Construction of adenovirus-mediated eukaryotic expression vector co-expressing mutant hypoxia-inducible factor-1 alpha target protein and humanized Renilla reniformis green fluorescent protein reporter molecule under normoxic conditions / 中国组织工程研究

BACKGROUND: Hypoxia-inducible factor-1 (HIF-1) can regulate the co-expression of various genes, and can induce angiogenesis with integrated physiological function.OBJECTIVE: To construct a novel adenoviral eukaryotic expression vector that can co-express mutant hypoxia-inducible factor-1 alpha (HIF-1a) target protein and humanized Renilla reniformis green fluorescent protein (hrGFP) reporter molecule under normoxic conditions.METHODS: The human HIF-1α gene carried by target gene donor plasmid pCMV6-XL5-HIF1α was sequenced and the site of restriction enzyme in above gene was analyzed. Site-directed mutagenesis of three amino acids including the 402 location, the 564 location, and the 803 location in gene coding region in HIF-1α were performed by polymerase chain reaction and sequencing was also done for monitoring mutation. The HIF-1α gene mutated correctly (HIF-1αmu) was coupled to adenoviral shuttle vector pShuttle-CMV-IRES- hrGFP-1. The recombinant adenovirus shuttle vector carrying HIF-1αmu gene was transferred to BJ5183-AD-1 electroporation competent cells after sequencing identification and Pme I restriction enzyme linearization.HIF-1αmu and hrGFP gene as well as hemeo-expression elements of hrGFP gene were reconstructed into adenoviral genome plasmids using homologous recombination mechanism in bacterium. Recombinants were obtained by Pac I restriction enzyme digestion and sequencing identification.RESULTS AND CONCLUSION: Amino acids including the 402 location, the 564 location and the 803 location in gene coding region in HIF-1α had become alanine after site-directed mutagenesis. Recombinant adenoviral expressing vector was successful as confirmed by restriction enzyme digestion and sequencing. These findings demonstrate that a novel recombinant adenoviral mutant eukaryotic expression vector pAd-HIF1αmu-IRES-hrGFP-1 was successfully constructed.

Construction of adenovirus vectors carrying VEGF121-FLAG and hrGFP-1 and their expressions in bone marrow stromal stem cells / 中国组织工程研究

BACKGROUND: Vascular endothelial growth factor (VEGF) can promote angiogenesis, and has been extensively used in treatment of bone defect. However, few studies have addressed its isomer VEGF121. OBJECTIVE: To construct adenovirus vector carrying VEGF121-FLAG and humanized Renilla reniformis green fluorescent protein 1(hrGFP-1) and observe its expression in bone marrow stromal stem cells (BMSCs). METHODS: Using polymerase chain reaction technique, VEGF121 gene contained in the plasmid of pTG19T-VEGF121 was used to remove termination codon. NotI and Xho I restriction sites were added before and after gene sequence. Obtained gene subclone was moved onto pMD19-T plasmid. The pMD19-T-VEGF121 and pShuttle-CMV-IRES-hrGFP-1 plasmids underwent double enzymatic digestion. Small fragment and big fragment were retrieved utilizing gel. Subsequently, coupled reaction was conducted to complete the construction of shuttle plasmid. After measuring virus titer, BMSCs were transfected and the fluorescence intensity was observed under fluorescence microscope.RESULTS AND CONCLUSION: Recombinant adenovirus plasmid was successfully constructed by enzymatic digestion determination and gene sequence. Fluorescence microscope has shown that BMSCs transfected with recombinant adenovirus presented significantly green fluorescence expression. Thus, adenovirus vector carrying VEGF121-FLAG and hrGFP-1 gene can express in eukaryotic cells, which can be used for gene therapy for ischemic disease.

Integrating Clinical Knowledge into Pharmacology Teaching: Investigation and Estimate / 中华医学教育探索杂志

Objective:The purpose of this study was to improve teaching quality of pharmacology by integrating different form of clinical knowledge.Methods:We tried to add the clinical documents such as some related videos and pictures,many common drugs' trade-names and analysis of typical cases into teaching of pharmacology in undergraduate course in our college.Results:Both the theory examination scores and ability of aggregate analysis of cases were higher in the experiment groups than in the controlled ones.And the students considered that the teaching mode with clinical knowledge could stir up their interest in learning pharmacology and increase teaching effect.Conclusion:The teaching mode of incorporating clinical knowledge into pharmacology would be helpful to improve teaching quality of pharmacology.