Objective: To explore the molecular mechanisms underlying the acute myeloid leukemia(AML)HL-60 cell differentiation induced by natural compound vibsanin A combined with tyrosine kinase inhibitors(TKI). Methods: Cell surface marker CD11b expression was detected by flow cytometry in HL-60 cells after treatment of the cells with vibsanin A in combination with imatinib or saracatinib for 72 h,and the cell morphology was examined by Wrigh-Giemsa staining. qRT-PCR and Western blot were used to examine the expression of differentiation-related C/EBPα,C/EBPβ, and c-Myc at both mRNA and protein levels in HL-60 cells after the cells were treated with the two drug combinations for various time(0-24 h). The recombinant lentiviral vector expressing c-Myc was constructed and used to transfect HL-60 cells in which the c-Myc cDNA was ectopically over expressed. The effect of c-Myc expression on the HL-60 cell differen-tiation induced by vibsanin A combined with TKI was investigated using the transfected HL-60 cells. Results: Vibsanin A combined with imatinib or saracatinib significantly enhanced HL-60 cell differentiation. Both drug combinations downregulated the expression of c-Myc at both mRNA and protein levels(P<0.01)in the HL-60 cells. In the transfected HL60 cells,the ectopic c-Myc overexpression could significantly counteract the down-regulated c-Myc expression and inhibit the cell differentiation induced by the vibsanin A/TKI combination. Conclusion: The combination of vibsanin A with imatinib or saracatinib could induce the HL-60 cell differentiation,probably via the downregulation of c-Myc expression

Objective To systematically evaluate the diagnostic value of magnetic resonance cholangiopancreatography (MRCP) in the investigation of bile duct anatomy of liver transplantation living donors.Methods A search in Cochrane library,MEDLINE,EMBASE,CBMdisc (China Biology Medicine disc) was performed to identify relevant English and Chinese-language abstracts,supplemented by Springer,OVID,Sciencedirect full text database,etc.Criteria for inclusion were based on validity criteria for diagnostic research published by the Cochrane collaboration.With Meta analysis package for Stata10.1,heterogeneity of the included articles was tested,which was used to select proper effect model to calculate pooled weighted sensitivity and specificity,positive likelihood ratio,negative likelihood ratio. Summary receiver operating characteristic (SROC) curve was performed and the area under the curve (AUC) was calculated. Finally,sensitivity analysis was performed.Results Seventeen articles with 34 studies were included.Heterogeneity analysis revealed heterogeneity between studies and the source was MRCP imaging methods spotted by meta-regression analysis. Subgroup analysis according to MRCP imaging methods showed homogeneity within subgroups.The pooled sensitivity,specificity,positive likelihood ratio,negative likelihood ratio,diagnostic odd ratio of breath-holding thick slice MRCP,3D MRCP,the combination of the prior two methods,contrast enhance MRCP were 0.89,0.78,4.1,0.14,29; 0.92,0.80,4.5,0.10,45;0.95,0.82,5.2,0.06,85; and 1.00,0.76,4.1,0,1228,respectively with fixed effect model analysis.The area under the SROC curve was 0.83,0.92,0.96 and 0.99 respectively.Conclusion The combination of thick slice and 3D MRCP is a practical and effective method with good sensitivity and specificity to investigate bile duct anatomy of living liver transplantation donors,which fully meets the requirements of the preoperative assessment of bile duct structure.

OBJECTIVETo investigate the migration of fluorescent dye PKH26-labeled BM-MSC in the Alzheimer's model rats.

METHODSNormal human bone marrow extracted for isolation of BM-MSC was cultured in vitro. The 5th passaged BM-MSC was labeled with PKH26, and observed under a fluorescence microscope for PKH26 labeling efficiency, and using flow cytometry BM-MSC surface markers was checked. The PKH26 labeled BM-MSC injected into the tail vein of the normal control group and AD animal model group, 14 days after finding the PKH26-labeled BM-MSC cells in the rat hippocampus using fluorescence microscopy. Using the Morris water maze experiment comparison of AD model and BM-MSC transplantation group of spatial learning and memory ability.

