasis rates for stage Ⅰ B,Ⅱ,Ⅲ and ⅣA patients were 16.7 ％ (1/6),48.2 ％ (68/141),57.4 ％ (31/54) and 87.5 ％ (7/8),respectively.Variate analysis showed that FIGO stage,anemia before treatment,para-aortic lymph node metastasis,and the number (≥3) and size (maximum diameter ≥ 2.0 cm) of CT positive lymph node were significant unfavorable prognostic factors for OS (P ＜ 0.05).FIGO stage,the number of lymph nodes metastasis,pathologic diagnosis and treatment regimen were identified to be independent prognostic factors in multivariate analysis.Conclusion Under current therapeutic strategy of concomitant chemoradiotherapy,lymph node metastatic status evaluated by pretreatment CT is still a significant and independent prognostic factor for cervical cancer.

A HPLC method for simultaneous determination of uridine, inosine, guanosine, adenosine and cordycepine
in Cordyceps sinensis was developed. The sample solution was prepared with 0.5% phosphoric acid solution by ultrasonic extraction. The separation was performed on a ZORBAX SB-AQ (150 mmí4.6 mm, 5 μm)column with gradient elution by 0.1% formic acid solution and acetonitrile, with column temperature 30℃, at a flow rate of 0.8 mL/min, and detected at wavelength of 260 nm. The result of method validation showed that the developed method had high accuracy and good repeatability. This method has been successfully applied for analysis of 5 kinds of nucleosides in different parts of C. sinensis. The results indicated that content of nucleosides in stroma is higher than that in insect body and whole C. sinensis.

OBJECTIVE:To adopt gel method for the determination of bacterial endotoxin in Fat emulsion(10%)/amino acid (15)/glucose (20%) injection. METHODS:According to the gel method in term ofbacterial endotoxin test methodin Chinese Pharmacopeia(2015 edition),the maximal valid dilution(MVD)of samples were determined through interference test and the vali-dated. The results were compared with chromogenic method. RESULTS:In gel method,the interference to agglutination reaction of TAL and bacterial endotoxin can be excluded when samples were diluted 24 times or less. In chromogenic method,the samples should be diluted 76 times or less. CONCLUSIONS:Gel method can be used for bacterial endotoxin test of Fat emulsion(10%)/amino acid(15)/glucose(20%)injection.

Objective: To observe the effect of electroacupuncture (EA) at different time points during the perioperative period on the recovery of gastrointestinal function after gastrointestinal malignant neoplasms surgery. Methods: Sixty-three patients who needed radical surgery for gastrointestinal tumors were randomized into a control group, treatment group 1 (postoperative EA group), and treatment group 2 (intraoperative and postoperative EA group). The control group received surgery and conventional Western medicine treatment, and treatment groups 1 and 2 received additional EA treatment at different time points. The initial flatus time after the surgery, visual analog scale (VAS) score at different time points after the surgery, the proportion of using patient-controlled analgesia (PCA) after the surgery, and the times of adding analgesics were observed in the three groups. Results: The initial flatus time after the surgery was earlier in treatment groups 1 and 2 than in the control group (P<0.05); the difference between treatment groups 1 and 2 was statistically insignificant (P>0.05). The VAS score was lower in treatment group 2 than in the control group at 6, 12, 24, and 72 h after the surgery (P<0.05); the VAS score was lower in treatment group 1 than in the control group only at 72 h after the surgery (P<0.05). There were no significant differences in the rate of using PCA among the three groups (P>0.05). Regarding the times of adding analgesics, it was less in treatment group 2 than in the control group at 12 h after the surgery (P<0.05). Conclusion: Either EA during and after the surgery or only after the surgery can hasten the initial flatus and boost the recovery of gastrointestinal function in patients after radical resection of gastrointestinal neoplasms. Successive EA during and after the surgery should be superior to postoperative EA regarding the analgesic effect after the surgery.

This paper conducted research on biomechanical characteristics and biological activity of concavity-convex amniotic membrane (CCAM) and discussed its superiority as ocular surface repair material. Folding and compression with vacuum of fresh amniotic membrane were used to prepare CCAM. After cutting the striga of CCAM, sixteen CCAM tissue section were chosen at random to test their tensile strength using electronic universal testing machine. The bilayer amniotic membrane (BAM), the double-deck amniotic membrane (DAM) and the monolayer amniotic membrane (MAM) were as controls. The test parameters included yield strength, tensile strength, elongation at break, elastic modulus and so on. The cytokines of fresh amniotic membrane (FAM), MAM and CCAM were analyzed by radioimmunoassay method. The CCAM was obviously thicker than MAM and DAM. After 15 min in PBS, the CCAM tissue can recover the normal shape. The tensile strength and the elongation at break of CCAM were higher than those of the MAM and the DAM (P < 0.05). The elastic modulus of the CCAM was smaller than that of the MAM and the DAM (P < 0.05). The content of 10 cytokines [epidermal growth factor (EGF), fibroblast growth factor (FGF), b-fibroblast growth factor b-FGF, hepatocyte growth factor (HGF), transforming growth factor-beta (TGF-beta), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), brain-derived nellrotrophic factor (BDNF), ciliary neurotrophic factor (CNTF)] of CCAM decreased significantly compared with the FAM and increased significantly compared with MAM and DAM in 6 cytokines (EGF, FGF, HGF, TGF-betap, PDGF, NGF; P < 0.05). The CCAM composites is thinner and has higher cytokine content than MAM, and better biomechanical properties than the MAM and the DAM, showing the superiority as ocular surface repair material.
Amnion ; chemistry ; physiology ; transplantation ; Biomechanical Phenomena ; Cytokines ; analysis ; Epidermal Growth Factor ; analysis ; Fibroblast Growth Factors ; analysis ; Hepatocyte Growth Factor ; analysis ; Humans ; Tissue Engineering ; methods ; Tissue Scaffolds

To explore the safety and efficacy of levosimendan in treating the patients with severe aortic stenosis and to analyze the cardial function before and after medication in order to guide clinical treatment. Methods: A total of 20 patients admitted in our hospital from 2014-01 to 2015-12 were enrolled with the standard of echocardiography confirmed severe aortic stenosis, left ventricular ejection fraction (LVEF)≤45%, NYHA III-IV and inefficacy for conventional anti-heart failure drug therapy. The patients received intravenous infusion of levosimendan at 0.1μg/(kg·min) by persistent pumping for 24 hours. Echocardiography, LVEF, dyspnea condition, NYHA grading and plasma levels of NT-proBNP were recorded pre- and post-medication to compare the cardiac function and symptoms of levosimendan therapy. Results: After levosimendan treatment, NYHA grade was improved, P=0.025 and NT-proBNP was reduced (9101.6±7368.0) pg/mLvs (13776.5±9503.7) pg/mL, P=0.018. The following parameters were similar before and after levosimendan therapy: LVEF (31.1±7.5)% vs (33.1±8.5)%, P=0.078, the average heart rate (79.6±13.8) bmp vs (82.8±9.5)bmp, P=0.200 and systolic blood pressure (99.6±11.7) mmHg vs (97.2±12.1) mmHg, P=0.071. There were 40% (8/20) patients with obviously improved and 50% (10/20) with improved dyspnea symptoms after levosimendan treatment. Conclusion: Our preliminary study presented that levosimendan could improve NYHA grading, remit dyspnea symptom and reduce blood NT-proBNP level in patients with severe aortic stenosis and heart failure; it had safety and tolerability at certain degree in clinical practice.