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目的:比较差速贴壁技术与常规培养方法对窦房结梭形细胞比例,旨在建立一套可靠的窦房结梭形细胞取材分离、纯化培养技术。方法:实验于2004-05在解放军第四军医大学心血管外科实验室完成。选用出生24h内的新西兰乳兔15只。对新西兰乳兔的窦房结组织进行原代细胞培养,将培养容器置于37℃、体积分数0.05CO2孵箱中培养(常规方法)。将培养容器置于37℃、体积分数0.05CO2孵箱中培养90min后,采用单细胞悬液中夹杂一部分心房肌细胞和成纤维细胞,成纤维细胞与心肌细胞相比,其贴壁过程快,大部分成纤维细胞能在短时间内(约30～60min)完成贴壁过程;而心肌细胞在短时间内不附着或附着不稳定,轻微震荡就可以浮起,利用这一点得到纯化心肌细胞的差速贴壁分离技术,并采用5-溴脱氧尿核苷抑制除心肌细胞外其他生长比较快的细胞(如成纤维细胞的生长),从而造成更有利于心肌细胞生长的环境。比较两种方法处理后各形态窦房结细胞的比例。结果:①在窦房结的培养中,起搏细胞、过渡细胞、和心房肌细胞都存在,起搏细胞的比例是评价培养方法的重要指标。差速贴壁技术结合5-溴脱氧尿核苷处理的纯化培养中窦房结梭形细胞比例明显高于常规方法犤(65±4)%,(45±3)%,P<001犦。②差速贴壁结合5-溴脱氧尿核苷处理的纯化培养的窦房结细胞梭形细胞电生理特征符合窦房结起搏细胞的特点,即动作电位有舒张期自动去极化。记录的10个梭形细胞的动作电位中平均最大舒张电位为(-50.9±4.8)mV,动作电位幅度为(62.9±5.0)mV。结论:①梭形细胞动作电位的特性与分离的窦房结单细胞的记录结果相似,证实了梭形细胞就是窦房结起搏细胞。②常规原代培养过程中,成纤维细胞生长速度快,而心肌细胞生长偏慢,成纤维细胞的数量很快会超过心肌细胞,使培养细胞中混杂有大量的成纤维细胞,从而影响心肌细胞培养模型的可靠性。③5-溴脱氧尿核苷可显著抑制培养心肌细胞中的成纤维细胞生长,且对心肌细胞无毒性与抑制作用,可使培养细胞中心肌细胞所占比例提高明显。因此用差速贴壁结合5-溴脱氧尿核苷处理的纯化培养较常规培养可更明显提高窦房结梭形细胞的比例,是一种可靠的窦房结细胞培养技术。
OBJECTIVE: To compare the ratio of spindle-shaped cells in sinoatrial node with the technique of differential adherence and conventional culture, aiming at establishing a reliable spindle-shaped cell culture and isolation of sinoatrial node. Methods: The experiment was performed at the Cardiovascular Surgery Laboratory of the Fourth Military Medical University of PLA in 2004-05. Selection of New Zealand rabbits born within 24h 24h 15. The primary cultured cells of New Zealand rabbit were made into primary cell culture. The culture vessel was incubated in a 0.05CO2 volumetric flask at 37 ℃ (routine method). After incubating the culture container at 37 ° C. for 90 min in a 0.05CO2 volumetric chamber, a part of atrial myocytes and fibroblasts were mixed with a single cell suspension. Compared with cardiomyocytes, fibroblasts adhered rapidly, Most fibroblasts can complete the process of attachment in a short period of time (about 30 ~ 60min). However, in a short period of time, the cardiomyocytes do not attach or attach unstable, and they can float with a slight shock. Using this point, purified cardiomyocytes Differential adherent separation technology, and the use of 5-bromodeoxyuridine inhibition of myocardial cells in addition to other faster growth of cells (such as the growth of fibroblasts), resulting in more conducive to myocardial cell growth environment. The proportions of sino-atrial node cells in each form were compared between the two methods. Results: ① In the culture of the sinus node, pacemaker cells, transitional cells and atrial myocytes are all present. The proportion of pacemaker cells is an important index to evaluate the culture method. The ratio of spindle cells in the sinoatrial node in the purified culture treated with 5-bromodeoxyuridine was significantly higher than that of the conventional method (65 ± 4)%, (45 ± 3)%, P <001 犦. (2) The electrophysiological characteristics of spindle cells in purified cultured sinus node cells treated by differential adherent method combined with 5-bromodeoxyuridine meet the characteristics of pacemaker cells in sinoatrial node, that is, the action potentials have diastolic auto-depolarization. The mean maximal diastolic potential (-50.9 ± 4.8) mV and the action potential amplitude (62.9 ± 5.0) mV were recorded for the 10 spindle cells. Conclusions: ① The characteristics of spindle-shaped action potential are similar to those of isolated single-cell sino-atrial node, which confirms that spindle-shaped cells are the pacemaker cells of the sinoatrial node. ② routine primary culture, fibroblasts grow fast, and myocardial cell growth is slow, the number of fibroblasts will soon exceed the myocardial cells, so that cultured cells mixed with a large number of fibroblasts, which affect the myocardial cells Develop model reliability. ③ 5-Bromodeoxyuridine can significantly inhibit the growth of fibroblasts in cultured cardiomyocytes, and has no toxic and inhibitory effects on cardiomyocytes, which can significantly increase the proportion of cardiomyocytes in cultured cells. Therefore, the use of differential adherent culture combined with 5 - bromodeoxyuridine treatment of purified culture more conventional culture can significantly improve the ratio of spindle cells of the sinus node, is a reliable sinoatrial node cell culture technology.