| Retinal Degeneration（RD）is a type of serious and worldwide blinding diseases.It mainly includes Retinitis Pigmentosa（RP）and Age-related Macular Degeneration（AMD）,among others.Key clinicopathological characteristics of RD includes Retinal Pigment Epithelium（RPE）and/or photoreceptor cell death resulted from its pathological changes.In addition,due to RD,the dead photoreceptor cells are unable to regenerate.Although previous studies have focused on finding successful treatments for retinal degene ration by investigating the effects of gene therapy,stem cell transplantation and artificial visual prosthesis,their results have been inconclusive.However,because photoreceptor cells die as retinal degeneration progresses,a possible treatment of retinal degenerationdisease is to replace the affected photoreceptor cells.Retinal Progenitor Cells（RPCs）,which originate fetal retina tissue,have the capacity of proliferation and differential potential to various cells of retina.Previous research indicates that transplanted RPCs can differentiate and integrate into the host retina[1].MacLaren et al[2]have found that by transplanted to adult or degenerated retinas,RPCs can integrate into retina and form synaptic connections.Aftab et al[3]have found that following transplantation,RPCs can integrate within the rhodopsin-/-mice retina and differentiate to express rhodopsin.Multiple teams have found that by transplanting Nrl-GFP+rod-precursor cells to subretinal space of GNAT1-/-mice,the rod precursors can differentiate to acquire the features of mature photoreceptor cells,fo rm synaptic connections with neurons in the recipient retina,project light information to the visual cortex,and eventually improve vision[4,5].Liu et al[6]of our group have observed recovery of visual function and maintenance of the outer nuclear layer thichness of RCS rats for 6 weeks after transplantation of human fetal derived RPCs.The phase I clinical trial of transplantaion of RPCs in 8 RP patients have confirmed the long-time safety and feasibility of RPCs therapy in RP[6].During early stage of RD,Müller cells can be activated and protect retina by proliferation,release of cytokines,clearance of excitoxicity,and release of antioxidants[7].In advanced stage of RD,activated Müller cells can form gliar scar on surface of retina and in subretinal space,block the nutrients exchange between retina and choroid membrane,thus aggravate retina damage[8,9].Glial scar formed by Müller cells critically hinders the survival,migration and differentiation of transplanted stem cells,which decreases the survival rate and effect of transplanted cells[10].Damage of retinal pigment epithelium can lead to immunosuppression of transplanted stem cells[11],and ultimately reduce the effectiveness of stem cells treatment.Therefore,improvement of microenvir onment in degenerated retina is an important aspect to enhance the therapeutic effect of stem cells treatment.Olfactory ensheathing cells（OECs）are a unique type of glial cells found in the olfactory system,including olfactory mucosa,olfactory nerve and olfactory bulb.In the mature olfactory system,OECs mainly guide olfactory axons growth from the nasal mucosa to the olfactory bulb and form synapses in the brain[12].OECs form a continuous cell base or pathway to support the primary olfactory axons regenerate from the periphery to the central nervous system[13,14].Due to the unique function,OECs transplantation therapy of central nervous systemdiseases and injury has become a focus of research.As a part of the nervous system,the pathological process of damage repair of optic nerve is similar to that of central nervous system.Therefore,OECs have been used in treatment of optic nervous damage.Li et al[15]have found that,OECs could survive in the damaged area and promote the regeneration of axons of retinal ganglion cells in optic nerve crush rat model.In the glaucoma rat model,transplanted OECs can protect optic disc by migration to optic nervous head[16].Leaver et al[17]have found that OECs could promote axonal regeneration of optic ganglion cells in vitro.Our previous results indicated that OECs,transplanted to subretinal space of RCS rats,could migrate through all layers of retina,clear up outer segment fragments[10,18],and reduce the formation of Müller cells glial scar by inhibiting the NOTCH signaling pathway[19],thus protecting photoreceptor cells in RCS rats.Improving the microenvironment of degenerated retina can increase the long-time survival of transplanted stem cells,and enhance the effectiveness of stem cell transplantation.Therefore,we hypothesize that combining transplantation of h OECs and hRPCs can inhibit glial scar and improve microenvironment.It can also increase survival rate of h RPCs,extend the validity of stem cells transplantation therapy and delay the progression of RD.This paper has two parts:Part One:Study on the protective effect and mechanism of OECs in a rat model of light-induced damage.1.To study the effect of OECs in light-induced retina damage,rat OECs were transplanted into the subretinal space of light-induced damage rats,and were found to significantly reduce the loss of photoreceptors and maintain the outer nuclear layer thickness.OECs could improve the function of retina,and maintain the amplitude of FERG a-and b-wave 8 weeks after transplantation.OECs transplantation could reduce the oxidative stress induced by continuous exposure to strong light.2.To investigate the protective effection and mechanism of OECs on oxidative stress injury in photoreceptors,a model of H2O2 induced 661W photoreceptor cells damage was established in vitro.We found that OECs could protect 661W cells,improve the activity and reduce the apoptosis ratio of damaged cells through a transwell co-culture system.The results suggested that OECs could protect photoreceptor cells because OECs could impede the rise of i NOS and NOX4,while reactivating the expression of SOD1 and CAT,increasing the total antioxidant capacity,reducing ROS,thus minimize oxidative damage to photoreceptor.Part Two:Study on the effect and mechanism of combined transplantation of h OECs and hRPCs to treat retinal degeneration.1.We found that h OECs with better activity could be obtained by primary culture from human fetal olfactory bulb and purification.HRPCs isolated from human fetal retinas could express specific marker of precusors,and have the capacity to proliferate in vitro.2.We transplanted h OECs and h RPCs into the subretinal space of RCS rats,and found that the efficacy of hOECs/hRPCs combined transplantation maintained the FERG results much better than hOECs or hRPCs single transplantation.Transplanted hOECs could inhibit gliosis of Müller cells.Combined transplantation showed more protection to the outer nuclear layer,and could maintain the outer nuclear layer thickness to 16 weeks post-transplantation.The results showed that h OECs could induce hRPCs to differentiate and express mature photoreceptor specific marker,while inducing the expression of GFAP increased in hRPCs.Four weeks after transplantation,only some hRPCs in the combined transplantation group expressed bipolar and photoreceptor marker PKCα,recoverin,and rhodopsin.The mechanismof h OECs/hRPC combined transplantation in retinal degeneration therapy may be:hOECs can improve the microenvironment of degenerated retina by inhibiting excessive activation of Müller cells and regulating activation of microglia,and promote differentiation of hRPCs to photoreceptor precursor.Taken together,combined transplantation of hOECs and hRPCs is a more effective treatment strategy for retinal degenerative diseases.Due to above findings,this study concludes that:1.By examining OECs’effect on the rescue of acute photoreceptors damage,this study has found that OECs can protect damaged photoreceptors by reducing oxidative stress.2.This study is the first to conduct research regarding combined transplantation of h OECs and hRPCs in retinal degenerative disease,and it has prove that combined transplantation is more effective at improving visual function for rats with retinal degeneration than single transplantation.3.This study has found that after transplantation,hOECs can inhibit the activation and gliosis of Müller cells and regulate the activation of microglia which boosts t he survival of h RPCs in the subretinal space,and extends the effect of combined transplantation on protecting the outer nuclear layer to 16 weeks after surgery.This may help explan the success of combined transplantation in prolonging the effectiveness o f stem cells.4.This research has found that hOECs can promote hRPCs differentiation into photoreceptor precursor,which may also explain the success of combined transplantation in prolonging the effectiveness of stem cells. |
