乳猪窦房结细胞原代培养及与ColⅠ纤维支架复合培养实验研究

PRIMARY CULTURE OF SINOATRIAL NODE CELLS FROM SUCKLING PIGS AND ITS CO-CULTURE WITH Col I FIBER SCAFFOLD

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 locate sinoatrial node （SAN） in suckling pigs, to develop a reliable method for isolation, purification and cultivation of SAN cells and to observe the compatibility of SAN cells and Col Ⅰ fiber scaffold. Methods Five newborn purebred ChangBaiShan suckling pigs （male and female）, aged less than 1-day-old and weighing 0.45-0.55 kg, were used. Multi-channels electrophysiological recorder was applied to detect the original site of atrial waves. Primary SAN cells harvested from that area were cultured by the conventional culture method and the purification culture method including differential velocity adherent technique and 5-BrdU treatment, respectively. Atrial myocytes isolated from the left atrium underwent purified culture. Cell morphology, time of cell attachment, time of unicellular pulsation, and pulsation frequency were observed using inverted microscope. The purified cultured SAN cells （5 × 10^5 cells/mL） were co-cultured with prewetted Col Ⅰ fiber scaffold for 5 days, and then the cells were observed by HE staining and scanning electron microscope （SEM）. Results The atrial waves occurred firstly at the area of SAN. The purified cultured SAN cells were spindle, triangular, and irregular in morphology, and the spindle cells comprised the greatest proportion. Atrial myocytes were not spindle-shaped, but primarily triangular and irregular. The proportion of spindle ceils in the conventional cultured SAN cells was decreased from 73.0% ± 2.9% in the purified cultured SAN cells, to 44.7% ± 2.3% （P 〈 0.01）, and the proportion of irregular cells increased from 7.0% ± 1.7% in the purified cultrued SAN cells to 36.1% ± 2.6% （P 〈 0.01）. The proportion of the triangular cells in the purified and the conventional cultured SAN cells was 20.0% ± 2.1% and 19.2% ± 2.5%, respectively （P 〉 0.05）. At 5 days after co-culture, HE staining displayed lots of SAN cells in Col Ⅰ fiber scaffold, and SEM demonstrated conglobate adherence of the cells to the surface and lateral pore wall of scaffold, mutual connections of the cell processes, or attachment of cells to lateral pore wall of scaffold through pseudopodia. Conclusion With accurate SAN location, the purification culture method containing differential velocity adherent technique and 5-BrdU treatment can increase the proportion of spindle cells and is a reliable method for the purification and cultivation of SAN cells. The SAN cells and Col Ⅰ fiber scaffold have a good cellular compatibility.