RESULTSTFlow cytometry showed BM-MSC surface markers CD73 and CD105 were positive. In vitro, PKH26-labeled rate of BM-MSC was 100 %. The Morris water maze experiment comparison of BM-MSC transplantation group and AD group of animals, BM-MSC transplantation group at 13, 14 days of spatial learning and memory ability than AD animal group had significantly improved. 14 days after BM-MSCs in rat hippocampus could be found which were PKH26-positive, consistent with DAPI staining. PKH26-positive cells in animal models of AD were significantly more than those in the normal control group.

CONCLUSIONBM-MSC in AD rats not only migrates through the blood-brain barrier, but also mainly survives in the hippocampus of AD rats, and it can improve AD rat model of learning disabilities.

Alzheimer Disease ; pathology ; Animals ; Bone Marrow Cells ; cytology ; Cell Movement ; Cells, Cultured ; Disease Models, Animal ; Humans ; Injections, Intravenous ; Male ; Mesenchymal Stromal Cells ; cytology ; Organic Chemicals ; Rats ; Rats, Sprague-Dawley

Objective:Osteoprotegerin (OPG) is a key molecule negatively regulating osteoclast differentiation and activation; and the conserved mycobacterial heat shock 70 (HSP70) peptide p111-125 has also been found to inhibit inflammation reactions in chronic arthritis. BaculoDirectTM baculovirus expression system was selected to express recombinant OPG-HSP70 in insect cells.Methods:The human functional fragment (p22-194) of OPG and functional fragment (p111-125) of mycobacterial HSP70 gene were cloned into the transfer vector pENTRTM/SD/D-TOPO. The recombinant plasmid was performed an LR reaction with the BaculoDirectTM Linear DNA to generate recombinant baculovirus DNA. The cultured Sf9 insect cells were directly transferred with the recombinant baculovirus DNA,and the pure recombinant baculovirus was obtained. Then recombinant baculovirus was infected Sf9 insect cells again to express the OPG-HSP70 gene.Results:The target protein was detected at the time of 48h post infection,reached at highest yield at the time of 72h post-infection. A 28kDa protein immunostaining band was detected by Western blotting from lysate of those cells.And the purified protein was obtained by using Ni-NTA system. Functional stuies on the fusion protein showed it significantly reduce osteoclast cell number[(3.10?0.640) cells under each microscope field in treatment group by comparing to (10.70?0.817)cells in the control group] in the osteoclast inhibition test,and reduce the inflammation reaction in a delayed type hypersensitivity (DTH) mice model (P

OBJECTIVETo construct T vectors based on Xcm I recognition site and optimize the PCR fragments for its ligation.

METHODSWe firstly cloned the human histone H4 cDNA containing one Xcm I recognition site at both its 5' and 3' end into pCDNA 3.0 vector and then generated T vector with pCDNA 3.0 backbone by cutting the recombinant plasmid with Xcm I. To increase the ligation efficiency, the primers were firstly phosphorylated before DNA fragments amplification and then the PCR products were treated with Taq DNA polymerase and dATP after PCR amplification. Two DNA fragments with the length of 312 bp and 1 329 bp were ligated to it and the ligation mixture was transformed into E. coli DH5α competent cells and the positive rates of the transformants were evaluated by PCR and DNA agarose gel electrophoresis.

RESULTSOur results showed that the T vector produced by our method could ligate to the target DNA fragments with high efficiency. Besides, the phosphorylation state of the primers used for PCR amplification is also an important factor determining the cloning efficiency. What's more, as for longer DNA fragments, retreatment with Taq DNA polymerase and dATP after PCR amplification and purification could improve the ligation efficiency significantly.

CONCLUSIONOur protocol may overcome the dependence on blue/white screening to get positive clones and provide a potent way to generate T vectors and ligate them to the target PCR fragment.

Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; Histones ; genetics ; Humans ; Polymerase Chain Reaction ; methods

OBJECTIVETo assess the condition of myocardial injury after cardiopulmonary bypass (CPB) and the effects of breviscapine (BVC) on cardiac function in children undergoing open heart surgery.

