新生大鼠Klliker器支持细胞体外凋亡及增殖的研究

The study on the proliferation and the apoptosis factors in vitro of Klliker organ supporting cells in the cochlea of newborn rat

目的:研究体外Klliker器支持细胞的增殖及凋亡情况,并探讨促进Klliker器支持细胞在体凋亡的可能因素。方法:采用机械分离和酶消化结合的方法提取新生大鼠Klliker器支持细胞并体外培养,通过流式细胞仪检测细胞纯度、凋亡率及凋亡周期,通过MTT法检测该细胞的生长曲线,并运用Realtime PCR及Western blot方法观察不同时间点Caspase-3、Caspase-8、Caspase-9及Bcl-2的表达规律;改变体外培养Klliker器支持细胞培养液中Ca2+、K+及谷氨酸浓度,采用MTT法检测Klliker器支持细胞的存活率。结果:Klliker器支持细胞纯度高达96.56%,呈明显的线性增长;各组细胞的Caspase-3、Caspase-8、Caspase-9及Bcl-2的mRNA及蛋白的表达稳定。提高Klliker器支持细胞外液中Ca2+浓度导致细胞活性降低,而细胞外K+对Klliker器支持细胞活性的影响表现为浓度依赖性,高浓度的谷氨酸对Klliker器支持细胞有一定的保护作用。结论:大鼠Klliker器支持细胞在体外无明显凋亡,呈线性增长趋势。高浓度的Ca2+可能是引起在体Klliker器支持细胞逐渐凋亡的因素,而高浓度的K+及谷氨酸对在体Klliker器支持细胞有一定的保护作用。

Objective：To study the apoptosis/proliferation of Kolliker organ supporting cells and to understand the prompting apoptosis factors in vivo in the supporting ceils in the Kolliker organ by changing the environment of the cultured supporting cells in the Kolliker organ in vitro, via the separation, culture and purification of the sup- porting cells in the Kolliker organ. Method：A combinatorial approach of enzymatic digestion and mechanical sepa- ration was employed to isolate and culture in vitro pure Kolliker organ supporting cells. The purity was tested by flow cytometry assay. And Kolliker 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 Bcb2, 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 Kolliker 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 Cae＋ might contribute to decrease the cell viability of supporting cells. And K＋ participated regula- tion 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 Kolliker or- gan 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.