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ObjectiveTo identify Dendrobium flexicaule and its related species， and analyze the differences in polysaccharide composition and D-mannose content， so as to provide theoretical basis for the accurate identification and quality control of Dendrobium medicinal materials. MethodNine samples of Dendrobium （S1-S9） were identified by DNA barcoding and infrared spectroscopy， and the contents of polysaccharides and D-mannose were determined by ultraviolet spectrophotometry （UV） and high performance liquid chromatography （HPLC）， respectively. UV detection condition was 488 nm， HPLC detection conditions were the mobile phase of 20 mmol·L-1 ammonium acetate solution-acetonitrile （81.5∶18.5） and the detection wavelength at 250 nm. ResultDNA barcoding results showed that samples S1-S3 were D. nobile， samples S4-S5 were D. officinale， sample S6 was D. huoshanense， and S7-S9 were D. flexicaule. One-dimensional infrared spectroscopy showed that only D. nobile had stable characteristics at the wavenumber of 1 570-1 467 cm-1， showing a "W" shape， while no absorption peak was found at the wavenumber of 842-740 cm-1， but the other Dendrobium samples had stable absorption peaks at the wavenumber of 842-740 cm-1. In the first derivative spectrum， at the wavenumber of 785 cm-1， D. huoshanense presented a "V" shape， while the rest of Dendrobium presented a "W" shape. At the wavenumber of 1 110 cm-1， D. flexicaule had a stable characteristic peak. In the second derivative spectrum， at the wavenumber of 1 125 cm-1， D. officinale presented an "M" shape， and the rest of Dendrobium was approximately "W" shape. The results of determination showed that the contents of polysaccharides in samples S1-S9 were 9.35%， 9.12%， 32.78%， 49.38%， 48.97%， 32.48%， 32.95%， 39.41% and 25.32%， and their contents of D-mannose were 1.39%， 0.47%， 13.57%， 3.04%， 33.85%， 23.57%， 16.64%， 17.47% and 19.49%， respectively. Among them， D. flexicaule had high polysaccharide and D-mannose contents. ConclusionBoth DNA barcoding and infrared spectroscopy can be used to identify D. flexicaule and its related species， and infrared spectroscopy is cost-effective and easy to operate. At the same time， D. flexicaule has high contents of polysaccharides and D-mannose， which can provide a scientific basis for rapid identification of D. flexicaule and its relatives， and provides a reference for its quality control， and resource development and utilization.
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OBJECTIVE:To establish a method for the content determination of berberine hydrochloride in Fufang huangge ji-angtang tablet. METHODS:UPLC was performed on the column of Acquity UPLC BEH C18 with mobile phase of acetoni-trile-0.1% H3PO4(21∶79,V/V)at flow rate of 0.3 ml/min,the detection wavelength was 345 nm,column temperature was 30℃, and the volume injection was 1.0 μl. RESULTS:The linear range of berberine hydrochloride was 0.008 16-0.081 6 μg(r=0.999 9);RSDs of precision,stability and reproducibility were lower than 1%;average recovery was 97.05%-98.96%(RSD=0.66%,n=6). CONCLUSIONS: The method is simple,precise,stablity and reproducible,and can be used for the quality control of Fufang huangge jiangtang tablet.
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BACKGROUND:Hip and knee replacement is a common surgery in the clinic; the procedure is relatively complex; and the risk of surgery is relatively high, so that postoperative analgesia is not satisfactory. Perioperative pain management has been a clinical concern. To find safe and effective analgesia has become one of the important tasks of joint surgeons. OBJECTIVE:To investigate the effect of celecoxib on multi-mode analgesia after hip and knee replacement. METHODS: A total of 80 cases undergoing hip and knee replacement in the Chongqing Dongnan Hospital from September 2012 to September 2013 were enroled in this study. These patients were randomly divided into two groups. In the control group, celecoxib was not used for analgesia after replacement. In the experimental group, celecoxib was used after replacement. The pain was observed at 1-5 days after surgery in the two groups. When the analgesic pump was removed, the drug dosage and opioid analgesics use were recorded. Side effects of drug use were also recorded. RESULTS AND CONCLUSION:In terms of analgesic efficacy, the analgesic effect was better in the experimental group than in the control group (95%, 85%, P 0.05). Pain visual analogue scale score was significantly lower in the experimental group than in the control group at 1, 2 and 3 days post surgery (P< 0.05). The drug dosage was significantly more in the experimental group than in the control group after surgery (P < 0.05). The frequency of opioid use in the experimental group was significantly lower than in the control group (P < 0.05). The complication rate was significantly lower in the experimental group (8%) than in the control group (18%) (P < 0.05). These findings demonstrate that the analgesic effect of celecoxib was ideal after hip and knee replacement using multi-mode analgesia, which can reduce the dose of analgesic drugs and have smal adverse reaction.
