OSBPL2介导RhoA/ROCK2信号通路参与听觉细胞肌动蛋白细胞骨架调控的实验研究
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1.南京医科大学医学遗传学系;2.南京医科大学

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国家自然科学基金项目(面上项目,重点项目,重大项目)


Experimental study on the role of OSBPL2-mediated RhoA/ROCK2 signaling pathway in the regulation of actin cytoskeleton in auditory cells
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Department of Medical Genetics,Nanjing Medical University

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    摘要:

    目的:探索氧化固醇结合蛋白样蛋白2 (Oxysterol Binding Protein-like Protein 2, OSBPL2)对听觉细胞肌动蛋白骨架形态和功能的影响及其相关分子机制。方法:采用Osbpl2基因敲除(Osbpl2-KO)HEI-OC1细胞探讨OSBPL2缺陷对听觉细胞肌动蛋白细胞骨架形态和功能的影响;扫描电镜观察Osbpl2-KO小鼠毛细胞静纤毛形态;Western blot实验检测HEI-OC1细胞和耳蜗内Rho/ROCK信号通路关键效应因子Rho激酶 2(Rho-associated coiled-coil-forming kinase 2 ,ROCK2)、Lim 激酶 1(LIM Domain Kinase 1, Limk1)、肌动蛋白解聚因子1(Actin depolymerization factor 1, ADF/CFL-1)的表达水平。结果:Osbpl2-KO HEI-OC1细胞微丝骨架中纤维肌动蛋白 (F-actin)的分布明显下调,细胞外周微刺突和丝状伪足明显减少,细胞迁移能力明显减弱;扫描电镜结果表明Osbpl2敲除导致小鼠耳蜗内毛细胞静纤毛变短且排列紊乱;Western blot检测结果表明,在HEI-OC1细胞和小鼠耳蜗中Osbpl2-KO均导致RhoA/ROCK2信号通路的抑制。结论:在听觉细胞中OSBPL2作为RhoA/ROCK2信号通路的重要效应因子参与了肌动蛋白细胞骨架的调控。

    Abstract:

    Objective: Exploring the effect of oxysterol-binding protein-like protein 2 (OSBPL2) on the morphology and function of the actin cytoskeleton in auditory cells and its related molecular mechanisms. Methods: Osbpl2 knockout (Osbpl2-KO) HEI-OC1 cells was used to investigate the effect of OSBPL2 deficiency on the morphology and function of the actin cytoskeleton in auditory cells. Stereocilia morphology of hair cells in Osbpl2-KO mouse was observed by scanning electron microscopy. Western blot assay was used to detect the expression level of Rho-associated coiled-coil-forming kinase 2 (ROCK2), Lim Domain Kinase 1 (Limk1) and Actin depolymerization factor 1 (ADF/CFL-1) in HEI-OC1 cells and mouse cochleae. Results: The distribution of F-actin in the microfilament skeleton of Osbpl2-KO HEI-OC1 cells was significantly down-regulated, the peripheral microspikes and filopodia were significantly reduced, and the cell migration ability was significantly weakened. In vivo, Osbpl2 deficient resulted in shortened and disordered arrangement of stereocilia in mouse inner hair cells. Western blot assay showed that Osbpl2-KO in HEI-OC1 cells and mouse cochlea led to the inhibition of RhoA/ROCK2 signaling pathway. Conclusion: OSBPL2 is involved in the regulation of actin cytoskeleton as an important effector of RhoA/ROCK2 signaling pathway in auditory cells.

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  • 收稿日期:2022-09-27
  • 最后修改日期:2022-11-11
  • 录用日期:2023-05-10
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