OBJECTIVETo construct a recombinant lentiviral vector (pXZ208-BDDhFVIII) mediating B-domain-deleted human coagulation factor VIII (BDDhFVIII) gene and investigate its expression in HLF, Chang-Liver and MSC cells.

METHODSBDDhFVIII gene fragment was separated by endonuclease digestion and was cloned into the multiple cloning sites of pXZ208 to construct a recombinant lentiviral vector pXZ208-BDDhFVIII. Viral particles were prepared by means of three-plasmid cotransfection of 293T package cells by calcium phosphate precipitation. After infection, the coagulant activity of human FVIII in the culture medium of 293T, HLF, Chang-Liver and MSC cells was assayed by one-stage method. The gene transduction efficiency was assayed by flow cytometry (FCM). Furthermore, PCR was performed to test the integration of BDDhFVIII.

RESULTSThe infection rates of HLF, Chang-Liver and MSC were (74.52 +/- 7.57)%, (27.24 +/- 6.53)% and (42.34 +/- 5.84)% respectively. The activities of FVIII in supernatants of HLF, Chang-Liver and MSC were (54.1 +/- 5.6)%, (22.5 +/- 2.9)% and (12.5 +/- 2.7)% respectively. BDDhFVIII gene integration was detected in all the infected cells.

CONCLUSIONThe recombinant lentiviral vector pXZ208-BDDhFVIII was successfully constructed and efficiently integrated into target cells to express human FVIII activity in vitro.

Cell Line ; Factor VIII ; biosynthesis ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; Plasmids ; Transfection

AIM:To investigate the effects of evodiamine on the growth and apoptosis of human hepatocellular carcinoma Huh7 cells,and to illustrate the molecular mechanism that evodiamine enhances antitumor activity of tumors nec -rosis factor-related apoptosis-inducing ligand(TRAIL)in Huh7 cells.METHODS: The cell viability was measured by MTT assay.The cell cycle distribution was analyzed by flow cytometry.The apoptosis rate was determined by TUNEL stai-ning.The protein levels of cell cycle-and apoptosis-related proteins were detected by Western blot analysis.RESULTS:Treatment of Huh7 cells with evodiamine reduced the cell viability(P<0.05).Evodiamine induced cell cycle arrest in G2/M phase by upregulation of p27,cyclin B1, cell division cycle protein 2(Cdc2)and p-Cdc2.Evodiamine triggered apoptosis accompanied by cleavage of caspase-3 and poly(ADP-ribose)polymerase(PARP).Combination of evodiamine with TRAIL significantly reduced the cell viability and increased cleavage of caspase -3 and PARP as compared with the use of each agent alone.Moreover,evodiamine increased the expression of death receptor 5(DR5)in the Huh7 cells.CON-CLUSION:Evodiamine inhibits the cell growth by reducing the cell viability and inducing cell cycle arrest.Evodiamine also triggers cell apoptosis and enhances the sensitivity of Huh 7 cells to TRAIL by upregulating the expression of DR5.

AIM: To investigate antioxidant activities and life span prolonging effects of the extracts from the roots of Incarvillea younghusbandii Sprague, and to study the correlations between these activities and the polar intensity of the extracts. METHOD: Five extracts (IYS1, IYS2, IYS3, IYS4 and YS5) with different polar intensity were prepared. Antioxidant activities in vitro were determined by LPO inhibitory and free radicals scavenging experiments. Life span prolonging effects in vivo were evaluated by feeding Drosophila melanogaster. RESULT: Total phenolic content in extracts were solvent-dependent and decreased in the order of IYS4 > IYS1 > IYS3 > IYS5 > IYS2. Organic extracts (IYS1 and IYS4) showed excellent LPO inhibitory activity, O(2)(· -) and ·OH scavenging activity compared to ascorbic acid (or benzoic acid, or BHT), while aqueous extracts (IYS2, IYS3 and IYS5) did not. The antioxidant activities (in vitro) were solvent dependent and decreased in the order of IYS4 > IYS1 > IYS3 > IYS5 ≥ IYS2. Drosophila melanogaster was fed with organic extracts (IYS1 or IYS4) at 5.0 mg mL(-1). The mean life span were increased by 24.4% (IYS1) or 23.0% (IYS4) in female and 15.3% (IYS1) or 16.9% (IYS4) in male; the maximum life span were increased by 8.4% (IYS1) or 11.2% (IYS4) in female and 9.7% (IYS1) or 15.8% (IYS4) in male, and the survival curves were significantly shifted to the right after fifteen days in both sexes survival period. Feeding aqueous extracts (IYS2, IYS3 or IYS5) at 5.0 mg·mL(-1), the significant life span prolonging effects were not achieved. The life span prolonging effects of the extracts were solvent-dependent and decreased in the order of IYS4 ≥ IYS1 > IYS3 > IYS2 > IYS5. CONCLUSION Extracts from the roots of Incarvillea younghusbandii Sprague showed excellent antioxidant activities and significant life span prolonging effects in Drosophila melanogaster. Positive correlations existed between the antioxidant activities and total phenolic content. Life span prolonging effect was positively correlated with the total phenolic content or antioxidant activities. The extracts possess better life span prolonging effect in females than in males.
Animals ; Antioxidants ; isolation & purification ; pharmacology ; Bignoniaceae ; chemistry ; Drosophila melanogaster ; drug effects ; Female ; Lipid Peroxidation ; drug effects ; Longevity ; drug effects ; Male ; Phenols ; chemistry ; isolation & purification ; pharmacology ; Plant Extracts ; chemistry ; pharmacology ; Plant Roots ; chemistry ; Sex Factors

