| Optic nerve injury is common in clinical,mang diseases can lead to optic nerve injury,which eventually result in blindness. The progressive death of RGCs is a major cause ofirreversible visual impairment following optic nerve injury. So far, few effective treatmentshave been discovered to restore visual function. Similar to the responses for the injury inthe central nervous system (CNS), the optic nerve cells will be necrocytosis immediatelywhen suffering from injury and the remnants would be eventually death or apoptosis due toischemia, excitatory neurotransmitter, calcium overloading and inflammation. It is knownthat retinal ganglion cells show branch buds at early stage after axon injury, but whichatrophy gradully, and fail to regeneration. It has been long thought that the optic nervecould not be regenerated and repaired. However,recent studys have revealed that the retinalganglion cells have intrinsic regenerating activity showing branch buds at early stage afteraxon injury, which was atrophied gradully and failed to regeneration. In fact, impairedRGCs can escape cell death and promote axonal regeneration in the correct environment.Therefore, the repair and regeneration of impaired RGCs for the functional recovery of theoptic nerve is currently a hot topic of research. he discovery and application of stem cells isof great beneficial for promoting nerve regeneration. However, there are ethical andpractical problems associated with the derivation and use of embryonic mesenchymal stemcells and neural stem cells.Mesenchymal stem cells (MSCs) derived from the earlydevelopment of mesoderm and ectoderm are refined as undifferentiated cells that arecapable of self renewal, hematopoiesis support, immune regulation and differentiationinto several cell types such as chondrocyte, adipocyte,osteocyte, myocyte and neuron-like cells.For these characteristics, MSCs have been attracting the greatest interest inregenerationn of the aging and tissue or organ damage disease in recently decades. Inrecent years,mesenchymal stem cells have become a hot for the use of the study of centralnervous system diseases such as the spinal cord disease and cerebral ischemicinjury. Studies have shown that mesenchymal stem cells have neuroprotective effects.Inthis study, we transplanted hunman umbilical cord blood mesenchymal stem cells(hUCB-MSCs) to the vitreous cavity in optical nerve injury rats to assess whetherhUCB-MSCs could protect and promote regeneration of axotomised neurons within the ratoptic acute injury model. Base on this study, we try to provide new ideas and makecompensation for the deficiencies of the existing clinical treatment of optical nerve injury.We also expect to improve function of the impaired vision and the life quality of patientswhich could be important for the development of the neuroscience and the clinicalophthalmology.This study involves two parts, the first part is cell experiments: isolated, purified andcultured of mesenchymal stem cells from human umbilical cord blood and indentified. Thesecond part is animal experiments which include: fluorescence gold (FG) retrogradetracing retinal ganglion cells(RGCs) and counting; hematoxylin-eosin (HE) stain toobserve the morphological changes of the retina; the express of nerve growth associatedprotein43(GAP-43) was detected by immunofluorescence,western blotting and real-timePCR; ATP ligand gated purinergic receptor P2X7was researched by real-time PCR andimmunofluorescence; the pro-apoptotic protein Bax and antiapoptotic protein Bcl-2, andCaspases-3was studied by immunofluorescence; and Tunel staining was used to detect thelevel of apoptosis. We used above mentioned methods to compare the differences betweenthe therapy group and control group. The results are as follows:1. Isolation, purification and cultivation of hUCB-MSCs in vitro Data showed thatin vitro cultured hUCB-MSCs are spherical and small in light microscope, consistent withthe previous report. Flow cytometry showed strong positive for CD13, CD29, CD44,CD105and CD90, and weak positive for CD34, CD45and HLA-DR, which furtherconfirmed the cultured cells were mesenchymal stem cells.2. Fluorogold retrograde tracing and counting RGCs Results showed that retinalganglion cell number was gradually decreased with the time course in both control andtherapy group, but the number of ganglion cells of the therapy group is higher than thecontrol group at the same time point.3. HE staining Results revealed that the number of RGCs was gradually reduced, theretinal layers were gradually disordered, the nucleus appeared deformation, and someganglion cells loss for a long fragment both in the control group and therapy group withtime course. But in therapy group, there were more RGCs, and the degree of disorder ofthe retinal layers is better than that in the control group.4. The expression of GAP-43Immunofluorescence showed that,in normal retinalthe GAP-43protein expression was scarce,rarely in inner plexiform layer.In control group,GAP-43protein was a little increased after optic nerve injury, obviously in the7th and14th days, then began to decline. In mesenchymal stem cell therapy group, the GAP-43protein expression was significantly higher in the7th and14th days,obviously differentfrom the control group.Western boltting showed that there was no obvious difference ofGAP43between normal group and control group at3d post-transplant. At7d and14d,GAP43expression was significantly increased, but the protein level of14d was slightlyless than the7d, beganning to decline. However, GAP43expression in therapy group wassignificantly elevated compared to control group at each time point,which was coincidewith the immunofluorescence detecting. Real-time PCR showed that the mRNA ofGAP-43in therapy group was highest at3d post-transplant, from then on, the mRNA ofGAP43began to fall sharply, and21d gene level was very low, significantdifferences compared control group and normal group (p<0.01). In control group, therewas no obvious diffierence in3d after injury compare with therapy group and normal group (p>0.05), and then declined sharply, showing significant diffierencecompare with therapy group and normal group in7d and14d (p <0.01), but no diffierencecompare to therapy group (p>0.05), and had obvious diffierece vs normal group (p <0.01)in21d.5. The expression of ATP ligand gated purinergic receptor P2X7Immunofluorescence showed that the expression of P2X7was gradually increased withtime after optic nerve injury both in the control group and therapygroup, but the expression level in therapy group was lower than the control group;themRNA of P2X7, in control group,began to rise in3d after injury (in3d p>0.05vsnormal group and therapy group; in7d p <0.05,vs normal group, p>0.05vs therapy group;in14d and21d p <0.01vs normal group and therapy group),while in therapy group,therewas not obvious diffierence compare to normal group in3d,7d and14d after injury(p>0.05), and from then on, it began to rise (p <0.05,vs normal group).6. The pro-apoptotic protein Bax and antiapoptotic protein Bcl-2, andCaspases-3The expression of Bax, and Caspase-3was gradually increased with time afteroptic nerve injury both in the control group and therapy group, but in therapy group it waslower than the control group; as for the Bcl-2, in control group the expressionlevel was higher in3d after injury, then gradually decreased, and in therapy groupthe express level of Bcl-2was higher in3th,7th,14th days after injury, but in21d the levelwas slightly lower.7. Tunel staining Results showed that the apoptotic cells were gradually increasedwith time course in both control group and therapy group, and in therapy group theapoptotic cells were less than control group at each time point.Conclusions:hUCB-MSCs may have beneficial effects of anti-apoptosis and promoting regenerationon acute optic nerve injury in rats. |
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