| BackgroundRetinal degenerative disease is a subgroup of clinical common and refractory eye disease, characterized by progressive loss of vision, which can be divided by inhereditary and acquired. Retinal degenerative disease is consisted of age related macular degeneration, retinal pigmentosa, Stargardt’s disease, etc. Among them, retinal pigmentosa is the most common hereditary retinal degenerative disease. Its inheritance patterns and disease-causing genes vary among different patients. The mechanism under these remains unclear and there is still no effective treatment. Since the retinal specimen is hard to achieve among patients, it is almost impossible to establish in vitro cellular model to study the pathogenesis of retinal pigmentosa. In2007, Japanese scientist Shinya Yamanaka reported the new bio-technique, to induce the human somatic cell into pluripotent stem cell which has the identical potential to differentiate into all three layers of cells as human embryonic stem cell. This breakthrough sheds light on the field of retinal diseases, especially retinal degenerative diseases. In vitro cellular model can be established through RP patient-specific induced pluripotent stem cell and all kinds of retinal cells deviating from it. More importantly, human induced pluripotent stem cell can also be used as the new source of the stem cell therapy treating retinal degenerative diseases. These applications bring hope and future to solve the refractory eye diseases.ObjectiveThis study took an example of an IFT140gene mutated retinal pigmentosa patient, and took the dermal fibroblast of adult patients. In the study, four different stem cell genes, OCT4, Klf4, Sox2and c-Myc, were transfected into the human dermal fibroblasts via lentivirus. And the human dermal fibroblasts were induced to express stem cell related genes and to transformed into human induced pluripotent stem cells(hiPSC). The study sought to explore the mechanism under the reprograming process of patient’s somatic cells, and to lay the foundation of further differentiation into different retinal cells and the mechanism under it.Material and MethodsThe study took the dermal tissue specimen of two IFT140gene mutated retinal pigmentosa patients and two normal control subjects and established the human dermal fibroblast cell line. The13.5-day embryos of CF-1mouse were taken to separate and culture the mouse embryonic fibroblast(MEF). After processed with mitomycin C, the P3MEF cells were made into feeder cells to support the stem cell growth.The study used four vectors containing recombinant Oct4, Klf4, Sox2and c-Myc genes respectively and the package vector PVSVG and△8.91to produce four lentivrus particles containing the recombinant genes in human embryonic kidney cells(293HEK-T).We mixed the lentivirus particles with the P1human dermal fibroblasts and polybrene and reprogramed the fibroblasts into induced pluripotent stem cells. On the5th day after transfection, we moved the transfected fibroblasts onto the feeder cells made of MEF and kept culture, until different type of cells which has the round shape similar to human embryonic stem cells emerged. We examined the fibroblasts and the cells20days after transfection using real-time PCR to identify the change of stem cell specific mRNA level between two kinds of cells.Results(1) We successfully established the human dermal fibroblast cell line of IFT140gene mutated retinal pigmentosa patients and normal control subjects. The human dermal fibroblasts were long spindle shaped, and intensively arrayed. The thawing rate after cryopreservation reached50to75percent.(2) We processed the CF-1mouse embryonic cells using mitomycin C and made the feeder cells. The thawing rate after cryopreservation reached75%.(3) The study packaged four lentivirus particles containing recombinant Oct4, Sox2, c-Myc and Klf4genes respectively in293HEK-T cells, using lentivirus vectors and package vectors. The tilters reached1.4×107TU/ml,1.2×107TU/ml,2.0×107TU/ml and3.5×106TU/ml, respectively.(4) We observed under microscope that the human dermal fibroblasts turned round and gathered into clones similar with human embryonic stem cells after transfected with lentiviruses.(5) Comparing with the original fibroblasts, the FSP-1mRNA, which is specific for the fibroblasts, was significantly down regulated after transfected; and the mRNA level of stem genes, endo-Oct4/Sox2/Klf4/c-Myc and Nanog, were significantly upregulated(p<0.05). ConclusionThis study first used the IFT140gene mutated retinal pigmentosa patient’s dermal fibroblasts, transfected four recombinant factors, Oct4, Klf4, Sox2and c-Myc, via lentiviruses. The transfected fibroblasts demonstrated the morphological changes similar with human embryonic stem cells, and expressed the stem cell specific genes.
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