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OBJECTIVE@#To discuss a effective method of nasal septum deviation.@*METHOD@#One hundred and sixteen subjects with nasal septum deviation were divided into treatment group (69 subjects) and control group (47 subjects) randomly. The combination therapy of correction of deviated nasal septum and plasma radiofrequency ablation were used in the treatment group. The combination therapy of deviated nasal septum and partial inferior turbinectomy were used in the control group. The data were analyzed by statistical method.@*RESULT@#The effective rate of physical signs and symptoms of the treatment group was 100.0%, while the control group was 85.1%. There was significant difference of two effective rates (P < 0.05). Furthermore, hyperventilation and nasal adhesion were not happened in treatment group 6 months after treatments.@*CONCLUSION@#The correction of deviated nasal septum and plasma radiofrequency ablation combination therapy had the satisfied and safety treatment effect, which was and plasma radiofrequency ablation combination therapy had the satisfied and safety treatment effect, which was easy for observation as well as the minimal tissue damage. The combination therapy method was according with the principle of functional minimally invasive surgery.
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Adult , Female , Humans , Male , Middle Aged , Young Adult , Catheter Ablation , Combined Modality Therapy , Nasal Septum , Congenital Abnormalities , General Surgery , Plastic Surgery ProceduresABSTRACT
A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients with hepatitis B-related hepatocellular carcinoma (HCC) and 3 cases of normal liver tissues. The integration sites and flanking sequences in human genome were sequenced and blasted, and the expression of integrated HBV genes was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The influence of the up-regulated expression of integrated genes on hepatocarcinogenesis was analyzed. Nineteen integration sites of HBV DNA into HCC tissues were obtained by RT-PCR and sequencing. These genes encoding proteins were: LOC51030, LOC157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in HCC tissues, matched surrounding liver tissues and normal liver tissues were in a descendent order. The ratio of MCM3AP mRNA to the GAPDH mRNA in these three tissues was 1.07375, 0.21573, 0.06747 respectively, with the difference being statistically significant among them (P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from HCC tissues in which HBV DNA integrated into MCM3AP were still significantly higher than those without HBV DNA integrated into MCM3AP. It was concluded that the HBV DNA integration sites into human genome were random, and MCM3AP was a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocarcinogenesis.
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ostic sensitivity and specificity of EUS are high in distinguishing benign and malignant character of upper digestive tract GIMTs. EUS plays an important role in guiding the clinical management of upper digestive tract GIMTs.
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The crystal structural data of TACE, MMP-1, MMP-2, MMP-3 and MMP-9 were obtained from PDB database, and then their catalytic domains' properties including conformation, molecular surface hydrophobicity and electrostatic potential were analyzed and compared by using Insight II molecular modeling software. It was found that the conformation and molecular surface hydrophobicity of catalytic domains of TACE and MMPs were not obviously different, but the molecular surface electrostatic potential of catalytic domain of TACE and MMPs had obvious differences. The findings are helpful in the Rational Drug Design of TACE selective inhibitor.
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The crystal structural data of TACE, MMP-1, MMP-2, MMP-3 and MMP-9 were obtained from PDB database, and then their catalytic domains' properties including conformation, molecular surface hydrophobicity and electrostatic potential were analyzed and compared by using Insight Ⅱ molecular modeling software. It was found that the conformation and molecular surface hydrophobicity of catalytic domains of TACE and MMPs were not obviously different, but the molecular surface electrostatic potential of catalytic domain of TACE and MMPs had obvious differences.The findings are helpful in the Rational Drug Design of TACE selective inhibitor.
