| The repair and regeneration of CNS injury has been the field of neuroscience research priorities and difficulties,There are two main causes for injured CNS hard to regeneration,which includes two aspects: 1.ganglion cells secondary apoptosis;2 the new axons is difficult to through the damage zone.In which the reborn axon needs to pass through the damaged zone which contains activate glial cells and the inhibition of the micro-environ ment produced by the scar.The formation of glial scar can reduce extent of neuronal toxicity which caused by acute inflammation,but its mechanical barrier formed hinders axonal regeneration through the damaged area,in addition to the mechanical barrier,glial scar can still produce some inhibitory factors of ganglion cells to damage the injured axon.After injuring,The central nervous system will cause a series of cellular and molecular changes when glial scar changes that will be leading glial scar in a few days,the main cell types involved in these changes are astrocytes,microglias and oligodendrocyte precursors.the final structure of the main glial scar cells are astrocytes,which will be division and migration to the central nervous system damage,eventually fill the vacant space.Many scholars do a lot of work trying to help the injured axons: apply some neurotrophic factor;the use of the material to interfere apoptosis signal pathway;use antibody or RNA interference blocking myelin inhibition;try to transplant peripheral nerves to the damaged zone or use stem cells to repair.These methods can maintain the survival of cells in a certain extent,but they can not make the regeneration of axons through the damaged area to form a functional connection.After years of research,scholars agree that there is a need to find a multiple-targeted material and also meet the two conditions(1.keep survival cells and promote axon regeneration and axonal regeneration through the glial scar area 2 to establish functional connectivity between the right cell axons)is the development trend of the treatment of central nerve repair and regeneration.Fischer with his colleages inadvertently found that when lens with optic nerve both injury simultaneously,something can promote the RGCs survival and the optic nerve axons regrowth.The regenerated axonal can be achieved on the hill as well as formating functional synapses,this protective effect was significantly stronger than the known neurotrophic factor or neural inhibitors.effect of this material is consistent with the above two conditions.Thesubstance was isolated and detected as alpha crystal protein.Crystallin take responsibilty 90% of total lens protein,the determinants of the maintenance of lens transparency and cataract development.alpha crystallin accounted for 40% of the total proteins which is a member as a molecular chaperonbelonged to the small heat shock protein(sHSP),it have been confirmed that alpha crystallin exists not only in the lens,but also have been found in the brain,heart,liver and other important organs.it play an important role in the protective efforts:In the visual system,alpha crystallincould in the regulation of many retinal related diseasesincluding optic nerve mechanical injury,ischemia,age-related macular degeneration,uveitis,diabetic retinopathy.It also participates in the regulation of AD and Parkinson syndrome disease.In the preliminary study,the experimental group also confirmed that the alpha crystallin can not only promote the survival and axon regeneration of cells,but also inhibit the activation of microglia and the release of inflammatory cytokines in the early stage of.This finding affirms the positive role of alpha crystal proteins in protection of optic nerves,but it remains a quetion of how it could go through the glial scar which main made of astrocytes.According to the above questions,we speculate that the alpha-crystallin has a certain inhibitory effect on the astrocytes in addition to RGCs,which can promote the regeneration of axons go through the glial scar.In this study,we isolated the astrocytes cultured them in vitro.We use lipopolysaccharide to imitate The activated state of optic nerve astrocytes which response to acute inflammatory after injuring to obverve the effect of alpha crystallin on the proliferation and expression of GFAP in the optic nerve astrocytes and detectie concentration of Inflammatory factors.On the other hand,the use physical scratch stimulation to active the optic nerve astrocytes for imitating the induces for astrcytes when optic nerve injury,we obverve the effects on the secretion of astrocyte migration and the expression of CSPGs and inflammatory factors with alpha-crystallin to explore whether crystallinby acting on astrocyte injury to promote axon regener ation.urreviou sresearch of t his field is divided into two parts:1.the effect of the α-crystallin on optic nerve astrocytes proliferation、activation and secretion stimulated with lipopolysaccharide2.the effect of alpha-crystallin for optic nerve astrocytes migration,activation and secretion function during wound healingMethod:Part I:the effect of the α-crystallin on optic nerve astrocytes proliferation、activation and secretion stimulated with lipopolysaccharide1.Primary divide optic nerve and culture the astrocytes,use immunohistochemical staining to test cell purity;2.using CCK-8 to observe the effect of α-crystallin on the proliferation of optic nerve astrocytes induced by lipopolysaccharide(lipopolysaccharide,LPS);3.using immunohistochemical staining and Western blot(WB)to observe the effect of α-crystallinbytesting the changes in the protein levels of astrocytes glial acidic protein(GFAP)induced by LPS;4.detecte the effect of α-crystallin by testing optic nerve astrocytes secretion of IL-1beta 、 TNF-alpha concentration which induced by LPS with usingELISA(Enzyme-Linked Immunosorbent,Assay).Part2:the effect of α-crystallin for optic nerve astrocytes migration,activation and secretion functionduring wound healing1.observe the α-crystallineffect on migration ability during wound healing;2.using immunohistochemical staining to observe the α-crystallin effect by testing the changes of astrocytes glial acidic protein(GFAP)and CSPGs during during wound healing;3.detecte the effect of α-crystallin by testing optic nerve astrocytes secretion ofIL-1beta、TNF-alphaconcentration which induced by LPS with usingELISA during wound healing.Result:Part I:the effect of the α-crystallin on optic nerve astrocytes proliferation、activation and secretion stimulated with lipopolysaccharideα-crystallin protein could successfully inhibit the activation and proliferation of the optic nerves astrocytes which induced by LPS.WB showed a significant decrease the level of GFAP in α-crystallin group.As well as,the concentration of TNF-α and IL-1β in cell culture supernatant of LPS group were highter compare with α-crystallin group(P<0.01).Part2:the effect of alpha-crystallin for optic nerve astrocytes migration,activation and secretion function during wound healingWe found the migration of astrocyte was delayed by α-rystallin treatment.The slower migration velocity was observed in α-crystallin treatment group.ICC showed a significant decrease the concentration of GFAP and CSPGs in α-crystallin group as well as the levels of TNF-α and IL-1β in cell culture supernatant during wound healing which detected at 24 hours,48hours dependly by ELISA were showed a significant decrease(P<0.01).Conclusion:Optic nerve injury induces astrocytes to be activated,proliferated and secreted inhibit moleculars to form glial scar and inhibitor environment prevented the RGCs axon regrowth and elongation.We stimulate optic nerves astrocytes by LPS and wound scratch to imitate the effect of the microenvironment on astrocytes after optic nerve injury,we have found that alpha crystallin can effectively not only inhibit the activation and proliferation of astrocytes also reduces the inhibitory expression of CSPGs molecules.It drops the secretion of inflammatory factor TNF-alpha and IL-1 beta.alpha crystallin may provide a broad application prospect to cure optic nerve injury diseases. |
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