新生大鼠K&#246;lliker器支持细胞体外凋亡及增殖的研究

何圆圆; 杨军

doi:10.13201/j.issn.1001-1781.2015.02.015

新生大鼠Kölliker器支持细胞体外凋亡及增殖的研究

何圆圆^1,2,3,
杨军^1,2, ,

- 上海交通大学医学院附属新华医院耳鼻咽喉-头颈外科上海交通大学医学院耳科学研究所(上海, 200092)
基金项目:
国家自然科学基金资助项目(No:81170919,81470689)

上海交通大学博士创新基金(No:BXJ201223)

详细信息

通讯作者: 杨军,E-mail:yangjun67@hotmail.com

中图分类号: R764.35

收稿日期: 2014-05-07

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

HE Yuanyuan^1,2,3,
YANG Jun^1,2, ,

- Department of Otorhinolaryngolory-Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai Jiaotong University Ear Institute, Shanghai, 200092, China

More Information

Corresponding author: YANG Jun, E-mail: yangjun67@hotmail.com

Received Date: 07 May 2014

摘要

摘要: 目的:研究体外Kölliker器支持细胞的增殖及凋亡情况,并探讨促进Kölliker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Kölliker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Kölliker器支持细胞培养液中Ca²⁺、K⁺及谷氨酸浓度,采用MTT法检测Kölliker器支持细胞的存活率。 结果:Kölliker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Kölliker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Kölliker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Kölliker器支持细胞有一定的保护作用。 结论:大鼠Kölliker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Kölliker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Kölliker器支持细胞有一定的保护作用。
- 耳蜗 /
- Kö /
- lliker器 /
- 支持细胞 /
- 凋亡 /
- 增殖
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 Kölliker organ in vitro, via the separation, culture and purification of the supporting cells in the Kölliker 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 Kölliker 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 Kölliker 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.
- cochlea /
- Kö /
- lliker organ /
- supporting cells /
- apoptosis /
- proliferation

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