Diagnosis, Differential ; Female ; Humans ; Infant, Newborn ; Nasopharyngeal Neoplasms ; pathology ; surgery ; Polyps ; pathology ; surgery ; Teratoma ; pathology

AIM: To investigate the potassium channel gene expression of myocardial sleeves of pulmonary vein and effects of amiodarone on rabbits with rapid atrial pacing.METHODS: Rabbits were divided into three groups(n=10),(1) the control group with sham operation and placebo;(2) the right atrial pacing(RAP) group at 600 beats/min with the placebo;(3) the amiodarone group treated for seven days with oral amiodarone at 100 mg ? kg-1 ? d-1.Based on RAP simultaneously,the messenger ribonucleic acid(mRNA) of specimen was measured by reverse transcription-polymerase chain reaction.RESULTS: Compared with the control group,Kv4.3(transient outward K+ current,Ito1) mRNA expression in RAP group was reduced by 51%(P

Dibenzothiophene (DBT) was used as a model compound. A bacteria strain, which can degrade dibenzo-thiophene efficiently, was obtained. This strain was identified as Pseudomonas stutzeris UP-1 according to its morphological, physiological and biochemical characters, and 16S rDNA sequence. The strain exhibits strong degradation capacity of DBT, and the end product of degradation is a kind of soluble compound. After the analysis of product of DBT degradation, it was deduced that the degradation of DBT by Pseudomonas stutzeri UP-1 is in accordance with the Kodama mechanism.

AIM: To study the effect of vascular endothelial growth factor(VEGF) on hematopoietic differentiation from mouse embryonic stem cells(ESC) in vitro.METHODS: ES-D3 was allowed to grow on mouse fetal fibroblast feeder layer,and then was developed into embryoid bodies(EB).EB cells were transferred into medium supplemented with different concentration of VEGF and VEGF+SCF for 1 week.Six groups,including.VEGF 5 ?g/L,VEGF 10 ?g/L,VEGF 20 ?g/L, VEGF 5 ?g/L+SCF,VEGF 10 ?g/L+SCF and VEGF 20 ?g/L+SCF,were designed.The group of spontaneous differentiation without cytokine(s) was used as control.Hematopoietic transcription factor GATA-2 and early hematopoietic differentiation genes(c-kit and ?-H1) were detected by RT-PCR.The content of CD34~+ cells in each group were measured by flow cytometry.The cells derived from ESC were incubated in semisolid methycellulose cultures.The numbers of total colony-forming units in culture(CFU-C) were counted by reverse microscope.RESULTS: ES-D3 grew and formed EB at day 4.VEGF had a stimulatory effect as a single factor on the expression of genes associated with early hematopoietic differentiation(GATA-2,c-kit and ?-H1),the generation of CD34~+ cells and CFU-C in EB.The effects of VEGF+SCF were the most potent in the experimental groups according to the percent of CD34~+ cells and the numbers of hematopoietic colonies.The most highest inducing efficacy was achieved in VEGF 20 ?g/L or VEGF 10 ?g/L combined with SCF.CONCLUSION: VEGF promotes the differentiation of ESC into hematopoietic cells in vitro.The strongest effect was achieved when VEGF was combined with SCF.

To produce antibodies capable of neutralizing botulinum neurotoxin type B(BoNT/B),We cloned the carboxy-terminal end of Hc containing the major determinants responsible for specific toxin,induced and purifed.The heavy-chain and kappa light-chain variable region gene repertoire of immunoglobulin were amplified individually from the spleen cell mRNA by RT-PCR and joined as a single-chain Fv(scFv)DNA fragment.These fragment were cloned into the phagemid pCANTAB5E and the phage display library was constructed.Results showed that the high affinity scFv was obtained after 4 rounds of panning,with its DNA sequence conforming to that of mouse antibody.

OBJECTIVETo explore the effect of arteriosclerosis obliterans (ASO) blood stasis syndrome (BSS) serum on vascular endothelial cell injury and to study the regulation of Taohong Siwu Decoction (TSD) on it.

METHODSUmbilical vein endothelial cell culture system was established. The serum endothelial cell injury model with ASO BSS was prepared. Low, medium, and high concentrations TSD containing serums were respectively added. The endothelial cell proliferation activity was observed by MTT method. Ultrastructures of endothelial cells were observed under transmission electron microscope. Changes of intracellular calcium ion concentration and the cytoskeleton were observed under laser confocal microscope. Contents of ET, NO, and transforming growth factor beta1 (TGF-beta1) in endothelial cell culture supernatant were detected by ELISA.

