| Retinal degeneration is one of the main causes of the permanent vision loss, which including age related macular degeneration (AMD) and retinitis pigmentosa (RP).RP is a disease which is characterized by retinal pigment deposit and resulted in photoreceptor loss.AMD is a disease which is characterized with drusen or choroid neovascularization.Currently there is no treatment for RP and AMD. The present treatment strategy mainly aims to slow down the cell apoptosis and the loss of vision. Drugs as lutein and anti-oxidants, anti-VEGF or laser therapy are used to slow down the progression, whileas all measures of which can not effectively prevent or reverse the progress of these diseases.Gene therapy and cell replacement therapy are wildly studied. A large number of studies have shown that RPE or retinal stem cells transplantation will be an ideal application for the treatment.The Muller cells extend through the entire retina, support the structure of the retina and transfer information between neurons as messengers. Additionally, Muller cells are critical for the maintenance of homeostasis in the retinal extracellular milieu. In teleost fish, when retina constantly expanding, progenitors in the retinal periphery can produce all neurons except rods. The rods in the outer nuclear layer are generated by a dedicated lineage of cells known as the rod lineage with the apex of Muller glia. Furthermore, when retina injured,Miiller cells will dedifferentiate into multipotent progenitors and responsible for regenerating all of the major retinal neurons. However, in mammals, Muller cells have very limited potential for cell cycle reentry, and do not contribute to a rod lineage. When retinal injured, Muller cells are activated through transiently de-differentiation process, ultimately gliosis happened, which is a double edged sword for retina. Many studies have been carried out to address how to promote the generation of retinal neurons from Muller cells in mammals. These studies suggest that under appropriate conditions, Muller cells can be reprogrammed to multipotential stem cells to generate retinal neurons. However, the mechanism of action remains unclear.Lin28a and Lin28b (collectively referred to as the RNA-binding protein Lin28) have been shown specifically to block Let-7 microRNAs to regulate the pluripotency of stem cells. During development of the central nervous system,expression of Lin28b completely blocks gliogenesis, while neurogenesis is increased. Additionally, in mouse hematopoietic stem cells (HSCs), self-renewal activity and Hmga2 levels are elevated after Lin28 over-expression. Furthermore, in teleost fish, pluripotency factor Lin28 is necessary for Muller cells de-differentiation, and lin28 is up-regulated within 6 h when the retina is injured. Our recent results demonstrated that ectopic expression of Lin28b in the rat retinitis pigmentosa model can stimulate Muller cells de-differentiation.Based on the above research status, this study constructed an overexpression system of Lin28b gene with Adenovirus as the vector. Through the way of infection of Muller cells derived from LE rat in vitro, to explore the relationship between Lin28b and Muller cells reprogramme and the related mechanism. Forthermore The retinitis pigmentosa rat was used to examine the protection property of the reprogrammed Muller cells. The main results are as follow:Lin28b stimulates the reprogramming of rat Muller glia to retinal progenitor cells.After Ad/Lin28b transfection of Muller cells, Lin28b can stabilitily express among the Muller cells. When the transfected Muller cells cultured in the de-differentiation medium for three days,the somatic of which gradually became round,synaptics gradually reduced.Meanwhile,the GFAP expression of Muller glia decreased,which indicated that Lin28b had the ability to repress GFAP expression of Muller cells. In addition, we found that, Lin28b stimulated Muller cells increased expression proliferation maker Ki67, at the same time,induced more Muller cells getting into S phase and G2 / M phase of the cell cycle obviously.Further we found that Lin28b could stimulate Muller cells to express the retinal progenitor cells markers such as Pax6, sox2, Nestin, CHX10, etc. Also, we found that Lin28b could repress microRNA Let-7 and induced ascl1a expression during the reprogramming of Muller cells. Taken together, these results suggest that Lin28b could stimulate the reprogramming of Muller cells through the way of repression of microRNAs Let-7 and increase the expression of ascl1a.The study of Lin28b reprogrammed Muller cell derived - retinal progenitor cells(LRMD - RPC) transplantation to rescue the retinal function of RCS rats.We observed that when LRMD-RPC was transplanted into the RCS subretinal space,LRMD-RPC survived and migrated laterally and longitudinally, and maintained its retinal progenitor cell characteristics. At the same time, we observed that when LRMD-RPC was transplanted into the RCS subretinal space,it was able to exert a protective effect of the outer nuclear layer of the retina at an early stage. ERG test showed that, after LRMD-RPC transplantation, b wave amplitude has been restored. Furthermore, we found that LRMD-RPC significantly inhibited the glialization of Muller cells after transplantation and differentiated to pkca-positive bipolar cells. Our data suggest that LRMD-RPC has a significant protective effect on the retina of RCS-p + rats and can improve the function of the retina.To sum up, we draw the following conclusions:1. Lin28b stimulates the reprogramming of LE rat derived Muller cells in vitro.2. Lin28b can repress the expression of MicroRNA let-7 and increase expression of asclla during the reprogramming of Muller cells.3. Transplantation of LRMD-RPC plays an important role in protecting the thickness of outer nuclear layer and visual function in RCS - P+ rats.4. Transplantation of LRMD-RPC can significantly inhibit the glialization of Muller cells and differentiate into PKC ? positive cells. |
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