Study On The Culture Vitro Of The Mice Induced Pluripotent Stem Cells And On The Vitro Cultivation,Isoiation And Cocultivation Of The Rabbit Corneal Endothelial Cells

Objective:Mice induced pluripotent stem cells are cultured and separated in vitro, together with the rabbit corneal endothelial cells to find out the characteristics of proliferation in vitro of these two cells. And experiment is also conducted to induce the differentiation from the induced pluripotent stem cells to the corneal endothelial cells by co-culture. Intensive attention are also paid to the morphological changes of the mice induced pluripotent stem cells with the rabbit corneal endothelial cells both before and after the co-culture, and the expression of its functional protein-Aquaporinl is also detected. All these provide experimental basis for the study of differentiation from induced pluripotent stem cells to corneal endothelial cells, and the feasibility of induced pluripotent stem cells as substitute of the corneal endothelial cells as the seed cells involved in the transplantation is also explored.Methods:1. The feeder cell method was utilized to culture, proliferate and passage the mice induced pluripotent stem cells in vitro, and the state of growth of the cells were observed. The P2cells were taken and identified by means of Western blotting.2. The distrip method was derived from the descemet membrane and endothelial layer of the white rabbit from New Zealand, the cells were placed in the cell culture container and the cell growth state of the corneal endothelial cells was observed. The immunohistochemistry was used to test the the relative specificity protein neuron-specific enolase (NSE) of the corneal endothelial cells while the P1cells were taken out.3. The induced pluripotent stem cells and the rabbit corneal endothelial cells were planted on the upper and lower room of the Transwell coculture system respectively, the morphology of the induced pluripotent stem cells and the expression of the AQP1against immunohistochemistry after two weeks were observed.Results:1. The mice induced pluripotent stem cells after recovery conducted vitro amplification, and tiny cloning could be seen in two to three days after recovery, then the cell volume expanded, which took on the typical shape of cloning, likewise the cloning took the round or oval shape which had clear boundary with high refractivity, and nest shape under the high power lens. The cells were closely packed, small in size, big in cell nucleus, clear in nucleolus, and high in the ratio of the nucleus/cytoplasm. The bottle bottom were almost covered up after four to six days and80%of them could go down to posterity, it was also clear that the proliferation ratio of subculture cells is much faster than that of primary cells. Cell amplification grew after three to four days, which could be regarded the most active. The transcription factor protein including Sox2、Oct4and Nanog were tested positive by means of Western blotting after the P2cells were taken out.2. The primary rabbit corneal endothelial cells cultured by the distrip method would have been adherent after48hours, groups of cells that had not been blowed began to migrate to proliferate in the surrounding areas, after five days they began to form a good monolayer which took on a good shape of "paving stone", and after six to seven days the cell division entered into a vigorous stage, cellular morphology took on the shape of long and flat fusiform, which took on the shape of the morphology of fibroblast, and vortex after ten days with the central cells taking on the shape of short fusiform or triangle, the surrounding cells losing their original morphology and on irregular forms. Furthermore after two weeks the cells reached80%fusion with the cells taking on the shape of hexagon, and finally after being closely packed the cells could go down to posterity. After24hours of passage, corneal endothelial cells had been largely adherent, and cell growth curve was "S" shaped. The cells were actively growing in the days from the second to the fifth which could be considered as a logarithmic phase. Populated state would be present after a period of one week and the cells took the shape of oval or tendency of polygon. Detection of cells’NSE was positive according to immunohistochemistry.3. After two weeks of culture in the Transwell co-culture system of mice induced pluripotent stem cells and rabbit corneal endothelial cells, there was no obvious change in the cellular morphology, but the expression was tested as positive after the immunohistochemical test of AQP1of mice induced pluripotent stem cell.Conclusion:From the methods above mice induced pluripotent stem cells and rabbit corneal endothelial cells of high purity and good activity can be attained. The mice induced pluripotent stem cells can be induced express the functional proteins of corneal endothelium AQP1in the Transwell co-culture system. The induced pluripotent stem cells have the potential to differentiate towards corneal endothelial cells.The induced pluripotent stem cells act as the seed cells which conduct the transplantation therapy of corneal endothelial lesion and damage on behalf of the corneal endothelial cells.