RESULTSIn ASO BSS serum group endothelial cell proliferation activities decreased, the cell structure was obviously destroyed, calcium ion concentration increased, contents of ET, NO and TGF-beta1 increased significantly (P < 0.01), and ET/NO ratio was imbalanced. After incubating with TSD drug containing serum, endothelial cell proliferation activities and injured cell structures were obviously improved; ET, NO and TGF-beta1 levels decreased (P < 0.05, P < 0.01), ET/NO ratios approximated to the normal level.

CONCLUSIONThe main mechanism of TSD for treating ASO ASS lied in improving injured vascular endothelial cells and endocrine disorder.

Arteriosclerosis Obliterans ; Cell Proliferation ; Drugs, Chinese Herbal ; therapeutic use ; Endothelial Cells ; Humans ; Medicine, Chinese Traditional ; Serum ; Transforming Growth Factor beta1 ; metabolism ; Umbilical Veins

OBJECTIVEMesenchymal stem cells (MSCs) from bone marrow are capable of differentiating into cells of different tissue lineages such as bone, cartilage, and adipose tissue and are the best candidates for tissue engineering. It is well accepted that umbilical cord blood (UCB) is a source for hematopoietic stem cells. However, controversy exists as to whether UCB contains MSCs and can serve as a source of MSCs. Therefore, the aim of this study was to explore the biological characteristics and inducing differentiation ability of in vitro expanded UCB MSCs.

METHODSUCB was collected on normal full term delivery of infants with informed consent (n = 35) obtained from the mothers. Mononuclear cells (MNCs) were isolated from UCB by gravity centrifugation and cultured with DMEM including 10% fetal bovine serum. The morphology was observed under microscope per day. Cytochemical staining was carried out and flow cytometry was used to examine the surface antigen phenotype. Fifth passage cells were transferred into a different medium and osteogenic differentiation, adipogenic differentiation, and neurogenic differentiation were assessed.

RESULTSMSCs could be isolated and cultured from MNCs of a few UCB sources. These cells displayed fibroblast-like morphology. They withstood over 20 passages without significant structural changes. These MSCs were negative for alkaline phosphatase (ALP) staining and positive for alpha-naphthol butyric acid esterase (NBE) staining. Expression of CD(29), CD(44)and CD(105), especially the human MSCs-specific markers SH-2 and SH-3 were observed, but CD(3), CD(14), CD(19), CD(34) and CD(45) could not be found, indicating that these cells were not of hematopoietic origin. Exposure of these MSCs to serum-free osteogenic condition, they could differentiate into bone cells and form mineralized matrix as evidenced by Alizarin red staining 2 weeks later. When these UCB-derived MSCs were cultured in adipogenic medium, morphologic changes in cells as well as the formation of neutral lipid vacuoles were noticeable as early as 1 week after induction and visualized by staining with oil-red O. Surprisingly, these MSCs were also able to differentiate into neuroglial-like cells. Morphology of these induced cells resembled that of neurons. Immunocytochemistry showed that they expressed Nestin and neuron-specific enolase (NSE), but not glial fibrillary acidic protein (GFAP).

CONCLUSIONUCB does contain MSCs. These MSCs, which are multipotent, could be isolated and cultured from a few UCB sources. UCB might serve as an alternative source of MSCs to bone marrow.

Alkaline Phosphatase ; metabolism ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Proliferation ; Centrifugation, Density Gradient ; Culture Media, Conditioned ; Fetal Blood ; cytology ; Flow Cytometry ; Histocytochemistry ; Humans ; Infant, Newborn ; Mesenchymal Stromal Cells ; metabolism ; Phosphopyruvate Hydratase ; metabolism

To compare the growth characteristics of non-hematopoietic adult stem cells (NASC) derived from rat fetal blood and rat bone marrow in vitro, and to study the differentiation of these stem cells into neuron-like cells in vitro, the fetal blood of pregnant rats and bone marrow of adult rats were sterilely collected; mononuclear cells (MNC) were isolated by using standard Ficoll-hypague techniques and then cultured in DMEM/LG containing 10% fetal bovine serum (FBS). The acquired NASCs were subcultured for passage. The immunophenotype of NASCs was detected by flow cytometry. The expanded NASCs were induced to differentiate into neurons-like cells by beta-mercaptoethanol (beta-ME), dimethylsulfoxide (DMSO), butylated hydroxyanisole (BHA). The specific markers of these neuron-like cells were detected by immunocytochemistry. The results showed that two kinds of subcultured NASCs showed homogeneous spindle-shaped and expressed antigens CD44 and CD54, but did not expressed CD11b and CD45. The both induced cells were similar to neuron in morphology and were positive for nestin and neuron-specific enolase (NSE), but negative for glial fibrillary acidic protein (GFAP). It is concluded that no significant difference of NASCs derived from pregnant rat fetal blood and adult rat bone marrow found in cell morphology and biological characteristics. NASCs of both origins can be induced to differentiate into neuron-like cells, so fetal blood can be regarded as another resource of NASC.
Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Embryonic Stem Cells ; cytology ; physiology ; Female ; Fetal Blood ; cytology ; Male ; Multipotent Stem Cells ; cytology ; physiology ; Neurons ; cytology ; Pregnancy ; Rats ; Rats, Wistar ; Stem Cells ; cytology

