DAPT对离体培养的基底膜的影响

In vitro effects of DAPT on Organ of Corti

目的观察新生大鼠体外培养的基底膜在Notch通路被阻断的情况下,毛细胞的生长变化情况。方法取新生(P0)SD大鼠耳蜗基底膜进行体外培养,采用自身对照的方式,实验组基底膜加入含Notch通路阻断剂DAPT(终浓度为5μM)的高糖培养基,对照组不加DAPT,培养5天(P5)后,用激光共聚焦显微镜观察毛细胞的生长变化情况。P0时采用逆转录PCR检测Notch通路的存在与否,P5时采用实时定量PCR检测阻断Notch通路后下游基因的变化情况。结果实验组经DAPT的处理后有毛细胞的增多,逆转录PCR证实了出生第一天时尚有Notch通路的存在,实时定量PCR证实DAPT处理后Hes1和Hes5表达降低,而Math1表达升高。结论新生大鼠中Notch通路活性尚存,加入Notch通路阻断剂DAPT后,抑制毛细胞生成的Hes1和Hes5表达降低,而促毛细胞生成的Math1表达升高,并且,通过形态学观察和统计学分析发现毛细胞数目增多。

Objective To study hair cell changes in new born rats when the Notch signal is inhibited by DAPT in vitro. Methods Cochleae were dissected from new born rats. The Organ of Corti was freed from surrounding tissues and explanted inextenso onto the surface of 6 well culture plates and randomly divided into two groups： an experiment group in which specimens were treated with DAPT （a Notch signal inhibitor） at 5 μM, and a control group in which specimens were treated with DMEM containing DMSO with a final concentration lower than 0.1%. The Organ of Corti was incubated at 37 degree in a humidified atmosphere with 5% CO2 for 5 days with daily replacement of the culture medium. The number of hair ceils and morphology of the basement membrane were then examined after immunohistochemical staining. RTPCR was used to confirm presence of Notch signal on P0, and quantitative RT-PCR was used to determine changes in downstream genes involved in Notch signaling on P5. Results There was an increase of hair cells in the experiment group compared to the control group. Notch signaling was present on P0 but Notch signaling genes changed after DAPT treatment. Conclusions Notch signaling is present in new born mammals. Increasing hair cells may be possible through inhibiting Notch signaling.