Objective To explore the effectiveness and safety of intravenous metoprolol by two injection methods in treatment for patients of atrial fibrillation anti rapid ventricular rates complicated with heart failure.Methods Patients of atrial fibrillation and rapid ventricular rates complicated with heart failure were administrated regular drug therapy for their heart failure,and then they were observed for half an hour.If their ventricular rates were above 100 beats/min and blood pressure equal to or above 100/60 mm Hg (1 mm Hg=0.133 kPa),they were randomly divided into three groups,the first one administrated with metoprolol 10 mg by minipump in an hour,the second one administrated with metoprolol 5 mg in 10 minutes by direct injection,and repeated by 5 mg 10 minutes later if their heart beats were still above 100 beats/min and blood pressure equal to or above 100/60 mm Hg,and the third one administrated with normal saline as control group.As either ventricular rates were equal to or lower than 60 beats/min or blood pressure lower than 90/60 mm Hg,drug administration would be terminated.Symptoms,physical signs, heart rate,blood pressure,rale on auscultation of the chest,parameters of haemodynamics,serum levels of brain natrium peptide (BNP) and atrial natrium peptide (ANP) were observed at 0 h and 1 h after drug injection.Results Clinical symptoms and physical signs of heart failure were improved significantly,heart rates and serum levels of BNP (F=15.929,14.534,all P

This study was aimed to construct a lentiviral vector carrying human coagulant factor VIII (FVIII) and to investigate its expression in 293T cells. B-domain-deleted factor VIII gene fragment (BDDhFVIIIcDNA) was obtained by enzyme digestion and cloned into lentiviral vector pXZ208 to establish the expression vector pXZ208-BDDhFVIII. Recombinant viral particles were prepared by cotransfection with packaging plasmid delta NRF and envelope plasmid VSV-G using calcium phosphate precipitation method. 293T cells were transfected by viral supernatant. Coagulant activity of FVIII, BDDhFVIIImRNA and genome integration were assayed by one-step method, RT-PCR and PCR after transfection. The results showed that 293T cells could be transfected by recombinant virus. The transfection rate of 293T was 59.57%. After transfection, the cells expressed FVIII efficiently. Detection confirmed that the activity of FVIII was 12%, 43% and 87% respectively at 24, 48 and 72 hours after infection. BDDhFVIII transcription was detected by RT-PCR from the infected cells. The gene integration in the targeted cells was also observed. It is concluded that the successfully constructed lentiviral vector is able to generate high level expression of human FVIII in 293T cells, which may provide a potential application of gene therapy to haemophilia A.
Cell Line ; Factor VIII ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Lentivirus ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism

