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

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

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 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

Objective·To investigate safety of laparoscopic surgery in diagnosis and treatment of gynecologic diseases in elderly women over 70 years old. Methods·A total of 420 cases of elderly patients over 70 years old from January 2009 to December 2016 were retrospectively analyzed in Shanghai Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. According to ages, the patients were divided into elderly group (70-80 years old,including 70 years old) and advanced age group (80 years old and above); according to surgical methods, the patients were divided into laparoscopy group and laparotomy group. The ages, American Society of Anesthesiology (ASA) classifications, body mass indexes (BMI), preoperative complications, surgical methods, intraoperative circumstances, postoperative pathology results and postoperative complications were analyzed. Results·There were no significant differences in age, ASA classification and BMI between laparoscopy group and laparotomy group in the same age group (P<0.05). Most patients had various chronic diseases before operations. Taking total hysterectomy as an example, laparoscopy groups in the two age groups were superior to laparotomy groups in the comparison of surgical time, bleeding volume, anal evacuation time and length of hospital stay (P<0.05). In the two age groups,laparoscopic malignant tumor radical surgery took less time, less bleeding and shorter anal evacuation time than laparotomy (P<0.05). Elderly laparoscopy group had less poor wound healing cases than elderly laparotomy group (P<0.05), while there was no statistical difference in other postoperative complications (P>0.05). All the patients completed the surgeries without perioperative deaths. Conclusion·Laparoscopy is safe in the diagnosis and treatment of gynecological benign and malignant diseases in elderly women with shorter operation time, less bleeding and faster postoperative recovery compared with laparotomy.

Animals ; Global Health ; History, 20th Century ; Humans ; Influenza A virus ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; history ; mortality ; virology ; Orthomyxoviridae Infections ; epidemiology ; veterinary ; virology ; Swine ; Swine Diseases ; epidemiology ; virology

Animals ; History, 20th Century ; Hong Kong ; epidemiology ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; epidemiology ; history ; virology ; Molecular Sequence Data ; Phylogeny

OBJECTIVETo investigate the cell-killing effect of adenovirus-mediated TK-ganciclovir (GCV) gene therapy in combination with tumor necrosis factor-alpha (TNF-alpha) against murine bladder carcinoma cells in vitro.

METHODSMurine bladder carcinoma MB49 cells were transfected with the adenoviral vector containing TK gene and green fluorescent protein (GFP) gene. The transfection efficiency was observed and the TK gene expression in the transfected cells was detected by RT-PCR. The survival rate of MB49 cells in response to TNF-alpha treatment and that of the TK gene-transfected cells after treatment with GCV and GCV+TNF-alpha were determined by MTT assay. The apoptosis of the cells after the treatments was analyzed by flow cytometry.

RESULTSIn cells transfected with TK gene, the cell inhibition rate increased gradually with the increment of GCV and TNF-alpha concentration. GCV in combination with TNF-alpha resulted in significantly increased killing efficiency of the cells as compared with GCV or TNF-alpha treatment alone, and the effect of the combined treatment was enhanced as the TNF-alpha concentration increased. GCV treatment (50 microg/ml) alone produced a cell killing rate of (24.39-/+1.10)%, and when combined with 5 microg/ml TNF-alpha, the rate was increased to (40.05-/+0.97) %, and further to (65.47-/+0.67) % when TNF-alpha concentration increased to 20 microg/ml. Flow cytometry revealed obvious apoptosis of the cells 8 h after treatments with TK/GCV, TNF-alpha, or TK/GCV+TNF-alpha, and the combined treatment resulted in the highest cell apoptotic rate.

CONCLUSIONTK/GCV in combination with TNF-alpha can enhance the effect of suicide gene therapy against murine bladder carcinoma cells and effectively induce apoptosis of the cells.

Adenoviridae ; genetics ; Animals ; Antiviral Agents ; pharmacology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Ganciclovir ; metabolism ; pharmacology ; Genetic Therapy ; methods ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Reverse Transcriptase Polymerase Chain Reaction ; Thymidine Kinase ; genetics ; metabolism ; Transfection ; Tumor Necrosis Factor-alpha ; pharmacology ; Urinary Bladder Neoplasms ; genetics ; metabolism ; pathology