| Optic nerve injury affected recovers and reconnections of patient's visual function significantly,so how to protect the nerve especially in the survival of RGCs and promote axonal regeneration after optic nerve injury has become a focus all over the world.Although it has been proved that crystallins can promote their infiltration in local tissue.And as small heat shock proteins,alpha-A and alpha-B,two alpha-crystallin subunit ofα-crystallin,can inhibit the activation of caspase-3,and promote the survival of retinal pigment epithelial cells,photoreceptors and lens epithelium cells.But whether crystallin can promote the survival of RGCs or not have still few reported.Recently research show the induction of lens injury results in release of lenticular factors which promote survival of RGCs and stimulate axonal regeneration throughout the optic pathway,and crystallins can promote the survival and growth of rat RGCs in vitro.Although it has reported thatα,β-H crystallin could promote the survival and growth of rat RGCs in vitro,but the existence ofα,β crystallin always keep subunit state throughout life,so it is still unknown which element of crystallin have the main power of neruroprotective and neurite-promoting activities.Purpose:To establish culture model and evaluation technique of retinal ganglion cells in SD rats in vitro.Study the effect of crystallins on survival and growth of rat RGCs in vitro,and investigative whether the protective effects of developing crystallin on axonal regeneration and survival of retinal ganglion cells is different following the different age of crystallin,then accomplish the comparative analysis of the proteome of lens in developing SD rat,to figure out the subunit of crystallin which promotes the survival and growth of rat RGCs in vitro.Quotes a new method for the treatment of optic nerve injury and provide experimental basis on the regeneration of optic nerve injury.Mthods:RGCs in SD rats were cultured in DMEM/F12, DMEM/F12 containing crystallins of different age rats,The cells activity and numbers and length of the longest processes of RGCs with processes were measured when RGCs were cultured for 1d,3d,5d,7d,and 9d.The crystallin from 1d,2w,6m and 1year were separated using immobilized pH gradient,two-dimensional electrophoresis(2-DE)and colloidal Coomassie Brilliant Blue staining.Image analysis was carried out using PDQuest software package.Partial significant differentaially proteins in gel were identified by matrix laser adsorption/ionization-time of flight-tandem(MALDI-TOF-TOF)Results:1 The model of RGCs cultured in vitro was complished succefully,RGCs has three type according shape in vitro,Survival time of RGCs in vitro is 7～10d,RGCs can be identified by Thy 1.1 monocolon antibody by immunehistochemistry.2 The best consentration of soluble crystallin is 10-5g/L after comparing three different concentrations.The length of the longest processes of RGCs:When RGCs had been cultured for 1d,3d,5d,7dand 9d the length of the longest processes of RGCs of crystallins group is lyear group,there was no difference between the two groups of culture media of 1d group and 2w group about their effect on the length of the longest processes of RGCs.3 2-DE were performed succefully three times to observe the reproducibility.We had detected 126±6 spots in ld group,138±8 spots in 2w group,143±7 spots in 6m and 149±8 spots in lyear group.2 chosen spots were identified by MALDI-TOF-TOF,where includeβb2 andαA crystallin.Conclusions:1.Developing crystallins can promote the survival and growth of rat RGCs in vitro significantly and directly.And crystallins of 10-5g/L is the most effective concentration among various concentrations.2.Different group from developing crystallin have different activity in promoting the survival and growth of rat RGCs,lyear group has the most strong neuroprotecive activity among 4 groups.3 The quantity ofβb2 andαA has significant change from 1d～1year, Tney may be the main neuroprotecitve factors among crystallin. |
PDF Full Text Request