The Yellow Fever (YF) vaccine, an attenuated yellow fever 17D (YF-17D) live vaccine, is one of the most effective and safest vaccines in the world and is regarded as one of the best candidates for viral expression vector. We here first reported in China the construction and characterization of the recombinant expression vector of yellow fever 17D which contained the proteinase 2A fragment of foot-and-mouth disease virus (FMDV). Three cDNA fragments representing the full-length YF-17D genome, named 5'-end cDNA (A), 3'-end cDNA (B) and middle cDNA (C), were obtained by reverse transcription polymerase chain reaction (RT-PCR), together with the introduction of SP6 enhancer, necessary restriction sites and overlaps for homologous recombination in yeast. Fragment A and B were then introduced into pRS424 in turn by DNA recombination, followed by transfection of fragment C and the recombinant pRS424 containing A and B (pRS-A-B) into yeast. A recombinant vector containing full length cDNA of YF-17D (pRS-YF) was obtained by screening on medium lack of tryptophan and uracil. A recombinant YF-17D expression vector containing FMDV-2A gene fragment (pRS-YF-2A1) was then constructed by methods of DNA recombination and homologous recombination in yeast described above. In vitro transcription of the recombinant vector pRS-YF-2A1 was then carried out and introduced into BHK-21 cells by electroporation. Results of indirect immunofluorescence assay (IFA) and titer determination showed a stable infectious recombinant virus was gotten, whose features such as growth curve were similar to those of the parental YF-17D. The results suggest that the recombinant vector pRS-YF-2A1, by introduction of heterogenous genes via 2A region, is potential to be an effective live vaccine expression vector.
Animals ; Cell Line ; Cloning, Molecular ; Cricetinae ; Epitopes ; immunology ; Foot-and-Mouth Disease ; prevention & control ; Foot-and-Mouth Disease Virus ; genetics ; immunology ; Genetic Engineering ; Genetic Vectors ; Recombination, Genetic ; Saccharomyces cerevisiae ; genetics ; metabolism ; Vaccines, Attenuated ; Viral Vaccines ; genetics ; immunology ; Yellow fever virus ; genetics ; immunology

5' nucleotides (5'NT), a purine degradative enzyme, is capable of hydrolyzing nucleotide and acting as a phosphotransferase simultaneously. It has critical role in maintaining nucleotide metabolism balance. The present study was aimed to investigate the expression of 5'NT in bone marrow granulocytes (BMGs) from patients with acute myeloid leukemia (AML) and healthy donors comparatively. The BMGs were isolated from bone marrow of 33 patients with AML and 6 healthy donors by using lymphocyte isolating solution. The reactivity of 5'NT was detected by electron microscope and cytochemistry of cytidine monophosphate (CMP). The positive BMG ratio and their index were calculated on the base of ultrastructural observation semiquantitatively. The results indicated that electron microscopy revealed plasma membrane reacting pattern of CMP. Most BMGs from normal donors were CMP negative or exhibited lower active degree. All cases of M(0), M(1), M(2) and t (8; 21) showed high positive percentages and high indexes of BMGs, but no statistic differences between them. APL of t (15; 17) shared lower percentages and indexes than other subtypes. There was no significant difference between APL and normal donors statistically. In conclusions, the results suggested the expression of 5'NT may be associated with BMG differentiation in AML, and APL of t (15; 17) may be a highly differentiated leukemia subtype.
5'-Nucleotidase ; metabolism ; ultrastructure ; Adolescent ; Adult ; Aged ; Bone Marrow Cells ; cytology ; enzymology ; Child ; Female ; Granulocytes ; enzymology ; Humans ; Leukemia, Myeloid, Acute ; classification ; enzymology ; Male ; Middle Aged ; Young Adult

OBJECTIVEThis study was to investigate the growth and proliferation characteristics of rat bone mesenchymal stem cells (BMSCs) isolated by the method of whole bone marrow culture and to explore the effect of cell inoculation density and incubation period on cell proliferation, with an aim to provide multipotential seed cells for preventing from degenerative disease.

METHODSBone mesenchymal stem cells were isolated by the method of whole bone marrow culture and then cultured in vitro. The cell morphologic features were observed by inverted microscope. The cell surface antigens were identified by flow cytometry. The effect of cell inoculation density and culture period on cell growth and proliferation was explored by analyzing the characteristics of a ten-day cell growth curve in 96-well plates.