METHODSThirty-six children (ASA II or III, aged 2-65 months) scheduled to receive ventricular septal defect repairing were randomly assigned to three groups, the control group treated with saline, and the BVC treated groups treated respectively with low dose (0.5 mg/kg) and high dose (1.0 mg/kg) BVC, 12 patients in each group. Saline or BVC (in volume of 15 mL) was administered intravenously after induction of anesthesia with micro-pump within 30 min. Blood levels of troponin I (cTn-I ) and malondialdehyde (MDA) were measured at different time points: pre-operation (T0), during aortic unclamping (T1), and 30 min, 1 h, 6 h, 24 h after aortic unclamping (T2, T3, T4, T5). And the time of operation, CPB, aortic unclamping, and the condition of drainage in 24 h after operation as well as the dosages of narcotics (midazolam, propofol and fentanyl) used were recorded.

RESULTSNo significant difference among groups was found in terms of sex ratio, age, body weight, time of aortic unclamping, CPB and operation, as well as the dosages of narcotics used and the volume of post-operation drainage. Compared with baseline (T0), levels of cTn-I at T1, T4 and T5 increased significantly in all three groups (P<0.01), with the peak revealed at T4; cTn-I in the control group were higher than those in the low dose BVC treated group at T1 and T4 (P<0.01), and those in the high dose BVC group at T1, T4, and T5, while it was insignificantly different between the two BVC treated groups. Level of plasmal MDA began to rise in all groups at T1 with the peak revealed at T2, it lowered after then, and reached the baseline at T5; comparison between groups showed that it was lower in the BVC treated groups than in the control group at T1-T4.

CONCLUSIONSDifferent degree of cardiac injury always happens after open heart surgery and CPB, showing high level of cTn- I within 24 h with the peak revealed at 6 h after aortic unclamping. Intravenous perfusion BVC before CPB at the dose of 0.5 or 1 mg/kg could protect the cardiac function to some extent.

Cardiopulmonary Bypass ; adverse effects ; Child ; Child, Preschool ; Dose-Response Relationship, Drug ; Female ; Flavonoids ; administration & dosage ; therapeutic use ; Humans ; Infant ; Male ; Malondialdehyde ; blood ; Postoperative Period ; Troponin I ; blood

OBJECTIVETo investigate the effect of houttuyfonate sodium (HS) on eliminating adhesion of Psedomonas aeruginosa (Pa) and forming biofilms.

METHODPa biofilms were established in 96-hold plates. MTT assay was used to evaluate the changes in metabolism of biofilms and assess the minimum eliminating concentration and minimum biofilm inhibitory concentration for adherent Pa. The colony counting method was used to observe the effect of HS on Pa adhesion and biomass in biofilms. SEM was employed to examine the effect of HS on adhesion of tested Pa and morphology of biofilms.

RESULTMEC80 and MEC50 of HS for adherent Pa was 500 mg x L(-1) and 125 mg x L(-1), respectively. Meanwhile, its SMIC80 for either early or mature biofilms of Pa was 500 mg x L(-1), and SMIC50 for early and mature biofilms of Pa were 31.25, 1.95 mg x L(-1), respectively. At the concentration of 250 mg x L(-1), the number of viable bacteria in the state of adhesion and in initial and mature biofilms decreased significantly, compared with the control group (P < 0.05). The number of bacteria on adherent carriers notably reduced under SEM. Following the continuous administration, there were no visible biofilms on carriers in the mature biofilm phase, with the biomass remarkably shrinking and the bacterial morphology changing from bacillus into coccobacillus.

CONCLUSIONHS displayed powerful effect on eliminating adherent Pa, and can inhibit Pa biofilm from being formed through continuous administration.

Alkanes ; pharmacology ; Anti-Bacterial Agents ; pharmacology ; Bacterial Adhesion ; drug effects ; Biofilms ; drug effects ; Microbial Sensitivity Tests ; Pseudomonas aeruginosa ; drug effects ; Sulfites ; pharmacology

