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BACKGROUND:In vitro culture of tissue-engineered bone is an important method for bone repair. Three-dimensional (3D) printed bone stents combined with bioreactor culture are of significance in bone tissue engineering. OBJECTIVE:To study thein vivo repair effect of the 3D printed biomaterial scaffold with human mesenchymal stem cells (hMSCs) cultured in bioreactor. METHODS:The scaffold was constructed by poly(lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA)via 3D printing and freeze-dying techniques, and then hMSCs were seeded onto the scaffold and cultured in bioreactors. Al rabbits were numbered and divided into control (No.1 and 2), experimental 1 (No. 3 and 4) and experimental 2 (No. 5 and 6) groups, and each group had two subgroups positive and negative. The rabbit left distal femur in each group was modeled into bone defect and the single PLGA/HA scaffold, PLGA/HA scaffold carrying non-induced hMSCs were implanted in the positive and negative groups of the control group, respectively; the PLGA/HA-201405-1 and PLGA/HA-201405-2carrying induced hMSCs were implanted into the positive and negative subgroups of the experimental 1 and 2 groups, respectively. Additionaly, the right femur in the experimental 2 group was driled only. The osteogenesis ability and biodegradability were determined using electron microscope, thein vivorepair was observed through CT examination, and the histopathological examination was performed after bone healing. RESULTS AND CONCLUSION:The scaffold with topological structure suitable for cellseeding was prepared. A large number of new calcium nodules were observed under electron microscope in the experimental groups indicating overt achievement in bone healing. These results suggest that the prepared scaffold achieves a good repair effect preliminarily.
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Objective To analyze the VP1-VP4 genetic region of enterovirus 71 ( EV71 ) strains isolated from children with severe or mild hand, foot and mouth disease ( HFMD) in Shenzhen in 2012. Methods EV71 strains were isolated from five children with mild HFMD and five children with severe HFMD in Shenzhen in 2012.Reverse transcription-polymerase chain reaction ( RT-PCR) method was used to amplify the sequence of VP1-VP4 genes of EV71 strains.The sequences of the amplified products were analyzed by comparing with those of the EV71 reference strains ( A, B and C genotypes) published in Gen-Bank using nucleotide alignment, amino acid alignment and phylogenetic tree analysis.Results The homo-geneity between the EV71 strains isolated from severe and mild cases was 95.1%-98.2% in nucleotides and 99.2%-100% in amino acids.The VP1-VP4 nucleotide sequences of 5 strains isolated from severe cases and 5 strains from mild cases in Shenzhen shared 87.9%-97.8% homologies in nucleotides and 97.3%-99.9% homologies in amino acids with the genotype C EV71 reference strain.The EV71 strains isolated from children in Shenzhen were highly similar with the EV71 strain (FJ439769) isolated in Fuyang in 2008 and the one isolated in Jingdezhen in 2011 (JQ806378, C4a subtype) in nucleotide sequences.Mutations at the residue 31 in the VP1 region ( N→D ) were detected in 3 strains isolated from children with severe HFMD.Conclusion All of the 10 EV71 strains isolated in Shenzhen in 2012 belonged to the sub-genotype C4a.The mutation ( aa31 N→D) in the VP1 region of EV71 might be related to the different clinical mani-festations of HFMD cases in Shenzhen area.
ABSTRACT
Porcine reproductive and respiratory syndrome virus (PRRSV) has been recognized as one of the most important pathogens of pigs throughout the world. In 2006, more than 10 provinces of China have experienced an epizootic outbreak of pig diseases characterized by high fever, reddened skin and high morbidity and mortality. From June 2006 to April 2007, we have investigated some clinical samples in Hubei province by RT-PCR and cloned several major genes, N, GP5 and NSP2 gene, shown in this study. Phylogenetic analysis of these genes revealed that the highly pathogenic PRRSV variant, ZB, was responsible for 2006 emergent outbreak of pig disease in Hubei province similar with those variants isolated from other provinces in China in 2006, and belongs to the NA-type PRRSV. In the PRRSV variants, the N and GP5 shear about 90% identity with prototypic ATCC VR-2332 and some typical NA-type Chinese isolates, except the 2850bp NSP2 gene (only shares 65% identity with ATCC VR-2332). But they all shear more than and 97% identity with other highly pathogenetic Chinese PRRSV strains. Additionally, there are extensive amino acid (aa) mutations in the GP5 protein and 2 deletions in the Nsp2 protein when compared with the previous isolates. Most of the variants found in 2006 epizootic outbreak of pig diseases in China were the farthest variants from the typical NA-type PRRSV in phylogenetic distance, and these diversities may be responsible for the differences in the pathogenicity observed between these variants and original Chinese PRRSV strains.
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Objective:To investigate the immunogenicity of SARS-CoV N DNA vaccine and the feasibility of live-attenuated Salmonella typhimurium as the carrier to deliver the N DNA vaccine.Method:The recombinant attenuated salmonella strain CS022 harboring the pcDNA-N DNA vaccine was constructed.And mouse was immunized with the recombinant strain via intranasal and oral routes.Cellular and humoral immune responses were assessed by ELISA,lymphocyte proliferation assays,ELISPOT and FACS.Result:The oral immunization with the transformed salmonellae elicited strong immune responses mainly including high level of N-specific antibody,a dramatic activation of IFN-?-secreting cells,a high level of lymphocyte proliferation,and a high level of activated CD8+ T cells.Conclusion:Live-attenuated Salmonella typhimurium could effectively deliver the SARS-CoV N DNA vaccine in vivo.These encouraging pre-clinical data provide a rational basis for undertaking a new immune style to investigate SARS vaccine.
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Objective To establish a rapid,effective,convenient method for detecting human adenoviruses and other viral pathogens using magnetic bead separation and PCR amplification.Methods A biotinylated oligonucleotide primers were hybridized to adenovirus DNA in stool samples.Setreptavidin coated magnetic beads were then added to isolate the DNA-oligonucleotide hybrid.The procedure allows for the recovery of viral DNA suitable for amplification by polymerase chain reaction.Results Ten samples collected from clinical stool were detected; six of them were positive.Results indicated that this nucleic acid separation technology is very effective in concentrating and purifying adenoviruses DNA while removing PCR inhibitors in stool samples.It also effectively increase the sensitivity of PCR amplification.Conclusion This technique can rapidly,reliably detect adenoviruses in clinical samples,and it can be used to detect other viral pathogens.
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Using specimens spiked with adenovirus or porcine parvovirus, several DNA extraction methods were evaluated for their ability to remove polymerase chain reaction (PCR) inhibitors from stool samples. It was found that PCR inhibition could be partially overcome by extracting viral DNA with guanidine thiocyanate ( GuScN) or chelex resin methods, but PCR inhibition could be hardly overcome by extracting viral DNA with traditional proteinase-K phenol chloroform extraction method. For adenoviurs (dsDNA) in stool samples, GuScN, chelex resin, proteinase-K phenol chloroform methods respectively allowed 5 TCID50, 50TCID50, 50 TCID50 titer to be detected. For porcine parvovirus (ssD-NA) in stool samples, three protocols respectively allowed 10 TCID50, 50 TCD50, 100 TCID50 to be detected. . Their rapidity and low cost make GuScN and chelex resin extraction methods the suitable for routine diagnostic testing.