RESULTSFlow cytometry results showed the detection rates for CD29, CD34 and CD45 were 97.68% (7607/7788), 7.93% (661/8340) and 2.76% (215/7788) respectively, which was consistent with the expression characteristics of BMSCs surface antigens. BMSCs became uniform after three cell passages, existing in a typical shape of whirlpool or radial colony. The senescent cells started to appear at 7(th) passage, and more senescent cells were found at 10(th) passage. The growth curve for moderate inoculation density was typically S-shaped. Lag phase was found during the first two days, and logarithm growth phase was in the following three days. Plateau phase started from the 6(th) day and cell numbers decreased slightly from the 8(th) day.

CONCLUSIONThe whole bone marrow culture is an effective way to obtain BMSCs. A moderate inoculation density was more advantageous to cell proliferation, by which more seed cells could be obtained.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley

Objective: To compare the value of two 3D imaging reconstruction methods for left atria and pulmonary vein on guiding the catheter ablation for atrial fibrillation (AF). Methods: From January 2014 to January 2017, a total of 100 drug refractory paroxysmal AF patients were divided into left atria direct angiography group (n=50), and indirect angiography group (n=50). 3D CARTO system was applied for mapping and guiding the ablation procedure. Patients assigned to direct angiography group were treated as follows: intraoperative puncture of atrial septum, inject contrast agent directly into the left atrium, conduct left atrial and pulmonary venous rotation angiography, reconstruct three-dimensional image, integrate the image into real-time X-ray system to facilitate circumferential pulmonary vein isolation. Patients assigned into the indirect angiography group were treated as follows: inject contrast agent through the right ventricle, conduct delayed rotation angiography of the left atria and pulmonary vein to guide circumferential pulmonary vein fixation and ablation. The left atrial and pulmonary venous image acquisition, the operation and X-ray exposure time, the success rate and the incidence of complication of the two groups were compared. The patients were followed up for 3-6 months. Results: General clinical characteristics of the two groups were similar(all P>0.05). Ablation was successful in all 100 patients. The operation time[(112.0±21.4)min vs. (134.0±24.3)min]and X-ray exposure time((10.7±4.7)min vs. (15.8±5.2)min)were significantly lower in direct angiography group than in indirect angiography group (both P<0.01). There was no significant difference between the two groups in the immediate (86%(43/50) vs. 82%(41/50), P=0.59) and short-term (76%(38/50) vs. 72%(36/50), P=0.65) success rate and complication rate (1 aneurysm in the direct angiography group, 1 pericardial tamponade in the indirect angiography group). In-hospital mortality was zero percent. Conclusion It is safe and effective method to guide the radiofrequency catheter ablation of paroxysmal atrial fibrillation by reconstruction 3D image of left atrium and pulmonary vein. Compared with indirect angiography group, direct angiography group can improve the imaging quality of left atrium and pulmonary vein, decrease the X-ray exposure time of the ablation procedure.

OBJECTIVE: To study the relationship between osteonecrosis of femoral head after internal fixation of femoral neck fracture and serum bone metabolism, vascular active factors, and analyze the risk factors. METHODS: Total 150 patients with femoral neck fracture who underwent reduction and internal fixation from April 2016 to April 2019 were selected, including 83 males and 67 females. According to whether there was necrosis of femoral head after operation, they were divided into necrosis group(32 cases) and non necrosis group (118 cases). Before operation and 1, 3, 5 days after operation, the serum levels of beta-C terminal cross-linked telopeptides of typeⅠ collagen(β-CTX), N-telopeptide of typeⅠ procollagen(PINP), nitric oxide (NO), Endothelin-1 (ET-1) were measured. The clinical characteristics of the two groups were compared. The risk factors of postoperative femoral head necrosis were analyzed by logistic regression model. The value of serum indexes in predicting postoperative femoral head necrosis was analyzed by ROC curve. RESULTS: There was no significant difference in the levels of serum PINP and β-CTX between necrotic group and non necrotic group before operation and 1, 3 and 5 days after operation( CONCLUSION The content of serum NO and ET-1 on the first day after operation can predict the necrosis of femoral head.
Female ; Femoral Neck Fractures/surgery* ; Femur Head ; Femur Head Necrosis/etiology* ; Fracture Fixation, Internal/adverse effects* ; Humans ; Male ; Osteonecrosis ; Retrospective Studies ; Risk Factors