Study On Source And Regulation Mechanism Of ATP In The K?lliker’s Organ Supporting Cells In Newborn Rat Cochleae

PART 1: In Vitro Primary Culture of the K?lliker’s organ Supporting Cells of from Newborn Rat Cochleae Objective: This part of study was to establish in vitro culture systems of the K?lliker’s organ supporting cells from newborn rat cochleae and to investigate the characterization,growth pattern.Methods: Using a combinatorial approach of enzymatic digestion and mechanical separation to allow isolation and culture of the K?lliker’s organ supporting cells from P1 to P3 SD rat cochleae.In vitro tissue culture was performed and isolated the K?lliker’s organ supporting cells were identified by immunohistochemistry and RT-PCR amplification.Results: Immunohistochemistry indicated that the isolated the K?lliker’s organ was purely epithelial tissue.The proliferative K?lliker’s organ supporting cells were explanted and arranged into polygonal paving stone and grew in islands-like patches.The K?lliker’s organ supporting cells presented significant ability to proliferate in the condition.Conclusion: The K?lliker’s organ can be successfully performed by use of enzymatic digestion combined with microdissection.The K?lliker’s organ supporting cells cultures showed significant ability to proliferate and which can be used for further experimental research.PART 2: Study on the form of ATP in K?lliker’s organ supporting cells in cochlea of newborn ratsObjective: This study aims to observe the presence of ATP vesicles in K?lliker’s organ supporting cells cultured in vitro in newborn rat cochlea,and to prove that ATP is stored in lysosomes.Methods: K?lliker’s organ supporting cells were isolated and purified for primary culture in vitro.The double staining of K?lliker’s organ supporting cells with Quinacrine/LAMP1,quinacrine/Lyso-tracker or Mant-ATP/Lyso-tracker were identified by confocal laser scanning microscope.K?lliker’s organ supporting cells were loaded with quinacrine,Lyso-tracker or Mito-tracker and then were treated with GPN or FCCP and oligomycin.The change of fluorescence intensity was observed under confocal laser scanning microscope.The ultrastructure of K?lliker’s organ supporting cells and ATP vesicles were observed under transmission electron microscope.Results: Under confocal laser microscope,a large number of green fluorescence particles were found in K?lliker’s organ supporting cells stained by quinacrine,while no green fluorescence particles were found in 3T3 cells.In K?lliker’s organ supporting cells,quinacrine was labeled with LAMP1 and Lyso Tracker,but could not be labeled by Mito Tracker.Mant-ATP-labeled vesicles were stained with Lyso Tracker.The fluorescence intensity of K?lliker’s organ supporting cells treated with GPN and Lyso Tracker was significantly reduced.However,no significant change in fluorescence intensity was observed when the K?lliker’s organ supporting cells were treated with Mito Tracker.K?lliker’s organ supporting cells treated with quinacrine and Lyso Tracker were added with FCCP and oligomycin,and the fluorescence intensity was not significantly decreased.When K?lliker’s organ supporting cells were treated with Mito Tracker,the fluorescence intensity was significantly reduced.TEM showed that lysosomes were labeled by quinacrine,but mitochondria were not.Conclusion: ATP was present in K?lliker’s organ supporting cells of cochlea of newborn rats,and lysosomes were the specific labeled organelles observed by confocal microscope and transmission electron microscope.PART 3: Preliminary study on ATP release mechanism of K?lliker’s organ supporting cells in cochlea of newborn ratsObjective: This part of the study aimed to confirm that the K?lliker’s organ supporting cells in vitro cultured newborn rat cochlea can release ATP,and to explore the mechanism on ATP release mechanism of K?lliker’s organ supporting cells.Methods: The K?lliker’s organ supporting cells were isolated and purified from P1-3 SD rats.Bioluminescence assay to study the release ATP from these supporting cells in the K?lliker organ,by affecting ATP metabolism,changing intra-and extracellular Ca²⁺ concentration,and inhibiting intracellular phospholipase signaling pathway.Results: The standard curve of the detection of ATP by bioluminescence showed a significant linear relationship.We found that changing the intra-and extracellular Ca²⁺ concentration can affect ATP release from the cells,and inhibition of intracellular phospholipase signaling pathway can also affect ATP release.Conclusion: The release of ATP from K?lliker’s organ supporting cells was affected by changes in intra‐ and extracellular Ca²⁺ concentrations.In addition,changes in the intracellular Ca²⁺ caused by inhibiting the phospholipase signaling pathway affected the release of ATP from supporting cells.We demonstrated that ATP is stored in the lysosomes of K?lliker’s organ supporting cells within newborn rat cochleae and that ATP release from K?lliker’s organ supporting cells may be Ca²⁺‐dependent.