The Transdifferentiation Process Of Math1 Induced Hair Cell Regeneration And Cell Reproduction

Objects 1) To explore the feasibility of adenovirus vectors' administration in perilymph for hair cell regeneration research, through the studies on the permeability of the vestibular membraneous labyrinth.2) New ectopic hair cell-like cells were found in the cultured neonatal rats'cochlear induced by math1gene transfer. The study tried to find the counterpart effect in the vestibular epithelia.3) To explore the conditions, the morphological transition process during the formation of hair cell-like cells induced by mathl and also the characteristics of these new cells.Methods Ad-math1-EGFP vectors were constructed and then the efficiency of the vectors were examined; The experimental animals were neonatal rats, mice and adult mice. In vitro, the culture models of whole vestibular labyrinth and the vestibular end-organ were established respectively. The effects of the two models were tested when treated with Ad-math1-EGFP. In end-organ culture models, normal vestibular epithelia treated with Ad-math1-EGFP were fixed at different time points and part of them were sliced; Treated with Ad-math1-EGFP, vestibular epithelia with different cell proliferation capabilities were fixed at a constant time point. All these samples were analyzed by immunocytochemistry, were examined and photographed using a Leica epifluorescence microscope, SP2 or SP5 confocal microscope.Results 1) In vitro, the successful establishment of the whole vestibular labyrinth and the vestibular end-organ culture models. The former were firstly kept the integrity of the membrane labyrinth at most and attached it to the cover-slips and then added with the adenovirus vectors to the culture media, which simulated the process of adenovirus vectors'administration in perilymph in vivo; The latter were firstly removed the non-sensory membrane labyrinth and attached the remaining sensory epithelia to the cover-slips and then were added with the vectors to the culture media, which simulated the process of adenovirus vectors' administration in endolymph in vivo 2) In whole vestibular labyrinth culture models treated with Ad-math1-EGFP, very few hair cell-like cells were found in the non-sensory region of the epithelia; in the end-organ culture models, there were some cells spreading and crawling from the sensory to non-sensory region with cubical shape. Robust hair cell-like cells were found originating from these cells in the non-sensory treated with Ad-math1-EGFP.3) These ectopic hair cell-like cells always clustered, with different cell shapes, ranging from the polygon-shape, hair-cell-shape, to the column-shape. The new cells presented with hair bundles, and sometimes the bundles were shared by two or more cells.4) In mechanical lesion vestibular epithelia, the supporting cells in lesioned epithelia had high proliferation capability, and could be differentiated into hair cell-like cells induced by mathl gene transfer; In the normal vestibular epithelia, very few supporting cells had high proliferation capability, and no hair cell-like cells were found induced by math1 gene transfer; In gentamycin treated vestibular epithelia, almost no supporting cells had high proliferation capability, and no hair cell-like cells were found induced by mathl gene transfer.5) Hair cell-like cells always originated from high proliferated cells, and were independent of cell proliferation during the process of transformation. Some cells in division were found to have the properties of hair cells.Conclusion 1) The vestibular membraneous labyrinth is a barrier preventing the adenovirus vectors in perilymph permeating into endolymph space.2) There are some high proliferating cells in the non-sensory region that can spreading and crawling in cultured vestibular epithelia of neonatal rats or mice. And these cells can be transformed into hair cell-like cells induced by Ad-math1-EGFP.3) High proliferated progenitor cells are easy to be hair cell-like cells induced by math1 gene transfer.4) It is a transdifferentiation process that is the formation of hair cell-like cells.5) Mathl induced hair cell-like cells are of differed cell shapes and present stereocilium that makes the possibilities of the functional hair cell.