The study was purposed to prepare the recombinant lentiviral vector pTK161 and pTK162 carrying B-domain-deleted canine factor (BDDcFVIII) gene, and to investigate whether the canine FVIII (cVIII) can be expressed in vitro. The BDDcFVIII gene was ligated behind PUB and 2OH1 promotors to create lentiviral vectors pTK161 and pTK162. Meantime lentiviral vectors pTK161' and pTK161' were produced by cloning a green fluorescent protein (GFP) into pTK151 and pTK152, which was driven by PUB and 2OH1 promotors respectively. Vector supernatant were prepared by using transfer calcium phosphate mediated-cotransfection of 293T cells. The virus vector, DeltaNRF packaging-plasmid, and VSV-G envelope-plasmid was assayed by titers and cFVIII activity in cell culture supernatant after infection into 293T cells. pTK161, pTK162, pTK161' and pTK161' were identified by restriction enzyme analyzing. The results showed that the lentiviral vectors pTK161, pTK162, pTK161' and pTK161' were successfully constructed, and the titers of pTK161' and pTK161' reached to 1.54 x 10(6) U/ml and 2.83 x 10(6) U/ml; the activity of cFVIII could be detected at 24 hours after infection of 293T cells by pTK161 and pTK162, and achieved the highest level at 72 hours later. The higher level of cFVIII activity was achieved by transfected with pTK162 than that of pTK161 (p < 0.05), which closed to the cFVIII activity in normal dog plasma. 1/4 of the highest level could be detected 6 weeks later. It is concluded that the prepared HIV1-based lentiviral vectors can infect 293T cells to express cFVIII effectively. The results provide the basis for further studying HIV-1-based lentiviral vector gene therapy for hemophilia A.
Animals ; Dogs ; Factor VIII ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; HIV-1 ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

This study was purposed to constructe the three-plasmid system of the lentiviral vector carrying the green fluorescent protein (GFP) gene and to investigate the expression of GFP in T lymphocytes of the mouse. The polypurine tract (PPT) element, ubiquinone promoter (PUB) and GFP were ligated to plasmid pLO134 using subcloning technology to construct plasmid pTK153. Human kidney 293T cells were co-transfected with the three-plasmid system containing packaging plasmid DeltaNRF, plasmid pTK153 and envelope plasmid VSV-G by using calcium phosphate DNA precipation and the expression of GFP was observed under fluorescence microscope after 12 hours. The viral particles were collected after transfection 72 hours, were frozen at -80 degrees C and were used to infect mouse T lymphocytes at multiplicity of infection (m.o.i.) of 3. The expression of GFP in mouse T lymphocytes was observed by fluorescence microscopy and fluorescence-activated cell sorting (FACS). The results showed that the transfection efficacy was 63.04 +/- 7.24% in 293T cells analysed by FACS and the viral titer was (3.09 +/- 0.61) x 10(6) U/ml. The expression of GFP was also evident in mouse T lymphocytes and the transduction efficacy was (37.98 +/- 6.26)%. It is concluded that the three-plasmid system of lentiviral vector containing GFP gene is successfully constructed and the transduction efficacy is high in mouse T lymphocytes.
Animals ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Lentivirus ; genetics ; Mice ; Mice, Inbred BALB C ; RNA, Viral ; analysis ; T-Lymphocytes ; metabolism ; Transduction, Genetic

OBJECTIVETo screen for abnormal methylation in CpG islands and CpG sites through whole genome of congenital microtia to identify their associated genes. To discuss the relationship between abnormal methylation level of genes and the etiology of congenital microtia.

METHODSResidual ear cartilage of 50 patients with microtia was collected with ear cartilage of 34 patients without ear malformations as control. Nimblegen CpG promoter array was chosen to screen the 28,226 CpG islands in the whole genome of both experimental and control groups. The genes with differential methylated CpG islands were selected. SpectroCHIP array was chosen to detect the methylation level of each CpG site in abnormal methyletion CpG islands of both experimental and control groups. The CpG sites with differential methylation level were selected.

RESULTSThere were 36 CpG islands with differential methylated level in whole genome between experimental group and control group, among which 29 CpG islands were connected with 29 named genes. In the abnormal methylated CpG islands of COL18A1, MYH14, RBMY1A1 and ZIC3, 6 differentially methylated CpG sites were found with statistical significance. The methylation level of these 6 CpG sites in experimental group and control group were COL18A1_2_CpG_170.9783 +/- 0.0235 and 0.9526 +/- 0.0589; MYH14_CpG_170.9600 +/- 0.0414 and 0.9284 +/- 0.0655; RBMY1A1_1_CpG_3.40.9966 +/- 0.0055 and 0.9914 +/- 0.0069; RBMY1A1_1_CpG_130.9648 +/- 0.0118 and 0.9757 +/- 0.0127; ZIC3_3_CpG_150.0867 +/- 0.0212 and 0.0543 +/- 0.0399; ZIC3_2_CpG_270.3775 +/- 0.1816 and 0.472 3 +/- 0.0439.

CONCLUSIONSThe DNA methylation profile of the entire genome is initially established. The abnormal methylated CpG islands of COL18A1, MYH14, RBMY1A1 and ZIC3 might be related to the pathogenesis of microtia.