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To obtain the recombinant tumor necrosis factor-α converting enzyme (TACE) ectodomain and use it as a selective molecule for the screening of TACE peptide inhibitors, the cDNA coding catalytic domain (T800) and full-length ectodomain (T1300) of TACE were amplified by RTPCR, and the expression plasmids were constructed by inserting T800 and T1300 into plasmid pET28a and pET-28c respectively. The recombinant T800 and T1300 were induced by IPTG, and SDSPAGE and Western blotting analysis results revealed that T800 and T1300 were highly expressed in the form of inclusion body. After Ni2+-NTA resin affinity chromatography, the recombinant proteins were used in the screening of TACE-binding peptides from phage display peptide library respectively. After 4 rounds of biopanning, the positive phage clones were analyzed by ELISA, competitive inhibition assay and DNA sequencing. A common amino acid sequence (TRWLVYFSRPYLVAT) was found and synthesized. The synthetic peptide could inhibit the TNF-α release from LPS-stimulated human peripheral blood mononuclear cells (PBMC) up to 60.3 %. FACS analysis revealed that the peptide mediated the accumulation of TNF-α on the cell surface. These results demonstrate that the TACE-binding peptide is an effective antagonist of TACE.
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Objective To study the expressi on of thrombomodulin (TM) in endometrial carcinoma and explore its relation to the infiltration degree and histological grading of endometrial carcinoma. Methods The expression of TM was detected by immunohistochemistry in 50 cases of endometrial adenocarcinoma, 10 cases of endometrial complex hyper plasia and 10 cases of normal endometrium. Results The posi tive rate of TM in endometrial carcinoma was significantly higher than that in c omplex hyperplasia and normal endometrium (P 0.05). Conclusion TM can be used as a new index for judging progre ssion and prognosis of endometrial carcinoma.
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A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid-phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA-A PCR amplification system and a small HLA-A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe2O3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano-beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab-on-a-chip.
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Adsorption , Cell Separation/instrumentation , Cell Separation/methods , Genotype , Leukocytes/cytology , Magnetics , Microchemistry/instrumentation , Microchemistry/methods , Nanotechnology , Oligonucleotide Array Sequence Analysis/methods , Polymerase Chain Reaction , Templates, GeneticABSTRACT
A protocol for enrichment and adsorption of karyocyte from whole blood by using magnetic nanometer beads as solid-phase absorbents was presented. The PCR amplification could be accomplished by using the nanobeads with karyocyte as template directly and the PCR products were applied on an oligonucleotide array to do gene typing. The HLA-A PCR amplification system and a small HLA-A oligonucleotide microarray were applied as the platform and an experiment protocol of separating karyocyte from whole blood using the magnetic nanometer beads (Fe2O3) were set up. The experimental conditions were also discussed. It showed that pH level of PBS eluent, Taq enzyme quantity and fragment length of products could influent the amplification results, and the magnetic nano-beads could succeed in sample preparation in microarray to provide a promising way in automatic detection and lab-on-a-chip.
Subject(s)
Adsorption , Cell Separation , Methods , Genotype , Leukocytes , Cell Biology , Magnetics , Microchemistry , Methods , Nanotechnology , Oligonucleotide Array Sequence Analysis , Methods , Polymerase Chain Reaction , Templates, GeneticABSTRACT
The effects of inhibitors of TNF alpha converting enzyme (TACE) on TNF alpha secretion were studied to develop an approach to interfere inflammation processes. The HL-60 cell lines were stimulated in vitro with LPS intravenously for different time to establish the cellular model of inflammation and simultaneously induce in vivo inflammation animal model by LPS The cytotoxic effects of soluble TNF alpha were checked using MTT colorimetric method to determine the rate of cell proliferation. The level of expression of TACE was detected by using RT-PCR, FCM and immuno-histochemical technique respectively. It was found Chinese medicine Reduqing (RDQ) could inhibit the transcription of TNF alpha mRNA induced by LPS stimulation (P < 0.01, compared with the control). The antioligodeoxyribonucleotide (anti-ODN) of TNF alpha mRNA could inhibit 78.9% of TNF alpha secretion. The mimic peptides of TACE substrates with hydroxamine group showed potency in vivo and in vitro against converting of pro-TNF alpha. It was concluded that all the three types of TACE inhibitors can regulate the expression of TACE at different levels and inhibit sTNF alpha secretion, indicating TACE is a novel target for inflammation therapy.
