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目的对乳猪窦房结所在部位进行准确定位,建立一套可靠的窦房结细胞取材、分离、纯化培养技术;观察窦房结细胞与ColⅠ纤维支架的相容性。方法24h内新生纯种长白山乳猪5只,雌雄不拘,体重0.45～0.55kg。实验动物采用多导电生理记录仪标记房波最早出现的部位,在该位置取材行原代窦房结细胞培养,分别采用常规方法培养和差速贴壁分离技术、5-BrdU处理纯化培养;于左心房部位取材行心房肌细胞纯化培养作为对照。倒置显微镜观察细胞形态、细胞贴壁时间、单细胞出现搏动时间、搏动频率等。将纯化培养的窦房结细胞以5×105个/mL密度接种至经预湿处理的ColⅠ纤维支架复合培养5d,行HE染色观察及扫描电镜观察。结果房波最早出现的位置即为窦房结所在部位。纯化培养的窦房结细胞有3种形态,即梭形、三角形与不规则形,其中梭形细胞最多;心房肌细胞主要为三角形及不规则形,而无梭形细胞。窦房结细胞纯化培养与常规培养比较,梭形细胞所占比例分别为73.0%±2.9%、44.7%±2.3%,二者差异有统计学意义(P<0.01);不规则形细胞分别为7.0%±1.7%、36.1%±2.6%,二者差异有统计学意义(P<0.01);三角形细胞分别为20.0%±2.1%、19.2%±2.5%,二者差异无统计学意义(P>0.05)。窦房结细胞复合ColⅠ纤维支架培养5d,HE染色观察示ColⅠ纤维支架材料中有大量细胞;扫描电镜见细胞成团吸附于支架表面及孔隙侧壁,并可见细胞间通过突起相互连接,或伸出伪足贴附于支架孔隙壁上。结论通过准确窦房结定位,采用差速贴壁结合5-BrdU处理的纯化培养可明显提高乳猪窦房结梭形细胞比例,是一种可靠的窦房结细胞纯化培养技术。窦房结细胞和ColⅠ纤维支架有较好的细胞相容性。
Objective To determine the location of the sinoatrial node of the piglets and to establish a reliable sinoatrial node cell culture, isolation, purification and culture techniques; To observe the compatibility of sinoatrial node cells and Col Ⅰ fiber scaffolds. Methods Newborn purebred Changbai Mountain suckling pigs within 24h, five male and female, weighing 0.45 ~ 0.55kg. Experimental animals using multi-conductive physiology recorder marked the earliest part of the room wave, at this location of the line of primary sinoatrial node cell culture, respectively, by conventional methods of culture and differential adherent separation technology, 5-BrdU treatment purification; The left atrium was taken from the atrial myocytes purified culture as a control. Inverted microscope observation of cell morphology, cell attachment time, single cell pulse time appears, pulsatile frequency. The purified cultured sinoatrial node cells were seeded at a density of 5 × 10 5 / mL into ColⅠ-fibroblast scaffolds pre-wet treated for 5 days and observed under HE staining and scanning electron microscopy. Results The earliest position of the atrial wave is the site of the sinus node. Purified culture of sinus node cells have three kinds of morphology, namely spindle, triangle and irregular shape, of which spindle cells; atrial myocytes are mainly triangular and irregular, and no spindle cells. Compared with the conventional culture, the percentage of spindle cells was 73.0% ± 2.9% and 44.7% ± 2.3%, respectively, the difference was statistically significant (P <0.01); the irregularly shaped cells were 7.0% ± 1.7% and 36.1% ± 2.6%, respectively (P <0.01). The number of triangular cells was 20.0% ± 2.1% and 19.2% ± 2.5% respectively, with no significant difference (P > 0.05). The sinoatrial node cells were cultured for 5 days with ColⅠfibrous scaffolds. HE staining showed a large number of cells in the ColⅠfibrous scaffolds; cells were observed by scanning electron microscopy to adhere to the surface of the scaffolds and to the side walls of the pores, and the cells were connected with each other through protrusions, A pseudo-foot attached to the scaffold pore wall. Conclusion The accurate culture of sinoatrial node cells can be significantly improved by the accurate culture of sarcolemma with 5-BrdU treatment. Sinoatrial node cells and Col Ⅰ fiber scaffolds have better cell compatibility.