| Introduction：Retinitis pigmentosa (RP) is one of inherited retinal disorders characterized byprogressive loss of photoreceptors and eventually leads to retina degeneration andatrophy. It is the most common subtype of retinal degeneration, responsible for loss ofvision for one in3500-4000people worldwide. It is a disease which starts with therod photoreceptors apotosis, leading to poor vision and constriction of peripheralvision. New approaches for RP therapies include: cell transplantation therapy, genetherapy, cytokine therapy, nutrition therapy, and hyperbaric oxygen therapy.In recent years, Cell transplantation and gene therapy have made great progressin the treatment of retinitis pigmentosa. Vectors can transduce neurotrophic factorgene or anti-apoptotic genes, and have neuroprotective or anti-apoptotic effects. Thetransplanted cells can be used as a substitute for degenerated cells or release somegrowth factors to enhance retinal cells survival. BMSCs were uesd widely intransplantation therapy, it is also a good carrier, which can mediate stable expression ofexogenous gene. So we can combine gene therapy and cell therapy, modify BMSCs withexogenous gene, then transplante into the patient’s eye. It will be significant improvementin degenerative retinal diseasesThe neurosensory retina is composed of six types of neurons. Rod photoreceptorand cone photoreceptor; bipolar cells, about10to hundreds of photoreceptor cellsassociated with a ganglion cell through bipolar cells, responsible for connection; RGCis responsible for conducting visual signal; horizontal cells and amacrine cells,responsible for the horizontal linkages of visual information. Therefore thedegeneration and death of retinal cell in RP, equals to the changes of neuronalapoptosis.GAP-43is neuron growth-associated protein, related to neural development andregeneration closely. Our previous experiment found that GAP-43gene played animportant role in the differentiation of bone marrow mesenchymal stem cells intoneuron-like cells by suppression subtractive hybridization (SSH) analysis. AfterGAP-43gene was introduced into the primary BMSCs, in vitro and in vivo experiments have confirmed the GAP43gene can promote BMSCs to differentiateinto retinal neurons.Royal College of Surgeons rats (RCS rat) is a typical hereditary animal model ofRP. We transplanted GAP-43gene-modified BMSCs into the eyes of RCS rats, toexplore the differentiation of GAP-43gene-modified BMSCs cells in different eyemicroenvironment, to observe the retina cell morphology and function after celltransplantation, especially the protective effect on retinal photoreceptor cell, and toexplore a new therapeutic approach.Purpose:To observe the therapeutic effects of growth associated protein-43(GAP-43)gene modified rat bone marrow mesenchymal stem cells (BMSCs)on experimentalretinal degeneration.Methods:SD rat BMSCs were isolated and cultivated by adherence method, then identifiedcell surface markers by flow cytometry. Construct lentiviral-mediated GAP-43overexpression virus (LV5-GAP), after infection BMSCs, get GAP-43gene-modifiedBMSCs (BMSCs-LV5-GAP-43). A total of67male RCS rats were divided into3groups at postnatal21(P21) randomly. A cell suspension of5-104BMSCs modifiedwith GAP-43in2μl PBS was injected into the subretinal space of BMSCs+GAP-43group rats, BMSCs group animals received5-104BMSCs in2μl PBS and NC grouprats received2μl PBS. The expression of GAP-43, GS and RHO was analyzed byimmunofluorescence, western blot. The thickness of ONL was assessed by the methodof HE staining and photoreceptor apoptosis was assessed by the method of TUNELdetection. The expression of Caspase-8and Caspase-9was analyzed by western blot.Results:（1）The BMSCs were cultured and passaged stably in vitro, flow cytometryanalysis showed that: CD90and CD44expression was positive, CD11b and CD45expression was negative; GAP-43gene sequence went through digestion,transformation, packagement and transfection to get lentiviral vector LV5-GAP; afterinfection BMSCs, Western Blot and immunofluorescence results showed that theexpression of GAP-43in BMSCs-LV5-GAP-43was increased (P <0.05); aftertransplanted the cells into the subretinal spaces, western blot revealed the expressionof GAP-43was significantly increased during14days after subretinal transplantation in BMSCs+GAP-43group;（2）30d following subretinal injection,Western blot revealed the expression ofRho in BMSCs+GAP-43group was significantly upregulated(P＜0.05), but the GS inBMSCs group was upregulated compared with BMSCs+GAP-43group(P＜0.05);Histological analysis revealed that the thickness of outer nuclear layer (ONL) inBMSCs+GAP-43group compared with the other two groups was significantlyincreased (P <0.05);（3）After30d following treated, TUNEL positive cells in BMSCs+GAP-43group compared with the other2groups, were decreased (P <0.05);（4）Western blot revealed the expression of Caspase-8and Caspase-9inBMSCs+GAP-43group was significantly upregulated(P <0.05).Conclusions:（1）The rat BMSCs were successfully cultured, and we constructed GAP-43gene overexpression of lentiviral vectors successfully; Lentiviral-mediated GAP-43can efficiently infect BMSCs. BMSCs-LV5-GAP-43were transplanted into thesubretinal space of RCS rat cells,they were gradually disappeared in the degenerationretina environment, but the expression of GAP-43gradually increased.（2）GAP-43canreduceMüllerproliferationaftertransplantatedBMSCs-LV5-GAP-43into the subretinal space and prevent "glial blocked"; and BMSCs-LV5-GAP-43can protectphotoreceptor cells, maintain the integrity of photoreceptor cells.（3）The TUNEL method results showed that the apoptotic cells were mainlylocated in the ONL of the retina, BMSCs-LV5-GAP-43has the anti-apoptotic effectto photoreceptor cells, and it’s through blocking exogenous and endogenous apoptoticpathway mediated by Caspase-9and Caspase-8, to inhibit neuronal apoptosis andmaintainthe integrity of ONL.（4）TheresultssuggestthatGAP-43genemodifiedBMSCshavethetherapeuticeffecton the early2months of retinal degeneration, which might make RP retina preserved. |
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