The study on the proliferation and the apoptosis factors in vitro of Kölliker organ supporting cells in the cochlea of newborn rat / 临床耳鼻咽喉头颈外科杂志

ABSTRACT

OBJECTIVE@#To study the apoptosis/proliferation of Kölliker organ supporting cells and to understand the prompting apoptosis factors in vivo in the supporting cells in the Kölliker organ by changing the environment of the cultured supporting cells in the Kliker organ in vitro, via the separation, culture and purification of the supporting cells in the K6lliker organ.@*METHOD@#A combinatorial approach of enzymatic digestion and mechanical separation was employed to isolate and culture in vitro pure Kölliker organ supporting cells. The purity was tested by flow cytometry assay. And K6lliker organ supporting cells were harvested to detect the rate and cycle of apoptosis by flow cytometry after Annexin V/PI staining, to test the cell growth curve by MTT assay, and to observe the differential expressions of the Bcl-2, Caspase-3, Caspase-8 and Caspase-9 through the Realtime PCR and Western blot. The calcium, potassium and glutamate concentrations in the culture medium of these cells in vitro were changed to detect the survival rate of cells by MTT assay.@*RESULT@#The purity of K6lliker organ supporting cells by flow cytometry assay was 96. 56%. And these cells showed no significant difference in apoptosis, but an evident linear growth. The results of Realtime PCR and Western blot showed that the expression of Bcl-2, Caspase-3, Caspase-8 and Caspase-9 mRNA and protein in all different time points kept stable. Furthermore, the elevation of extracellular Ca2+ might contribute to decrease the cell viability of supporting cells. And K+ participated regulation of cell viability in a concentration-depending way. However, glutamate appeared to be a protective factor in high concentration.@*CONCLUSION@#There is no significant apoptosis in vitro of the supporting cells in the Kölliker organ of rats, showing a linear growth. The Ca2+ in high concentration might contribute to the apoptosis factor of these cells. However, the K+ and glutamate appear to be protective factors in high concentration.