| A wide range of diseases which can affect retina can cause photoreceptor and other retinal neuron loss. These diseases include hereditary disease such as retinitis pigmentosa, and some other widespread diseases such as macular degeneration, retina detachment, glaucoma and diabetic retinopathy. Because retina is the extension of brain, it is also lack of effective self-repair like the neurons in the brain; so these diseases usually cause irreversible vision loss. There are certain limitations in the treatment for these diseases, the treatment can save and recover vision is preferable. The fundamental cause of vision loss in these diseases is retinal functional cell death, so retinal functional cell replacement or regeneration may is a promise way to cure the disease and recover vision.Use retinal progenitor or precursor cells to do cell transplantation have been extensively researched recent years. There were studies in retinal degeneration animal model showed that cells can migrated and integrated into host retina; and expressed mature retinal cell markers after transplantation. And some studies even showed retina functional improvement after cell transplantation. So cell transplantation seems to be a feasible and promise way to treat the disease. The expansion of cells in vitro is one major problem we have to deal with before transplantation. The other problem is the characteristics of host animal model, a baseline of morphological and functional change of retina should be set for a better understanding for these diseases. And this characteristic work will also benefit the future study, it will help us to observe and evaluate the treatment effect.There were three parts in our study to illustrate the transplantation of hRPC in rhodopsin-/-mice. The first part:The low oxygen tension culture of human retinal progenitor cells. The second part:Monitoring Morphological and Functional Changes in the Retina of Rhodopsin-/-Mice. The last part:Transplantation of human retinal progenitor cells into subretinal space of Rhodopsin-/-Mice.PartⅠ The low oxygen tension culture of human retinal progenitor cellsObjective Culture human retinal progenitor cells in normal tension oxygen (20%) and in low tension oxygen (3%). Compare different characteristics between these two conditions, such as cell proliferation and sternness. Figure out the mechanism in these findings and which factor/factors play a key role.Methods Isolate retinal progenitor cells from human fetal eyes (12-18weeks gestational age). Expend cells in vitro, in normal tension oxygen (20%) and in low tension oxygen (3%) incubators. Harvest cells at different time points, and exam the mRNA level (quantitative PCR) and protein level (ICC) of certain factors of proliferation and stemness. And also run test (western blot) for hypoxia inducible factors to figure out their different role in low oxygen tension culture.Results Proliferation assay and MTT test both imply that hRPC proliferate better in low tension oxygen cultural condition. Real time PCR and ICC both confirmed it from the aspect of mRNA level and protein level:Ki67and Cyclin D1both expressed higher in low oxygen tension condition. Klf4and c-Myc also showed the same trend in this condition. Hypoxia inducible factor1a expressed in cells in low oxygen tension. Conclusions Low oxygen tension is a better cultural condition for human retinal progenitor cells expansion in vitro, because it can promote the proliferation and also can maintain the sternness of cells. Hypoxia inducible factor1a may play the key role in low oxygen tension culture. Low oxygen tension culture could be a routine way to provide enough cells for retina degeneration research and possible treatment.Part IIMonitoring Morphological and Functional Changes in the Retina of Rhodopsin-/-MiceObjective Retinitis pigmentosa (RP) and Age-related macular degeneration (AMD) are two major retinal degenerative diseases that cause irreversible blindness. Rodent models are very important tools for us to understand these diseases. Previous studies showed that rhodopsin is an essential element in photo-transduction and a structural protein in rod outer segment. Rhodopsin-/-mice do not express rodopsin and losing rod photoreceptors within a few months postnatal. We use Spectral Domain Optical Coherence Tomography (SD-OCT) to monitor the dynamic morphological changes in the retina of rhodopsin-/-mice.Methods Rhodopsin-/-C57B16mice and wild type C57B16mice at age3,6,9and12weeks were investigated using SD-OCT to obtain cross-sectional images of retina. Radial volume scan (centered on optic disc, diameter1.3mm) was used, each volume consisted100B-scans (1,000A-scans per B-scan). And the outer nuclear layer (ONL) thickness was measured from selected scans. Histological examination (semi-thin cross-section of retina, H&E staining) was performed at each time point to compare with the OCT data. Dark