ABSTRACT
Objective To investigate the expression of glucose transporter 1(GLUT 1) and its transcription activity in the liver of burned rats Methods The Wistar rats inflicted with 30% TBSA full thickness flame burn on the back were sacrificed at 0 5, 1, 2, 4, 8 and 16 h after burn GLUT 1 protein levels in the rat livers were determined by Western Blotting with the reference to those in 6 normal rats The liver cells were transfected with Construct A, and 24 h later subjected to hypoxia (1%O 2) to mimic the hypoxia environment The samples were harvested at 3, 6 and 12 h and determination of the reporter gene luciferase and pSV ? galactosidase activities was performed Results ①Compared with that in the normal control, GLUT 1 protein level in the liver was significantly increased( P
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Objective To construct a prokaryotic expression vector for a fusion protein, TAT protein transduction domain (PTD) and the PAS-B domain of hypoxia inducible factor 1?(HIF-1?), and then express and purifr the fusion protein. Methods The expression plasmids pTAT-PAS-B, pET-PAS-B and pTAT-EGFP were constructed respectively, and transformed into E. coli. BL21(DE3)pLysS strain to be induced by IPTG. The obtained proteins were analyzed by SDS-PAGE and Western blotting. The fusion protein were purified with Ni-NTA-His affinity chromatography. Results The three recombinant plasmids were constructed successfully. The objective fusion proteins were obtained by optimizing the conditions for expression and purification. Conclusion The successful expression and purification of the fusion protein TAT-HIF-1?PAS-B has laid the foundation for using it to modulate the activity of HIF-1? in vivo.
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Objective To explore the mechanisms of hypoxia-induced expression of glucose transporter 1 (GLUT-1) in rat liver cells. Methods Wild type hypoxia response element (HRE) plasmid (construct N) containing the potential HIF-1 binding site was constructed by using construct A which contains the full length of the 5′-flanking region of the rat GLUT-1 gene as a template of PCR, and the PCR product was subcloned into the reporter plasmid pGL3-Promoter. Mutation type HRE plasmid (construct M) was made using a two-step overlapping PCR strategy. Then the liver cells were transfected with constructs N and M, respectively. At 24 h after transfection, the cells were subjected to hypoxia (1% O 2) to mimic the hypoxic environment caused by burn for 12 h. The samples were harvested and the determination of the reporter gene luciferase activity and pSV-?galactosidase activity was performed. Results Constructs N and M were successfully constructed and the liver cells were successfully transfected with construct N or construct M. Hypoxia induced more enhanced luciferase activity of constructs N and M as compared with the control (P
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Objective To obtain differentiated osteoblast-specific inactivation of fgfr1 mice Methods To obtain fgfr1△/+/OC-CreTG/+ mice,fgfr1flox/flox mice obtained from NIH were crossed with OC-Cre mice To obtain fgfr1△/△/OC-CreTG/+ mutant mice,fgfr1△/+/OC-CreTG/+ further crossed with themselves or fgfr1flox/flox mice After fgfr1△/△/OC-CreTG/+ crossed with fgfr1flox/flox mice,half of their offspring were mutant mice Results Differentiated osteoblast-specific fgfr1 knockout mice were obtained Conclusion fgfr1△/△/OC-CreTG/+ mice were obtained through proper crossing strategy,which provides a suitable platform for studying fgfr1 function in bone development and fracture healing