蜗轴螺旋动脉平滑肌的培养及其鉴定

Cultivation and Identification of Vascular Smooth Muscle Cells from Spiral Modiolar Artery

目的:本研究通过联合两种常用的细胞培养方法,建立了一个蜗轴螺旋动脉平滑肌细胞原代培养的模型。方法:分离的蜗轴螺旋动脉的平滑肌来自于豚鼠。切碎血管组织并且将其放入37度的0.1%胰蛋白酶溶液中消化20分钟。消化后,将这些消化后的组织块在35-mm的培养皿中贴壁。运用这种培养方法,混杂的成纤维细胞通过与血管平滑肌细胞不同的贴壁能力,可在传代的时候被去除。在7-10天后,细胞从组织块中长出来。大约3周,细胞可长满培养皿。在第三代培养的血管平滑肌细胞中,纯的并且活性好的细胞可以被活得。经过形态学,免疫荧光化学和电镜对这种方法得到的血管平滑肌细胞进行了鉴别。结果:显示了典型的平滑肌的细胞的特点:形态学的"峰-谷"样的生长形态,免疫荧光化学显示这些细胞表达平滑肌细胞的特异性标志物(α-SM-actin和myosin。结论:通过本文研究方法得到的大量的纯的蜗轴螺旋动脉血管平滑肌细胞是一个很好的研究血管平滑肌细胞在内耳循环紊乱过程生理功能的一个体外模型。除此之外,还可以是一些作用于血管平滑肌药物评价的体外模型。

Objective：To establish a method for primary cultures of vascular smooth muscle cells（VSMCs） from the spiral modiolar artery（SMA） by combining two common cultural methods in vitro.Methods：VSMCs were isolated from SMAs of guinea pigs.The arterial tissues were minced and enzymatically digested at 37℃ for 20 min using a trypsin solution（0.1%）.After digestion,the fragments were explanted in a 35-mm culture dish.Contaminated fibroblasts were separated from VSMCs due to their different adhesion abilities.Results：The cells migrated from the explants within 7-10 d and grew to confluence in approximately3 weeks.Pure and viable VSMCs were obtained from the confluent third passage.The cells obtained based on this protocol were subsequently characterized by morphology,immunofluorescence and transmission electron microscopy analysis.The results demonstrated typical characteristics of VSMCs,including a ＂hill-and-valley＂ growth pattern and the expression of cell type-specific markers（α-smooth muscle actin and myosin） by morphological and immunofluorescence analysis,respectively.Conclusion：The multitude and purified cells obtained from this method could be a model to investigate the physiological mechanisms of VSMCs in the circulation disturbances of the inner ear.In addition,VSMCs were regarded to be an excellent model system for the evaluation of drugs in vitro.