Base Sequence ; Cartilage ; Case-Control Studies ; Congenital Microtia ; genetics ; CpG Islands ; genetics ; DNA Methylation ; Humans ; Promoter Regions, Genetic

OBJECTIVETo evaluate the effects of Tripterygium hypoglaucum (level) Hutch (THH) on cytokine production in acute graft-versus-host disease (aGVHD)mice and explore the mechanisms.

METHODS2 x 10(7) bone marrow cells mixed with 2 x 10(7) spleen cells from the same C57BL/6 mouse were transplanted into the myeloablative irradiated inbred BALB/c mouse to establish a aGVHD model. The experiments were designed as follows: control group (group A), CsA prophylaxis group (group B), THH prophylaxis group (group C), and combined THH with CsA prophylaxis group (group D). aGVHD was assessed by histologic changes of skin, liver and intestines. Chimerism was detected by H-2b molecular expression on recipient mice bone marrow cells with flow cytometer. Serum concentrations of IFN-gamma, IL-4 and IL-10 were determined by ELISA.

RESULTSThe serum concentrations of IFN-gamma in group B, C, D were significantly lower than that in group A, while those of IL-10 was significantly higher than that in group A (P < 0.05). There was no changes in concentration of IL-4 in all the groups (P > 0.05). The median survival time for group A was nine days, while that of group B, C, D each was more than 30 days being significantly longer than that of group A (P < 0.05). The recipient mice of group A displayed significant clinical symptoms of GVHD, and died within 20 days; whereas those of group B, C, D showed only ruffled fur and uplift posture. Histologic changes of liver and intestines in group B and C displayed a few lymphocytes infiltration while the histologic morphology of skin, liver and intestines in the survived mice of group D was normal. Allogeneic chimerism rates of group B, C, D at day 30 after allo-BMT were (99.18 +/- 0.58)%, (97.68 +/- 0.59)%, and (99.15 +/- 0.11)%, respectively.

CONCLUSIONTHH could regulate the production of cytokines and prevent aGVHD. THH and CsA at low dose combination showed synergic effect in preventing aGVHD.

Animals ; Bone Marrow Transplantation ; Disease Models, Animal ; Female ; Graft vs Host Disease ; blood ; prevention & control ; Interferon-gamma ; blood ; Interleukin-10 ; blood ; Interleukin-4 ; blood ; Lymphocyte Transfusion ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Phytotherapy ; Tripterygium ; chemistry

OBJECTIVETo observe the effects of the three-period treatment theory of bone fracture in TCM (Traditional Chinese Medicine) on VEGF and VEGF mRNA expression in the issues of outer periosteum, endosteum and bone marrow of rabbits, and to explore the rationality of phasing method in TCM in treating fracture.

METHODS3 mm bone defection were made at lower one third part of both radius in 140 male healthy rabbits. The rabbits were randomly divided into four groups, including three-period treatment group (TTG), one-period treatment group(OTG), positive medicine treatment group(PTG) and model control group (MCG). The rabbits in TG were treated with three-period treatment, rabbits in OTG were treated with one-period treatment, rabbits in PTG were fed by Guzhe-Cuoshangsan (a Chinese patent medicine which was used to treat bone fracture), rabbits in model control group were given no prescription or drug but distilled water as same dose as that of other groups. At day 3, 6, 9, 14, 28, 42 and 56, five rabbits from every group were randomly selected and were killed by aeroembolism. The left radiuses were taken out as the research object. Immunohistochemistry stain and in situ hybridization stain were performed to examinate the VEGF and VEGF mRNA expression in the outer periosteum, endosteum and bone marrow.

RESULTSThe VEGF and VEGF mRNA expression of all TCM treatment groups were enhanced in the outer periosteum, endosteum and bone marrow at different time points in fracture healing. The VEGF and VEGF mRNA expression in the three tissues of TTG had the tendency of higher than that of the other groups at the most time points after operation.

CONCLUSIONTreating fracture in stages has more predominant effect on the expression of VEGF and VEGF mRNA in the outer periosteum, endosteum and bone marrow than that of treating fracture with single prescription or drug.

Animals ; Bone Marrow ; metabolism ; Fractures, Bone ; metabolism ; therapy ; Male ; Medicine, Chinese Traditional ; Periosteum ; metabolism ; RNA, Messenger ; analysis ; Rabbits ; Vascular Endothelial Growth Factors ; analysis ; genetics