| Damages to photoreceptor cells can occur in retinitis pigmentosa, retinal detachment, retinal light damage, ocular trauma and other diseases, in which it can lead to irreversible damage to visual function, and even lead to blindness. Retinal detachment(RD), defined as the separation of the neurosensory retina from the underlying retinal pigment epithelium, is one of the commonest sight-threatening diseases in eyes. It also occurs in a variety of retinal disorders such as uveitis, diabetic retinopathy and retinopathy of prematurity. Although the anatomic reattachment rate has greatly increased with advances in surgical management, visual acuity is not always restored. Various mechanisms of cell death in photoreceptor cells, one of which is apoptosis, together with other structural changes in the retina, lead to the unsatisfactory outcome. Efforts have been attempted on photoreceptors protection, including suppressing factors participated in the apoptosis, applying neurotrophic factors and modulating inflammation and so on. However, as the photoreceptor death is mediated by multiple parallel pathways and proliferation, together with remodeling, is also involved in the pathological process, the measures aiming at single factor are not always completely valid. Bone marrow stem cells(BMSCs) are a kind of stem cell which acts mainly as the progenitors of all connective tissue cells and can be differentiated into several tissue-forming cells. BMSCs have been proposed as a potential source in cellbased therapy because of their pluripotency, relative ease of isolation and less ethical issues. BMSCs are proved to have the potential to differentiate into neural cells, retinal pigment epithelium cells or photoreceptors as they can express rhodopsin and opsin. When transplanted, the donor BMSCs can survive, migrate, differentiate and integrate within the retina. BMSCs are also known to express a variety of cytokines and neurotrophic factors, such as BDNF and b FGF which can protect injured retina.Therefore, this study intends to explore mechanism underlying the protective effects of BMSCs in the animal models of retinal detachment and cultured retinal cone 661 w cells under hypoxia.Methods1. In vitro study on BMSCs’ protective effects and mechanism(1) Cell damage model was prepared by hypoxic culture; the cellular morphological changes were inspected by microscopy, changes in cell vitality were detected by MTT reduction method, and at the same time, early apoptosis was detected using the Annexin V-PI double staining through flow cytometry(FCM); alteration in mitochondrial membrane potential(MMP) was detected by JC-1 staining though FCM; caspase 3 expression detection is carried out by Western blot; Western blot was also performed to observe cell autophagy after hypoxia; 1 h before hypoxia treatment, autophagy inhibitor 3 – MA was added into the medium, to observe the changes of cell vitality, apoptosis and autophagy;(2) Transwell culture system was applied to coculture the damaged 661 w cells with BMSCs, the profiles of cell vitality, apoptosis and autophagy of 661 w cells were detected suing the methods mentioned above.2. In vivo study of the BMSCs’ protective effect and its mechanism in detached retina(1) BMSCs were isolated, cultured and identified;(2) Preparation of retinal detachment in the rat model and intraocular transplantation of BMSCs, observation on the retina with multiple time points after transplantation; Blank group was kept untouched, while control group was made by subretinal injection of PBS;(3) Preliminary detection of the protective effect of BMSCs on retina; by cyrosection and HE staining to observe retinal morphology and outer nuclear layer thickness, and to detect the expression of rhodopsin which acts as a marker of photoreceptor with immunofluorescence and Western blot;(4) Study on the retinal protection mechanisms: TUNEL is used to count the number of apoptotic cells in the retina, and Western blot is performed to detect the expression profiles of proteins associated with apoptosis, as well as those of autophagyrelated proteins expressed in the retina tissue;(5) Cell labeling kit was applied to trace the BMSCs in the retina, and immunofluorescence was used to detect the migration and differentiation of BMSCs.Results1.661 w cells cultured under hypoxia for 2 h, 4 h, 8 h, 16 h, 24 h and 48 h showed reduced cell viability to different degrees, accompanied with increased number of apoptotic cells and abnormal cell morphologies. This study also found that cell autophagy began to increase after hypoxia, and peaked at 8 hours after hypoxia. After 3-MA was applied before hypoxia, which works as an autophagy inhibitor, cell viability declined;2. Cocultured with BMSCs, 661 w cells had better morphology, and the number of apoptotic cells decreased significantly. After the autophagy inhibitor was added, even with BMSCs treatment, the number of apoptotic cells in 661 w cells increased and cell viability still reduced.3. In the retina-detached eyes received BMSCs transplantation, the thickness of retinal outer nuclear layer is more close to that of the normal retina, and the photoreceptor cells were thicker and more well-structured compared to those in blank and control groups;4. After BMSCs transplantation, the apoptosis cells in the outer nuclear layer of retina decreased significantly, and the Caspase- 3,-8,-9 expressions were less than the control;5. In the short term after transplantation, the retina LC- 3II was highly expressed, while P62 expression was reduced, indicating the activation of autophagy in the BMSCs treated retina;6. During the observation period of 8 weeks, no obvious migration and differentiation of transplanted BMSCs was found in BMSCs injected eye;ConclusionsBMSCs was chosen as research object of this study, while retinal detachment and cellular hypoxia culture acted as experimental model in vivo and in vitro respectively, to explore the protective effect of stem cells and the possible mechanism. The results suggested that hypoxia culture in vitro causes damages to photoreceptor cells, while increased autophagy in early stage seems to facilitate the survival in hypoxic stress, reduce apoptosis and keep cell viability; coculture with BMSCs can reduce the damage to 661 w cells imposed by hypoxia and reduce apoptosis in 661 w cells and this kind of protective effect is fulfilled, at least to some degree, by autophagy mediated by BMSCs. After retinal detachment, retinal cells, especially the photoreceptor cells, undergoes a series of pathological changes(including proliferation, apoptosis, autophagy and necrosis), which leads to the incomplete structural and functional restoration of photoreceptors after the retina reattached. BMSCs transplantation can significantly reduce the intraocular photoreceptor cell death and preserve retinal morphological structure. The capacity of BMSCs to reduce retinal cell apoptosis and activate autophagy in short term after transplantation may help the retinal cells survive the low oxygen and poor nutrition status after retinal detachment. |
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