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Humans , ADAM Proteins , ADAM17 Protein , Drugs, Chinese Herbal , Pharmacology , HL-60 Cells , Inflammation , Metabolism , Lipopolysaccharides , Metalloendopeptidases , Oligodeoxyribonucleotides , Protein Precursors , RNA, Messenger , Genetics , Metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription, Genetic , Tumor Necrosis Factor-alpha , Genetics , Metabolism , Bodily SecretionsABSTRACT
In order to investigate the effects of LPS on the TACE gene transcription and expression and its regulating effect on the TM-TNF secretion, in vitro studies were carried out on HL-60 cells stimulated by LPS. TACE, TNF-alpha mRNA levels were detected by Dot-Elisa and the distribution of membrane molecules determined by flow cytometry assay and indirect immunofluorescence. The results showed that: (1) TACE was detected in or on HL-60 cells and it is predominantly localized on cell surface and to a perinuclear compartment. (2) LPS induced a time dependent increasement of TNF-alpha mRNA and enhanced TNF conversion with decreasing distribution of TNF in cell surface and increasing secretion of TNF protein. Such conversion could be inhibited by TACE ODN. (3) LPS also induced time-dependently increased expression of TACE gene and activation of its function. On the other hand, TACE protein in cell lysate and on cell surface was decreased. It was suggested that TACE molecular structure might change following its mediating membrane-anchored molecular secretion.
Subject(s)
Humans , ADAM Proteins , ADAM17 Protein , Gene Expression , HL-60 Cells , Lipopolysaccharides , Pharmacology , Metalloendopeptidases , Genetics , RNA, Messenger , Genetics , Transcription, Genetic , Tumor Necrosis Factor-alpha , GeneticsABSTRACT
AIM: To investigate the expression of vascular endothelial growth factor (VEGF) and tumor necrsis factor-alpha (TNF-?) in synovium of rats with adjuvant arthritis (AA) and the relationship between the pathological score and the expression of VEGF and TNF-? protein. METHODS: AA was produced in Wistar rats by inoculating complete Freund's adjuvant (CFA). The arthral pathological score was calculated, production of VEGF and TNF-? protein were assayed by histoimmunochemical staining at different stage after CFA inoculation. RESULTS: In AA group, the pathological score and expression of VEGF protein in synovium increased significantly (P
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Objective:To study the effects of TNF? converting enzyme(TACE)inhibitors on TNF? secretion and develop an approach to interfere inflammation processes.Methods:(1)Stimulate the HL-60 cell lines in vitro with LPS for different time to establish the cellular model of inflammation and simultaneously induce in vivo inflammatoin animal model by LPS.(2)Check the cytotoxic effects of TNF? secretion using MTT colorimetric method for cell proliferaton.(3)Detect the level of expression of TACE carrying out RT-PCR,FCM techniques and immuno-histochemical dying technique.Results:(1)PDQ had the inhibitive effect on TNF?mRNA expression induced by LPS stimulation( P
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AIM:To study functions of pro-domain in tumor necrosis factor-? converting enzyme (TACE) maturation and to develop an approach to interfere with inflammation processes. METHODS: The plasmid pIRES2-EGFP was used to generate the expression plasmids constructed with the signal peptide and pro-domain, full length or without the pro-domain(designated as pIRES2-EGFP/T648, pIRES2-EGFP/T2472, pIRES2-EGFP/T57-T1824,respectively). The plasmids were used to transfect the U937 cells.After the transfected cells were stimulated by LPS, the effect of expression plasmids on TNF-? secretion was detected by ELISA and flow cytometry(FCM).RESULTS: The plasmid pIRES2-EGFP/T648 inhibited 61.09% TNF-? secretion and mediated the accumulation of TNF-? on the surface of U937 cells.The sTNF-? secretion and the level of the mTNF-? in the plasmid pIRES2-EGFP/T57-T1824 transfected cells had no significant deviation compared with the pIRES2-EGFP transfected cells. The sTNF-? secretion of the cells transfected by the plasmid pIRES2-EGFP/T2472 increased and the level of the mTNF-? decreased.CONCLUSION: The pro-domain has dual effects in TACE maturation and might be applied to molecular design of anti-inflammation drug.