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

作　　者：李献帅 陈献国[1] 楼洋[1] 许博[1] 徐小义 

机构地区：[1]浙江省金华市中心医院心胸外科,金华321000

出　　处：《浙江中西医结合杂志》2018年第3期188-190,共3页Zhejiang Journal of Integrated Traditional Chinese and Western Medicine

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c Tn T;显微镜下计数比较两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(66.7%比20.8%,P<0.05);大量的自发跳动心肌细胞出现时间直接贴壁法早与悬浮法,差异有统计学意义[(13.0±1.1)天比(13.9±0.9)天,P<0.05];分化的心肌细胞c Tn T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两种方法相仿[(63.0±7.0)次/分比(63.8±5.6)次/分,P>0.05];跳动心肌细胞缺氧24h后检测到的凋亡比例比较差异无统计学意义[(8.0±0.5)%比(8.1±0.4)%,P>0.05]。结论两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;两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无明显差异。Objective to compare the time, efficiency, activity and anti-hypoxia ability of cardiomyocytes that are directionally induced from human embryonic stem cells by adherence method and suspension method respectively. Methods Human embryonic stem cells were induced into cardiomyocytes by adherence method and suspension method respectively. The specific marker c Tn T of cardiomyocytes was detected by immunofluorescence; the time, percentage and beating frequency of cardiomyocytes were observed under the microscope; the apoptosis ratio of beating cardiomyocytes after treating with hypoxia for 24 h was assessed with apoptosis-hoechst staining kit. Results The percentage of beating cardiomyocytes directionally induced from human embryonic stem cells by adherence method was higher than that of suspension method(66.7% vs 20.8%, P<0.05). The mean time of appearing beating cardiomyocytes in adherence group was 13 days, which was shorter than 14 days of suspension group(P<0.05). Beating cardiomyocytes of each group were positive to c Tn T staining, and spontaneous action potentials were detected in two group. The average beating frequency of differentiated cardiomyocytes was similar(63.0±7.0 times/min vs 63.8±5.6 times/min, P>0.05. There was no significant difference in the percentage of apoptotic cells detected by beating cardiomyocytes after hypoxia 24 h. Conclusion The two methods can 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.

关 键 词：人胚胎干细胞 分化 心肌细胞 贴壁法 悬浮法 

分 类 号：R54[医药卫生—心血管疾病]