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- 2017
成年大鼠窦房结活细胞分离方法的建立及钠通道各亚型表达的观察
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Abstract:
摘要：目的 建立急性分离成年大鼠窦房结细胞的方法，了解其形态结构特点，系统观察钠通道各亚型在窦房结细胞的表达情况，为进一步研究钠通道在窦房结功能中的作用及分子机制提供依据。方法 取成年大鼠窦房结组织，Ⅱ型胶原蛋白酶联合蛋白酶酶解分离活性窦房结细胞，行钠通道亚型Nav1.1、Nav1.2、Nav1.3、Nav1.5、Nav1.6、Nav1.7、Nav1.8、Nav1.9的免疫荧光染色，激光共聚焦显微镜观察各亚型在窦房结细胞的表达。结果 在急性分离的窦房结细胞中观察到梭形、弧形、细长弯曲形等细胞形态，以梭形为主，细胞横纹清楚、活性好，可存活6～8h；钠通道Nav1.1、Nav1.5、Nav1.6、Nav1.7、Nav1.8、Nav1.9在大鼠窦房结细胞呈阳性表达，Nav1.2、Nav1.3表达呈阴性。结论 Ⅱ型胶原蛋白酶联合蛋白酶酶解分离方法是可靠的成年大鼠活性窦房结细胞分离方法，钠通道各亚型在成年大鼠窦房结细胞呈差异性表达。
ABSTRACT: Objective To establish a method to isolate single sinoatrial node cells of adult rats and observe the cells’ morphological structure and the expression of sodium channel subtype so as to provide experimental basis for further research on the role of sodium channel in sinoatrial node function. Methods We isolated adult rat sinoatrial node tissue and cut it into slices about 2mm in width, digested the slices with type Ⅱ collagenase combinate protease, and observed the cells’ morphological structure. We then performed immunofluorescence staining with HCN4 and laser confocal imaging to identify cells and observe the expressions of Nav1.1, Nav1.2, Nav1.3, Nav1.5, Nav1.6, Nav1.7, Nav1.8 and Nav1.9. Results The isolated cells had spindle-like, arc-like, and slender and curved shapes, but were mainly spindle-shaped. The stripes of the cells were clear, had good bioactivity and could survive 6-8 hours. Sodium channels Nav1.1, Nav1.5, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 were positively expressed in adult rat sinoatrial node cells, while Nav1.2 and Nav1.3 were negatively expressed. Conclusion The method of digesting and isolating adult rat sinoatrial node cells with type Ⅱ collagenase combinate protease is reliable, and sodium channel subtypes are differently expressed in sinoatrial node cells
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