ObjectiveTo isolate and identify endothelial progenitor cells (EPCs) from human umbilical cord,and to study the cell proliferation and gene transfection of green fluorescent protein plasmid in vitro.MethodsEPCs were isolated from human umbilical cord in enzyme digestion method.The biological characteristics of EPCs were identified by flow cytometry and laser confocal microscope.The enhanced green fluorescent protein (EGFP) gene transfection mediated by EPCs was investigated using Lipofectamine 2000 as transfection reagent.ResultsEndothelial progenitor cells isolated from umbilical cord formed typical endothelial cell colony 9 days later.These cellsdisplayed an improved positive expression of CD133 and kinase insert domain receptor (KDR).The endotheliallineage characteristics of expanded cells were confirmed by fluorescein isothiocyanate (FITC)-UEA-1 binding and DiI-ac-LDL uptake assay with the aid of laser confocal microscope.The transfection results demonstrated high expression of EGFP taking EPCs as host cell.ConclusionEndothelial progenitor cells isolated from umbilical cord can be propagated and induced to differentiate into endothelial cells in the appropriate culture conditions.EPCs demonstrated to be an ideal carrier for gene and cell therapy.

ObjectiveTo investigate the cytotoxicity and gene transfection mediated by NMPCS/DNA nanoparticles.MethodsN-methylene phosphonic chitosan (NMPCS) was synthesized using one-step reaction under homogeneous conditions.The NMPCS/DNA nanoparticles were prepared using complex coacervation method.The cytotoxicity of NMPCS alone and its complexes with plasmid DNA were determined by MTT assay on HeLa cells.The gene transfection mediated by NMPCS/DNA nanoparticles were investigated using pGL3control vector as reporter gene.ResultsThe MTT results suggested that the NMPCS and NMPCS/DNA complexes showed significantly lower cytotoxicity than PEI and PEI/DNA complexes,respectively.The gene transfection mediated by NMPCS/DNA nanoparticles were greatly improved compared with unmodified chitosan.ConclusionNMPCS would demonstrate great potential as a novel,safe,efficient non-viral vector for gene delivery.

OBJECTIVETo evaluate the feasibility and stability of chemically conjugating IgM on collagen films.

METHODSIgM was labeled with 125I using the chloramine-T method. Six collagen films were randomly divided into two groups. In chemical coupling group 125I-labeled IgM was chemically coupled with the films through N-succinmiclyl-3- (2-pyridyl-dithio) propionate reaction. In control group 125I-labeled IgM was absorbed onto collagen films. The amount of IgM on the collagen films and the amount of IgM remained on the films after extensive rinsing with phosphate buffered saline were monitored by counting the radioactivity of 125I.

RESULTSThe amount of antibodies loaded onto collagen films in the chemical coupling group was 15 times higher than that on the control films, showing significant statistical difference (P < 0.01). And the stability of conjugation antibodies on collagen films was significantly better than the control films.

CONCLUSIONChemical coupling is an effective approach to immobilize antibodies on collagen for further plasmid DNA tethering.

Angioplasty, Balloon, Coronary ; instrumentation ; Animals ; Antibodies, Antinuclear ; metabolism ; Cattle ; Coated Materials, Biocompatible ; chemistry ; metabolism ; Collagen ; chemistry ; metabolism ; Genetic Vectors ; Immunoglobulin M ; metabolism ; In Vitro Techniques ; Mice ; Protein Binding ; Stents ; Surface Properties

OBJECTIVETo explore the feasibility of using an endovascular metal stent as a highly efficient and site-specific gene delivery system.

METHODSStents were formulated with a collagen coating. Anti-DNA monoclonal antibodies were covalently bound to the collagen surface by a cross linking reagent of N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Binding capacity and stability of antibody and plasmid DNA on stents were quantified by radioactive labeling. The gene transduction efficiency was evaluated in cell culture and in rabbits.

RESULTSThe amount of antibodies binding on collagen matrix through SPDP reaction was 15 times higher than that of through physical absorption (P < 0.005). The binding stability of plasmid was significantly better than the control groups (P < 0.01). There was no harmful effect on cell growth with the anti-DNA antibody modified stents. The stents retrieved from cell culture after 72 hours of incubation in A10 cells showed numerous transducted cells only infiltrating the surface coating indicating a highly localized and efficient gene delivery pattern. Results of in vivo gene transfer by this modified stent revealed (2.8 +/- 0.7)% of total cells transduction and the higher transduction location was neointimal layer (about 7%). No distal spread of vector was detectable in the anti-DNA antibody modified stent implantation animals.

