原代培养乳鼠窦房结细胞的形态学及表面抗原研究

Study on morphology and surface antigen of primary cultured neonatal rats' sinoatrial node cells

目的建立乳鼠窦房结细胞(SNC)的体外分离纯化培养方法,了解SNC的形态结构特点,观察表面抗原超极化激活环核苷酸门控阳离子通道基因4(HCN4)和缝隙连接蛋白45(Cx45)的细胞表达特点,为细胞生物起搏的深入研究提供一定实验依据。方法取新生SD乳鼠的窦房结组织,胰酶逐次消化,并予差速贴壁及5-溴脱氧尿嘧啶核苷(5-BrdU)纯化处理进行原代窦房结细胞的培养。结果在培养的窦房结细胞中可观察到3种形态的细胞,即梭形细胞、三角形细胞和多边形细胞,其中梭形细胞所占比例最大,搏动频率最快,细胞器不发达,肌原纤维稀少,表达HCN4和Cx45;三角形细胞微量表达Cx45。结论①胰酶消化法结合差速贴壁及5-BrdU处理,是可靠的窦房结细胞纯化培养技术;②搏动频率最快、HCN4和Cx45表达阳性的梭形细胞可能系窦房结起搏细胞。

Objective To understand morphological characteristics of sinoatrial node cells （SNCs） by taking purified isolation and culture of neonatal rat＇s SNCs in vitro and to understand the expression characteristics of surface antigens hyperpolarization activated cyclic nucleotide gated cation channel 4 （HCN4） and connexin 45 （Cx45） on SNCs. Methods The SNCs were isolated from sinus node tissues removed from neonatal rats by trypsin and purified cultured with differential attachment and 5＇-bromodeoxyuridine （BrdU） treatment. Results Three different types of ceils were observed among the purified cultured SNCs： spindle, triangular and irregular cells. The spindle cells took up the greatest proportion, had the fastest beats, and had less developed organelle and scarce muscular fibrils. Immunofluorescence staining was positive against HCN4 and Cx45 on the spindle cells. Small amounts of HCN4 was observed on triangular cells. Conclusion （1） Isolation by trypsin combined with differential attachment and BrdU treatment is a reliable approach to culturing sinoatrial node cells. （2） The spindle cells with the fastest beats and positive expressions of HCN4 and Cx45 may be the sinus node pacemaker cells.