To study immunophenotype and cytotoxicity of the immunocytes in bone marrow and peripheral blood after activation by combined cytokines, mononuclear cells (MNC) of bone marrow and peripheral blood were activated by IFN-gamma, IL-1, IL-2 and McAb-CD3 in vitro. The cell amount and morphology during culture were observed. Cytochemical staining and immunophenotype analysis were done before and after culture in two groups of MNC. Cytotoxicity was tested by MTT method. The results showed that the cell number of two groups increased obviously in culture (P < 0.05), while the peripheral blood mononuclear cells increased more markedly (P < 0.05). The cytochemical staining showed POX decrease, but PAS increase in two groups. The positive ratios of CD3(+), CD56(+) and CD38(+) cells in two groups increased obviously after culture (P < 0.05), but there was no significant difference between those two groups. CD3(+) CD56(+) cells increased obviously in peripheral blood mononuclear cells activated by cytokines (P < 0.05), but CD3(+) CD56(+) cells did not increase in bone marrow mononuclear cells. There was no significant difference between two groups' cytotoxicity. It was concluded that IFN-gamma, IL-1, IL-2 and McAb-C D3 increased cell number and cytotoxicity of both bone marrow and peripheral blood mononuclear cells that can be used in cell immunotherapy.
ADP-ribosyl Cyclase ; immunology ; ADP-ribosyl Cyclase 1 ; Antibodies, Monoclonal ; pharmacology ; Antigens, CD ; immunology ; Bone Marrow Cells ; cytology ; drug effects ; immunology ; CD3 Complex ; immunology ; CD56 Antigen ; immunology ; Cell Count ; Cell Division ; drug effects ; Coculture Techniques ; Cytokines ; pharmacology ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; drug effects ; HL-60 Cells ; Humans ; Immunophenotyping ; Interferon-gamma ; pharmacology ; Interleukin-1 ; pharmacology ; Interleukin-2 ; pharmacology ; K562 Cells ; Leukocytes, Mononuclear ; cytology ; drug effects ; immunology ; Membrane Glycoproteins ; Time Factors

OBJECTIVETo establish a multiplex RT-PCR-based reverse dot blot hybridization technique to detect influenza viruses.

METHODSObtain the HA nucleotide sequences of seasonal influenza H1N1, seasonal influenza H3N2, influenza H1N1 and human avian influenza H5N1 from GenBank. Design primers in conservative district and probes t in high variable region respectively, after analyzing the HA nucleotide sequences of influenza virus through the Vector NTI 9.0. Establish and optimize multiple RT-PCR system by comparing amplification efficiency and specificity at different primer concentrations. Establish the reverse dot hybridization system after optimizing the concentration of probes. To compare the sensitivity and specificity of this technique and the general RT-PCR Method through extracting the viral RNA of the mentioned influenza virus which are to be the reference substance.

RESULTSSuccessfully establish a multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses. This technique is 100-1000 times more sensitive than gel electrophoresis method, and it has a good specificity.

CONCLUSIONSuccessfully established multiplex RT-PCR-based reverse dot blot hybridization technique for detecting influenza viruses.

Humans ; Influenza A Virus, H1N1 Subtype ; genetics ; isolation & purification ; Influenza A Virus, H3N2 Subtype ; genetics ; isolation & purification ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; Influenza, Human ; diagnosis ; virology ; Nucleic Acid Hybridization ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

OBJECTIVETo establish a rapid quantitative analysis method for the quality control of Danhong injection extraction using near-infrared (NIR) spectroscopy.

METHODOnline collecting the NIR spectra during the mixed extraction process of Salvia miltiorrhiza and Carthamus tinctorius, partial least squares regression (PLSR) models were developed for the quality indicators rosmarinic acid (RA), salvia acid B (SaB), lithospermic acid (LA), hydroxysafflor yellow A (HSYA) and solid content (SSC), with HPLC and weight-loss method as reference methods.

RESULTThe correlation coefficients of the cross validation for RA, SaB, LA, HSYA and SSC were 0.909 3, 0.915 2, 0.901 9, 0.747 7 and 0.931 4, respectively. And the root mean square errors of cross validation (RMSECV) were 0.012 1, 0.251, 0.017 7, 0.038 1 g x L(-1) and 0.359%, respectively.

CONCLUSIONIn this study, NIR spectroscopy was successfully applied to achieve the real-time determination of the contents of RA, SaB, LA and SSC, while the performance of the HSYA calibration model needed to be improved.

Carthamus tinctorius ; chemistry ; Chemistry, Pharmaceutical ; methods ; standards ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; analysis ; isolation & purification ; Online Systems ; Salvia miltiorrhiza ; chemistry ; Spectroscopy, Near-Infrared ; methods