CONCLUSIONSAnti-DNA antibody modified stents represent a novel highly efficient and site-specific gene delivery system which can deliver various kinds of plasmid vectors. The release of plasmid DNA tethered on the stents could be controlled in some conditions. This novel system provided a novel platform for cardiovascular site-specific gene therapy.

Animals ; Antibodies, Antinuclear ; immunology ; Antibodies, Monoclonal ; immunology ; Cells, Cultured ; Coated Materials, Biocompatible ; Collagen ; DNA ; genetics ; Gene Transfer Techniques ; Genetic Vectors ; Male ; Mice ; Plasmids ; Rabbits ; Stainless Steel ; Stents

OBJECTIVETo investigate the cytotoxic effect of multi-walled carbon nanotubes (MWCNTs) on human liver L02 cells and its relevant mechanism.

METHODSMWCNTs, carboxyl modification MWCNTs (MWCNTs-COOH), and hydroxyl modification MWCNTs (MWCNTs-OH) were characterized by transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. The carbon nanotubes at concentrations of 12.5, 25, 50, 100, and 200 μg/ml were incubated with human liver L02 cells for 24, 48 and 72 hours, respectively. The cell viability was evaluated by water soluble tetrazolium salts assay and the intercellular reactive oxygen species induced by the carbon nanotubes were detected by 2', 7'-dichlorodihydrofluorescein diacetate method.

RESULTSTransmission electron microscope showed that the average outside diameters (10 to 20 nm) and the average length (10 to 30 μm) of the three MWCNTs were similar. Scanning electron microscope indicated that the three MWCNTs had a similar surface topography. X-ray photoelectron spectroscopy demonstrated that the MWCNTs-COOH and MWCNTs-OH had relatively high peak areas at 289 and 286ev, respectively,indicating that they have been modified by carboxyl and hydroxyl groups,respectively. Water soluble tetrazolium salts assay showed that the MWCNTs-COOH was less cytotoxic when compared to MWCNTs which demonstrated to be slightly more cytotoxic than MWCNTs-OH. The capability to induce increase in intracellular reactive oxygen species was in the following order: MWCNTs > MWCNTs-COOH > MWCNTs-OH.

CONCLUSIONSModification of MWCNTs with carboxyl group and hydroxyl group improves the biocompatibility of MWCNTs to some extents. MWCNTs-COOH has better compatibility than MWCNTs at the low concentration,and MWCNTs-OH showed better compatibility than MWCNTs after 48 hours. Different mechanisms may be involved in the interaction between cells and the MWCNTs with different chemical surfaces.

Cell Survival ; drug effects ; Cells, Cultured ; Hepatocytes ; drug effects ; metabolism ; Humans ; Nanotubes, Carbon ; chemistry ; toxicity ; Reactive Oxygen Species ; metabolism

OBJECTIVETo generate recombinant human tissue factor pathway inhibitor (TFPI) in Pichia pastoris.

METHODSTo improve the expression of TFPI, a silent mutation was generated at the specific site of TFPI cDNA. Both wild-type TFPI cDNA and mutated TFPI cDNA were cloned into the expression vector pPic9. The constructed plasmids were subsequently transformed into Pichia pastoris cells GS115 and KM71, and the transformants were confirmed by polymerase chain reaction and DNA sequencing. The expression of recombinant protein was induced by addition of 0.5% methanol in the culture medium. The cell culture medium after induction was concentrated through ultra filtration. The recombinant protein was further purified by a three-step process (Heparin-sepharose CL-6B affinity chromatography, DEAE-Sepharose Fast Flow affinity chromatography, and Sephadex G75-gel filtration). The amount of the recombinant protein was quantified with gel imaging system. The activity of the recombinant protein was analyzed by the chromogenic substrate assay.

RESULTSThe amount of TFPI expressed in the mutated clone (1 mg/L) was much higher than that in the wild type clone (0.1 mg/L). The TFPI activity in the recombinant GS115 cells could be detected 12 hours after induction and reached the peak at 36 hours, while the TFPI activity in the recombinant KM71 cells started to show up at 24 hours after induction and reached the peak at 72 hours. The expression of recombinant protein in the silent mutant was significantly higher than those of wild type clone in both GS115 and KM71 host cells. The relative molecular mass of recombinant TFPI was approximately 42 000.

