| 李献帅,陈献国.贴壁法与悬浮法诱导人胚胎干细胞分化为心肌细胞的对比研究[J].浙江中西医结合杂志,2018,28(3): |
| 贴壁法与悬浮法诱导人胚胎干细胞分化为心肌细胞的对比研究 |
| Comparative Study of Directional Differentiation into Cardiomyocytes of Human Embryonic Stem Cells by Adherence Method and Suspension Method |
| 投稿时间：2017-08-24 修订日期：2018-01-11 |
| DOI： |
| 中文关键词: 人胚胎干细胞 分化 心肌细胞 贴壁法 悬浮法 |
| 英文关键词:human embryonic stem cells differentiation cardiomyocytes adherence method suspension method |
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| 中文摘要: |
| 目的 对比研究直接贴壁法与悬浮法分别诱导人胚胎干细胞分化为心肌细胞所需的时间、分化成功率,以及分化后细胞的活性、抗缺氧凋亡刺激的能力。方法 人胚胎干细胞分别通过悬浮法和直接贴壁法诱导分化为心肌细胞。免疫荧光染色检测心肌细胞特异标志物cTnT；显微镜下计数比较2组细胞诱导分化出现跳动心肌细胞的时间、百分比和跳动频率；跳动心肌细胞经过24h缺氧刺激后,用凋亡试剂盒检测心肌细胞凋亡比例。结果 直接贴壁法诱导分化为心肌细胞的成功率更高(平均66.7%,悬浮法平均20.8%)；大量的自发跳动心肌细胞出现时间直接贴壁法更早一些(平均13天,悬浮法平均14天)；分化的心肌细胞cTnT染色均阳性,均检测到自发性动作电位；分化后的心肌细胞平均跳动频率范围相仿(63.0±7.0次/分VS 63.8±5.6次/分)；跳动心肌细胞缺氧24h后检测到的凋亡比例也没有明显差异((8.0±0.5)%VS(8.1±0.4)%。)。结论 2种方法均能成功诱导胚胎干细胞分化为心肌细胞,直接贴壁法分化成功率更高,所需分化时间较短；2种方法分化出的心肌细胞在细胞活性方面以及抗凋亡能力方面没有明显差异。 |
| 英文摘要: |
| Objective: Comparatively studying the time, efficiency, activity and anti-hypoxia ability of cardiomyocytes which are directionally induced from human embryonic stem cells by adherence method and suspension method respectively; Method: Human embryonic stem cells are induced into cardiomyocytes by adherence method and suspension method respectively;. Staining the specific marker cTnT of cardiomyocytes by immunofluorescence；Comparing the time, percentage and beating frequency of cardiomyocytes by counting under the microscope; Using apoptosis-hoechst staining kit to detect the apoptosis ratio of beating cardiomyocytes which have been treated by hypoxia with 24h. Result: The percentage of beating cardiomyocytes which are directionally induced from human embryonic stem cells by adherence method is obviously higher than that of suspension method ( 66.7% vs 20.8%); The group of adherence method results in that the mean time days of appearing beating cardiomyocytes is 13 days ,which is shorter than that of suspension method (14 days); Beating cardiomyocytes of each group are positive to cTnT staining; Spontaneous action potentials were detected in two group; The average beating frequency of differentiated cardiomyocytes is similar （63.0±7.0）times/min vs (63.8±5.6) times/min）；There was no significant difference in the percentage of apoptotic cells detected by beating cardiomyocytes after hypoxia 24h; Conclusion: The two methods both successfully induce the differentiation of embryonic stem cells into myocardiocytes, and the direct adherence method gain a higher differentiation rate and a shorter differentiation time; The cardiomyocytes differentiated by the two methods showed no significant difference in cell viability and anti apoptotic ability. |
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