AIM:To explore the effects of αA-crystallin in astrocyte gliosis after optic nerve crush(ONC) and the mechanism of α-crystallin in neuroprotection and axon regeneration.METHODS:ONC was established on the SpragueDawley rat model and αA-crystallin(10^(-4) g/L,4 μL) was intravitreously injected into the rat model.Flash-visual evoked potential(F-VEP) was examined 14 d after ONC,and the glial fibrillary acidic protein(GFAP) levels in the retina and crush site were analyzed 1,3,5,7 and 14 d after ONC by immunohistochemistry(IHC) and Western blot respectively.The levels of beta Tubulin(TUJ1),growth-associated membrane phosphoprotein-43(GAP-43),chondroitin sulfate proteoglycans(CSPGs) and neurocan were also determined by IHC 14 d after ONC.RESULTS:GFAP level in the retina and the optic nerve significantly increased 1d after ONC,and reached the peak level 7d post-ONC.Injection of αA-crystallin significantly decreased GFAP level in both the retina and the crush site 3d after ONC,and induced astrocytes architecture remodeling at the crush site.Quantification of retinal ganglion cell(RGC) axons indicated αAcrystallin markedly promoted axon regeneration in ONC rats and enhanced the regenerated axons penetrated into the glial scar.CSPGs and neurocan expression also decreased 14 d after αA-crystallin injection.The amplitude(N1-P1) and latency(P1) of F-VEP were also restored.CONCLUSION:Our results suggest α-crystallin promotes the axon regeneration of RGCs and suppresses the activation of astrocytes.
Wei-Yang ShaoXiao LiuXian-Liang GuXi YingNan WuHai-Wei XuYi Wang
AIM: To recombine the human alpha B-crystallin(αBcrystallin) using gene cloning technology and prokaryotic expression vector and confirm the biological activity of recombinant human αB-crystallin. METHODS: Cloning the human αB-crystallin cDNA according to the nucleotide sequence of the human αBcrystallin, constructing the pET-28/CRYAB prokaryotic expression plasmid by restriction enzyme digestion method, and stably expressing transformed into the Escherichia coli(E. coli) DH5 alpha. The recombinant human αB-crystallin was purified by Q sepharose. By enzyme digestion analysis, Western blotting and sequencing, the recombinant human αB-crystallin was identified and the activity of its molecular protein was detected.RESULTS: Compared with the gene bank(GeneBank), the cloned human sequence of human αB-crystallin cDNA has the same open reading frame. Identification and sequencing of the cloned human αB-crystallin cDNA in prokaryotic expression vector confirmed the full length sequence, and the vector was constructed successfully. The E. coli containing plasmid pET-28/CRYAB induced by isopropyl-β-D-thiogalactoside successfully expressed the human αB-crystallin. Insulin confirmed that the recombinant human αB-crystallin has a molecular chaperone activity. CONCLUSION: The prokaryotic expression vector pET-28/CRYAB of recombinant human αB-crystallin issuccessfully constructed, and the recombinant human αBcrystallin with molecular chaperone activity is obtained, which lay a foundation for the research and application of the recombinant human αB-crystallin and its chaperone activity.