CONCLUSIONIntroduction of the silent mutation at the specific site of TFPI cDNA can increase the recombinant protein expression in Pichia pastoris, which is much higher than that in insect cells or saccharomyces cerevisiae.

Humans ; Lipoproteins ; biosynthesis ; genetics ; Mutation ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the effect of the gene nanoparticles using chitosan (CNP), arginine modified chitosan (ANP), or hexadecylated chitosan (HNP) as carriers on the human normal liver cell line L02.

METHODSCNPs, ANPs, and HNPs were prepared using complex coacervation method. The size and zeta potential of the gene nanoparticles were measured using Zetasizer nanoZS. The nanoparticles at concentrations of 5, 10, 30, and 50 microg/ml (based on the content of DNA) were incubated with L02 cells, respectively. The cell viability was evaluated by MTT assay, and the effect of the gene nanoparticles on the cell apoptosis was analyzed by flow cytometry.

RESULTSThe zeta potential of the gene nanoparticles ranged from 12.10 to 14.63 mV, and their diameters ranged from 148.07 to 179.47 nm. MTT assay showed that the viability of L02 cells began to decrease when the concentration of CNPs reached 30 microg/ml and higher. Furthermore, the CNPs could induce cell apoptosis as the concentration of CNPs reached 30 microg/ml and higher.

CONCLUSIONCNPs can induce L02 cell apoptosis at relatively higher concentrations.

Apoptosis ; Cell Line ; Cell Survival ; Chitosan ; chemistry ; DNA ; chemistry ; genetics ; Gene Transfer Techniques ; instrumentation ; Humans ; Nanoparticles ; chemistry

Objective To investigate the prevalence and major risk factors of peripartum thromboembolic disease in different regions of Guangdong province.Methods Data from 169 218pregnant women in different regions of Guangdong province from January 2005 to June 2010 were analyzed retrospectively.The prevalence and epidemiological characteristics of thromboembolic disease during pregnancy or puerperium were investigated.Results Of the studied population,( 1 )20 l cases ( 1.3‰ ) suffered from thromboembolic disease during pregnancy or puerperium including 128 cases of deep vein thrombosis (DVT),68 cases of cerebral venous thrombosis (CVT) and 5pulmonary embolism,the prevalence rates were 0.8‰,0.4‰,and 0.02‰ respectively.(2) Risk factors in different regions showed that,in the Pearl River Delta area,the major risk factors for DVT would include previous or family history of thrombosis,pregnancy complications,with medically involved diseases,prolonged bed rest and pregnancy weight gain ＞ 15 kg etc.While in castern,western,northern parts of Guangdong,the major risk factors for DVT would include pregnancy weight gain ＞ 15 kg,prolonged bed rest,preeclampsia,cesarean section and complications during pregnancy.In Pearl River Delta region,the major risk factors for CVT would include eclampsia,preeclampsia,pregnancy complications,prolonged bed rest ＞3 days,past history or family history of thrombosis.While eclampsia,preeclampsia,advanced age or younger age,pregnancy weight gain ＞15 kg,complications during pregnancy were the major risk factors for CVT in the eastern,western or northem parts of Guangdong.Conclusion Prevalence and major risk factors of peripartum thromboembolic disease in different regions of Guangdong were different.It was crucial to take effective measures in pregnant women with different epidemiological characteristics and risk factors to prevent and reduce the incidence of peripartum thromboembolic disease.

Background:Transcranial alternating current stimulation (tACS) offers a new approach for adult patients with major depressive disorder (MDD).The study is to evaluate the efficacy and safety of tACS treating MDD.Methods:This is an 8-week,double-blind,randomized,placebo-controlled study.Ninety-two drug-naive patients with MDD aged 18 to 65 years will receive 20 daily 40-min,77.5-Hz,15-mA sessions of active or sham tACS targeting the forehead and both mastoid areas on weekdays for 4 consecutive weeks (week 4),following a 4-week observation period (week 8).The primary outcome is the remission rate defined as the 17-item Hamilton depression rating scale (HDRS-17) score ≤7 at week 8.Secondary outcomes are the rates of response at weeks 4 and 8 and rate of remission at week 4 based on HDRS-17,the proportion of participants having improvement in the clinical global impression-improvement,the change in HDRS-17 score (range,0-52,with higher scores indicating more depression) over the study,and variations of brain imaging and neurocognition from baseline to week 4.Safety will be assessed by vital signs at weeks 4 and 8,and adverse events will be collected during the entire study.Discussion:The tACS applied in this trial may have treatment effects on MDD with minimal side effects.