| Optic nerve injury is a frequently occurring disease. Because optic nerve can not regenerate after injury, how to protect the RGCs and promote axonal regeneration after optic nerve injury has always been a focus in neuroscience study. For nearly 10 years, research shows that?-crystallin can promote axon regeneration after optic nerve injury, which gives hope for treatment of optic nerve injury. Native?-crystallin consists of two types of homologous subunits,?A- and?B-crystallins.?B-crystallins is one of the most representative member of mammalian small heat shock protein family, shares the common features with other nine members. Meanwhile, it has a strong anti-apoptotic effect; its interaction with cytoskeleton participates in maintaining cell structure and plays an important role in cytoprotection. Recent studies reveal that heat shock protein has a strong relationship with neurological diseases acting as a protective molecular chaperone. The positive treatment effect of recombinant?B-crystallin for multiple sclerosis had been proved in animal experiment. However, the role of recombinant human?B-crystallin in optic nerve survival and/or regeneration had not been understood well up to now.ObjectiveTo establish recombinant protein expression system for recombining?B-crystallin and fusion protein Trx-?B-crystallin, and investigate the protective effect of recombinant human?B-crystallin on retinal ganglion cells after optic nerve injury with establishing injury models of rats. To analyze the curative effect of recombinant?B-crystallin and fusion protein Trx-?B-crystallin on optic nerve injury with electron physiologic examination, determine effect of recombinant?B-crystallin and fusion protein Trx-?B-crystallin on important organs of body with microhistologic examination, and provide experimental foundation of recombinant?B-crystallin for retinal ganglion cell injury and optic nerve injury.Methods1 Full length?B-crystallin cDNA was cloned into pET28a and pET32a vector. The expressive plasmid pET28a-?B-crystallin and pET32a-?B-crystallin which contained the cDNA of the?B-crystallin under a T7-promoter were transformed into competent Escherichia coli BL21(DE3) cells and then were induced with IPTG. The purified recombinant human?B-crystallin and fusion protein Trx-?B-crystallin were obtained by purification, and were identified by SDS–PAGE. Western blotting and protein spectrum analysis were used to determine the recombinant protein. Micromolecule heat shock protein insulinum experiment was used to validate the molecular chaperone activity of recombinant human?B-crystallin and the fusion protein Trx-?B-crystallin, and the difference of activity between two molecular companies were compared and analyzed.2 Optic nerve injury rat models were established in adult Long Evans rats. Intravenous injection of recombinant human?B-crystallin, fusion protein Trx-?B-crystallin, Trx protein and normal sodium were taken. Number of survived retinal ganglion cells was observed at 2 weeks and 4 weeks after injury by fluorescen marking and immunofluorescence staining. Numbers of retinal ganglion cells were observed and compared among groups.3 Optic nerve injury rat models were established in adult Long Evans rats. Intravenous injection of recombinant human?B-crystallin, fusion protein Trx-?B-crystallin, Trx protein and normal sodium were taken. Visual function of rats in each group was observed at 2 weeks and 4 weeks after injury with flash visual evoked potential. Effect of recombinant human?B-crystallin, fusion protein Trx-?B-crystallin on recovery of visual function after injury was investigated.4 Behavior changes of rats in each experimental group were observed at 2 weeks and 4 weeks after injury. Histopathologic examination was taken in heart, liver, spleen and kidney to evaluate safety of recombinant human?B-crystallin, fusion protein Trx-?B-crystallin.Results1 The recombinant human?B-crystallin and fusion protein Trx-?B-crystallin was cloned and expressed in Escherichia coli. The active recombinant proteins could be purified from the Escherichia coli.2 The recombinant human?B-crystallin and fusion protein Trx-?B-crystallin were with molecular chaperone activity, and the fusion protein Trx-?B-crystallin activity was with higher molecular activity.3 Flurogold retrograde tracing showed that intravenous injection of the recombinant human?B-crystallin and fusion protein Trx-?B-crystallin could rescue survival of retinal ganglion cells, and the survival rate of RGCs significantly increased at 2 weeks and 4 weeks after injury compared with control groups(P<0.05).4 Enhanced survive of RGCs was also observed after intravenous injection of the recombinant human?B-crystallin and fusion protein Trx-?B-crystallin by immunofluorescence staining method. RGCs was significantly increased compared with that of the untreated controls at 2 weeks and 4weeks after injury (P<0.05).5 Amplitude of P1 wave decreased and latency P1 wave increased at 2 weeks after injury. The P1 wave amplitude of the recombinant human?B-crystallin group and the fusion protein Trx-?B-crystallin group were higher than the control groups and the latency of the recombinant human?B-crystallin group and the fusion protein Trx-?B -crystallin group were shorter. But no difference was found in amplitude and latency of P1 wave at 4 weeks after injury.6 No difference was found in behavior changes of rats among recombinant human?B-crystallin group and the fusion protein Trx-?B-crystallin group, Trx protein and normal sodium group, and there was without obvious abnormal in histopathologic examination of organs of body at 4 weeks after injury.Conclusions1 Escherichia coli expression system can successfully express recombinant human?B-crystallin and the fusion protein Trx-?B-crystallin with molecular chaperone activity.2?Recombinant human?B-crystallin and the fusion protein Trx-?B-crystallin play an important role in repairing RGCs after optic nerve injury. Intravenous administration can significantly preserve injured optic nerve, and increase RGCs survival. All these experimental results indicate that the recombinant human?B-crystallin and the fusion protein Trx-?B-crystallin can effectively protect RGCs after optic nerve injury, and promote the recovery of visual function of RGCs. 3 Intravenous injections of recombinant human?B-crystallin and fusion protein Trx-?B -crystallin are safe, and can not cause histopathological changes of vital organs and behavioral changes of spontaneous movement.4 The fusion protein Trx-?B-crystallin exhibites higher molecular chaperone activity than the recombinant human?B-crystallin, but their efficacy of protection after optic nerve injury had no significant difference, which suggested that the molecular chaperone activity and protecting function was not a same functional domain. |
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