Over-expression of Hsp75 in neural stem cells reduced Aβ-mediated neu-rotoxicity / 中国病理生理杂志

AIM:To investigate the effect of heat shock protein 75 ( Hsp75 ) over-expression on Aβ-induced neurotoxicity in the neural stem cells and to explore its mechanism.METHODS:An adenovirus-mediated Hsp75 over-ex-pression vector was used in vitro.The mouse neural stem cell C17.2 was cultured in vitro and divided into control group, Aβgroup, negative adenovirus vector transfection group and Hsp75 over-expression adenovirus vector transfection group. The transfection and cellular immune identification were detected by fluorescence microscopy.The cell morphology was ob-served under inverted phase-contrast microscope.The cell viability and apoptosis were detected by MTT assay and flow cy-tometry, respectively.Hsp75 over-expression and cleaved caspase-3 protein level were measured by Western blot.RE-SULTS:Observation by fluorescence microscopy indicated that C17.2 cells were successfully transfected and Hsp75 gene was effectively expressed in the neural stem cells after transfection.In addition, the morphology and viability of the cells did not change and these cells did not differentiate after transfection.As compared with control group, the cell viability in Aβgroup and negative adenovirus vector transfection group was significantly decreased (P<0.05), and the cell apoptotic rate and cleaved caspase-3 level (P<0.05) were increased.As compared with Aβgroup and negative adenovirus vector transfection group, Hsp75 over-expression significantly increased the cell viability, and decreased the cell apoptosis and cleaved caspase-3 level ( P<0.05 ) .CONCLUSION: Hsp75 over-expression protects the neural stem cells against Aβ-induced injury.The mechanism may be related to inhibiting caspase-3 pathway-dependent apoptosis.

Construction of Adenovirus Vector Carrying Human HSP75 Gene and Expressing in C17.2 Neural Stem Cells / 中国康复理论与实践

Objective To construct recombinant adenovirus vector carrying the human HSP75 gene and detect its expression in C17.2 neural stem cells. Methods HSP75 gene was amplified from plasmid carrying the human HSP75 gene and inserted to the polyclonal site of adenovirus shuttle plasmid pHBAd-MCMV-GFP. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pH-BAd-MCMV-HSP75-GFP and large adenovirus helper plasmid pHBAd-BHGloxΔE1,3 Cre in mediation of LipofiterTM. The recombinant ad-enovirus was obtained and the viral titer was examined using the method of TCID50. The transfection and expression of HSP75 was detect-ed by fluorescence microscope, flow cytometer and Western blotting. Results Restriction digestion, sequencing analysis and PCR amplifica-tion revealed the successful construction of recombinant shuttle plasmid and recombinant adenovirus. The titer of recombinant adenovirus was 1.0×1010 PFU/ml. Western blotting indicated HSP75 gene could be expressed effectively in neural stem cells after transfection. Conclu-sion The recombinant adenovirus vector carrying HSP75 gene was successfully constructed and can be expressed after transfected in C17.2 neural stem cells.

Construction of Adenovirus Vector Carrying Human HSP75 Gene and Expressing in C17.2 Neural Stem Cells / 中国康复理论与实践

@#Objective To construct recombinant adenovirus vector carrying the human HSP75 gene and detect its expression in C17.2 neural stem cells. Methods HSP75 gene was amplified from plasmid carrying the human HSP75 gene and inserted to the polyclonal site of adenovirus shuttle plasmid pHBAd-MCMV-GFP. HEK293 cells were co-transfected with the constructed recombinant shuttle plasmid pHBAd-MCMV-HSP75-GFP and large adenovirus helper plasmid pHBAd-BHGloxΔE1,3 Cre in mediation of LipofiterTM. The recombinant adenovirus was obtained and the viral titer was examined using the method of TCID50. The transfection and expression of HSP75 was detected by fluorescence microscope, flow cytometer and Western blotting. Results Restriction digestion, sequencing analysis and PCR amplification revealed the successful construction of recombinant shuttle plasmid and recombinant adenovirus. The titer of recombinant adenovirus was 1.0×1010 PFU/ml. Western blotting indicated HSP75 gene could be expressed effectively in neural stem cells after transfection. Conclusion The recombinant adenovirus vector carrying HSP75 gene was successfully constructed and can be expressed after transfected in C17.2 neural stem cells.

Screening of the protein interacting with inhibitor of differentiation 1′ using yeast two hybrid method / 第三军医大学学报

Objective To obtain the protein interacting with inhibitor of differentiation1′(Id1′). Methods The recombinant bait plasmid pHybLex/Zeo Id1′ was constructed and transformed into yeast strain EGY48/ pSH18 34 to test pHybLex/Zeo Id1′ for non specific activation. Adult human lung cDNA libraries were screened to obtain true positive library plasmid. The true positive library clone was obtained by sequencing and basic local alignment sequence tool (BLAST). Results The recombinant bait vector, named as pHybLex/Zeo Id1′, was confirmed by sequencing. pHybLex/Zeo Id1′ was transformed into yeast strain EGY48/pSH18 34 and the transformants had no autonomously activated reporter genes. One true positive clone, obtained by screening of the adult human lung cDNA libraries, was confirmed to be Fyn by sequencing and BLAST. Conclusion Id1′ can interact with Fyn.