OBJECTIVETo apply the single cell nested multiplex polymerase chain reaction (PCR) to HLA typing, and analyze the influence factors on the amplification results.

METHODSSingle cell DNA templates were prepared with different methods. The exon 2, 3 and intron 2 of HLA-A, B, and exon 2 of DRBI were amplified using multiplex PCR. The second round of SSP-PCR HLA typing was carried out according to the large scale routine HLA typing results.

RESULTSEnzyme lysis method was the most efficient procedure for preparing the single cell DNA template, with a success rate (SR) of 93.3%, while the SRs of alkali lysis and freezing-thaw lysis methods were 83.3% and 73.3%, respectively. The second round amplification using enzyme lysis and SSP-PCR in 20 samples obtained a 95% success rate and a 15% allele drop out rate. The time for performing the whole procedure was less than 6 hours.

CONCLUSIONThe modified nested multiplex PCR technique is efficient for single cell HLA typing and might be applied to clinical preimplantation genetic diagnosis.

Histocompatibility Testing ; methods ; Humans ; Polymerase Chain Reaction ; methods

OBJECTIVECord blood (CB) provides a rich source of stem cells for transplantation. CB transplantation has been used widely after myeloablative therapy. One major disadvantage of CB transplantation is delayed platelet engraftment. The aim of this study was to hasten platelet engraftment by investigating the ability of different hematopoietic growth factor combinations to generate large numbers of megakaryocyte (Mk) from CB and by evaluating the biologic characteristics and function of the expanded Mk.

METHODSCB samples were obtained at the end of normal full-term deliveries with informed consent. Mononuclear cells (MNCs) were isolated from CB using Ficoll density centrifugation. MNC population was positively selected for CD(34) expression by magnetic cell sorting (MACS). CD(34)(+) cells were cultured in serum-free and stroma-free medium containing the following two different cytokine combinations: thrombopoietin (TPO) + stem cell factor (SCF) + interleukin (IL) -3 + IL-6 and TPO + SCF. Cultures were characterized after 3, 7, 10 and 14 days by flow cytometry, colony forming unit-megakaryocyte (CFU-Mk) and maturation evaluation (Mk ploidy). The expanded Mk function was examined by the platelet activation in vitro and severe combined immunodiffiency (SCID) mice transplantation in vivo.

RESULTSDifferent results were observed with different culture conditions. With the first cytokine combination optimal expansion of CD(41)(+) cells was observed on day 10, but the optimal expansion of Mk progenitors (CD(34)(+)CD(41)(+)) was observed on day 7, with a median 121 and 44-fold increase at the starting cell dose. This result was also proven by CFU-Mk. The largest numbers of CFU-Mk were also observed on day 7. The degree of maturation of Mk cells also increased as suggested by DNA content of CD(41)(+) cells, which means that CD(34)(+) cells cultured for 3 - 7 days were richer in primitive Mks, while those cultured for 10 - 14 days had greater numbers of more differentiated Mks. For the second cytokine combination, CD(41)(+) and CD(34)(+)CD(41)(+) cells were fewer than the first one, but it produced 36 and 85-fold CD(34)(+)CD(41)(+) and CD(41)(+) respectively on day 7. Platelet activation test confirmed that the expanded Mks had normal function. Therefore, the expanded Mks could be transplanted into the SCID mice bone marrow and produce human platelet in the peripheral blood of the mice.

CONCLUSIONEx vivo expanded Mk might facilitate CB transplantation and help shorten the period of post-transplant thrombocytopenia.

Animals ; Antigens, CD34 ; Cell Culture Techniques ; methods ; Cells, Cultured ; Culture Media ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; Megakaryocytes ; cytology ; Mice ; Mice, SCID