| With the development of cell and gene therapy, target cell is one of the crucial factors. The multipotent of mesenchymal stem cells (MSCs), such as the easy isolation and cultured property, as well as their high ex vivo expansive potential make MSCs an attractive candidate for cell-based therapy. To improve the treatment effect of MSCs, modification and inducement are neened.Viral vectors are widely used gene transfection vector, different type of viral vector has its own advantages, to select vectors can efficiently transduce MSCs, we compare the transduction and gene expression of different recombinant viral vectors in the rat mesenchymal stem cells(rMSCs) in vitro. This provides the experimental evidence of cell and gene therapy of MSCs.Hereditary retinal degeneration, which involves loss of retinal photoreceptor cells and consequently peripheral vision, constitutes major cause of blindness in young adulthood. At present there are no satisfactory therapeutic options for this disease. Transplantation of stem cell represents a promising, albeit challenging, approach to replace photoreceptor cells lost due to disease. However, there is no direct evidence to show the fate of grafted MSCs in retina after transplantation and dynamic change of the grafted MSCs number and location. In this study we took the benefit of gfp-labeled MSCs and fluorescent stereoscope to visualize and record the fate and the location of grafted MSCs in living retina after transplantation into normal rat subretinal space and approach the possibility of grafted MSCs to induce photoreceptor rescue effects in an animal model for retinitis pigmentosa rats.SECTION I. Transduction efficacy and gene expression among different recombinant viral vectors to rat mesenchymal stem cells in vitro[Objective] To investigate the transduction and gene expression of different recombinant viral vectors in the rat mesenchymal stem cells(rMSCs) in vitro.[Methods] rMSCs were isolated by Filcoll-hypaque gradient centrifuging method (lymphocytes separation) and encoding EGFP were infected into the cultured mesenchymal stem cells,GFP positive rate and mean intensity of GFP fluorescence were detected by flow cytometry and fluorescence microscopy.[Results] Data from flow cytometry showed that the percentage of positive cells by CD11b, CD45 and CD90 were 14.1%±3.3%, 1.1%±0.4% and 82.3%±5.7% respectively. The percentage of EGFP positive cells and mean intensity of EGFP fluorescence in rMSCs transduced by 10, 100 and 1000 MOI of Ad5-EGFP 2 days after transduction were 33.6%±2.7%,88.6%±1.0% and 99.9%±0.1%,and 4.4±0.3, 39.8±1.5 and 811.4±3.9 respectively; the EGFP positive cells and mean intensity of EGFP fluorescence in rMSCs transduced by 10, 100 and 1000 MOI of Ad5F35-EGFP 2 days after transduction were 96.9%±0.4%,99.9%±0.1% and 99.7%±0.1%, and 369.3±14.8, 895.4±7.5 and 703.2±38.4 respectively; the EGFP positive cells in rMSCs transduced by 1×104 and 1×105vg/cell of rAAV1/2-EGFP and rAAV2-EGFP 6 days after transduction were 0.94%±0.31% and 1.30%±0.36%, and 2.16%±0.38% and 3.90%±0.33% respectively; the GFP positive cells and mean intensity of GFP fluorescence in rMSCs transduced by 30TU/cell of LV-GFP 6 days after transduction were 60.5%±3.2% and 27.0±3.6 respectively. [Conclusion] rMSCs can be efficiently transduced by Ad5, Ad5F35 and Lentivirus and express exogenous gene . Transduction efficacy was correlated with the multiplicity of infection. MSC may be an effective platform for therapeutic gene delivery.SECTION II. Fate and protective effect of Mesenchymal stem cells after subretinal transplantationEngraftment of MSCs has been proposed as a therapeutic approach to degenerative diseases. In the present study we investigated the fate and the dynamic progress of grafted MSCs in living retina and the possibility for transplanting MSCs to treat retinal degeneration. Around 1×10~5 gfp-MSCs in 2μl PBS were injected to adult Sprague-Dawley rat subretinal space, 2 weeks later the transplanted gfp-MSCs were found to diffuse in subretinal space. There were still a certain amounts of transplanted cells survived in subretinal space around injection site even 9 weeks after transplantation. The same numbers of MSCs were transplanted into left eye subretinal space of 3-week-old hereditary retinal degeneration RCS rats, their right eyes were injected PBS as control. 5 weeks after transplantation, the amount of rudimentary photoreceptors in grafted eyes was significantly more than control eyes. The results from our study indicate that grafted MSCs could survive and rescue retinal degeneration.
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