adapted electroretinogram (ERG) was performed to correlate the morphological and functional changes in rhodopsin-/-mice.Results SD-OCT showed that the ONL thickness in rhodopsin-/-mice was gradually decreased through week3to week12postnatal. The average thickness of ONL at3,6,9and12week were40.6±1.61μm,27.9±1.65μm,14.5±0.7μm and6.0±0.78μm, respectively. The outer segment layer was not detectable in rhodopsin-/-mice. The histological examinations identified the same trend. At3weeks, Rhodopsin-/-mice had9-10nuclei in the ONL while wild type mice had11-12nuclei. At12weeks, the number of nuclei dramatically decreased to1-2in rhodopsin-/-mice but no significant change in wild type mice. The ERG in rhodopsin-/-mice showed no distinct a wave, and b wave amplitudes decreased gradually in concordant with anatomical changes. The average b wave amplitudes at3,6and9weeks were196.8±32.6μV,179.7±26.4μV and118.3±27.6μV. There was no apparent morphological and functional change in the wild type C57B16mice at all time points (the average ONL thickness was52.5±0.69μm, and the average b wave amplitude was673.9±7.0μV).Conclusions The findings of the SD-OCT observation identified that the thickness of the ONL obviously decreased from week3to week12in rhodopsin-/-mice, but no observable changes in wild type mice. This phenomenon was also confirmed using the histological examinations on the the number of nuclei in the ONL and quantified amplitude of ERG. Therefore, SD-OCT can be a useful and non-invasive way to monitor the disease progress and the morphological changes in retina degenerative animal models.Part III Transplantation of human retinal progenitor cells into subretinal space of Rhodopsin-/-MiceObjective Use subretina injection to transplant human retinal progenitor cells into immune suppressed rhodopsin-/-mice. Observe the viability, migration, integration and differentiation of transplanted cells. Evaluate the degenerated retina of rhodopsin-/-mice morphologically and functionally.Methods Expend hRPC in low oxygen tension to passage7to9for transplantation. Rhodopsin-/-mice were treated with ciclosporin (210mg/L in drinking water). Inject hRPC (50,000/ul) into subretinal space of rhodopsin-/-mice (9mice), and inject PBS for control group (9rhodopsin-/-mice). Use SD-OCT to monitor morphological change in retina at3days and3weeks post injection. Do ERG and histology after3week’s OCT observation.Results The transplanted site can be found through the observation of SD-OCT, and can be confirmed by later histological exam. Live hRPC can be found in subretinal space of rhodopsin-/-mice from retina section, but no obvious cell migration and integration into host retina. No obvious difference in ERG between mice injected with cells and PBS.Conclusions Ciclosporin pretreated rhodopsin-/-mice can promote the survival of transplanted cell. But the cell migration and integration into host retina remained challenging. SUMMARY and CONCLUSIONSMain findings:1. Proliferation assay and MTT test both imply that hRPC proliferate better in low tension oxygen cultural condition. Real time PCR and ICC both confirmed it from the aspect of mRNA level and protein level:Ki67and Cyclin Dl both expressed higher in low oxygen tension condition. Klf4and c-Myc also showed the same trend in this condition. Hypoxia inducible factor1a expressed in cells in low oxygen tension.2. SD-OCT showed that the ONL thickness in rhodopsin-/-mice was gradually decreased through week3to week12postnatal. The histological examinations identified the same trend. The ERG in rhodopsin-/-mice showed no distinct a wave, and b wave amplitudes decreased gradually in concordant with anatomical changes. There was no apparent morphological and functional change in the wild type C57B16mice at all time points.3. The transplanted site can be found through the observation of SD-OCT, and can be confirmed by later histological exam. Live hRPC can be found in subretinal space of rhodopsin-/-mice from retina section, but no obvious cell migration and integration into host retina. No obvious difference in ERG between mice injected with cells and PBS.Conclusions:1. Low oxygen tension is a better cultural condition for human retinal progenitor cells expansion in vitro, because it can promote the proliferation and also can maintain the sternness of cells. Hypoxia inducible factor la may play the key role in low oxygen tension culture. Low oxygen tension culture could be a routine way to provide enough cells for retina degeneration research and possible treatment.2. SD-OCT can be a useful and non-invasive way to monitor the disease progress and the morphological changes in retina degenerative animal models.3. Ciclosporin pretreated rhodopsin-/-mice can promote the survival of transplanted cell. But the cell migration and integration into host retina remained challenging. |
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