The Study Of The Target Protein-α-crystallin In Binding To RGCs

Objective:To study the binding site of exogenousα-crystallin in RGCs and explore the target proteins of exogenousα-crystallin, investigate the molecular mechanism ofα-crystallin stimulating RGCs axon regeneration so as to provide theoretical basis for clinical treatment and research after optic nerve injury.Methods:1.the exogenousα-crystallin is biotin labeled by the means of biotinylating protein,then to study the binding site of exogenousα-crystallin in RGCs and RGC-5, and the identification of RGC-5 by the means of immunohistochemistry study.2. To study whether exogenousα-crystallin acts on RGC-5 membrane receptor integrinα6 by the means of immunohistochemistry and blocking the binding sites of receptor integrinα6 . And for the further verification of which, we use sbiotinylation proteininteraction Pull-Down technology and Western-Blot.3. Using SDS-GAGE and mass spectrometry to verify what is the target protein ofα-crystallin .Results:1. exogenousα-crystallin bind to membrane of RGCs .2. The exogenousα-crystallin and integrinα6 have no combination.3.There is a 50KD protein bands on SDS-GAGE gel electrophoresis . the results of mass spectrometry analysis is that the ratings of glutathione S-transferase significantly is greater than the criteria of system evaluation.Conclusion:1.Exogenousα-crystallin stimulates RGCs axon regeneration by binding on membrane of RGCs.2. although integrinα6 mediates laminin-1 to stimulates RGCs axonregeneratio n ,besides, integrinα6 mediatesα-crystallin to resist the apoptosis of crystal epithelial cells .But our experiment confirmed that exogenousα-crystallin and integrinα6 have no effective relationship in RGCs.3. Glutathione S-transferase may be the target protein of exogenousαcrystallin. However, whetherαcrystallin exactly target on the glutathione S-transferase, needs further verification.