目的 对比研究人和小鼠胚胎干细胞体外定向诱导分化为心肌细胞的方法、效率以及抗缺氧凋亡刺激的能力,为体外胚胎干细胞诱导分化为心肌细胞提供基础实验依据.方法 按照加或不加诱导剂分为4组:人胚胎干细胞(hESC)加诱导剂组、hESC不加诱导剂组、小鼠胚胎干细胞(mESC)加诱导剂组、mESC不加诱导剂组.hESC分别通过不加诱导剂的悬浮法和加诱导剂的直接贴壁法诱导分化为心肌细胞;mESC分别通过加和不加诱导剂的悬滴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心肌肌钙蛋白T(cTnT);显微镜下计数比较4组诱导分化细胞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24h缺氧刺激后,用凋亡试剂盒检测心肌细胞凋亡比例.结果 hESC不加诱导剂组分化出现自发跳动心肌细胞的平均时间为(13.9±0.9)天,百分比20.8％,平均跳动频率(63.8±5.6)次/min; hESC加诱导剂组分化出现自发跳动心肌细胞的平均时间为(13.0±1.1)天,百分比66.7％,平均跳动频率(63.0±7.0)次/min;mESC不加诱导剂组分化出现自发跳动心肌细胞的平均时间为(14.3±1.0)天,百分比12.5％,平均跳动频率(80.2±3.9)次/min; mESC加诱导剂组分化出现自发跳动心肌细胞的平均时间为(12.2±1.2)天,百分比81.3％,平均跳动频率为(79.9±7.7)次/min.各组跳动心肌细胞cTnT染色阳性,缺氧24h后检测到不同的凋亡比例.结论 4组均能成功诱导胚胎干细胞分化为心肌细胞,其中hESC加诱导剂的直接贴壁法诱导成功在国内尚属首次.两种细胞系加诱导剂均比不加诱导剂分化效率明显提高;mESC诱导成心肌细胞更简单快速,效率更高;在无其他保护因子存在的情况下,hESC诱导的心肌细胞抗缺氧凋亡刺激能力更强,体外维持跳动的时间也更长.%Objective Comparatively studying the method,efficiency and anti-hypoxia ability of cardiomyocytes which are directionally induced from human embryonic stem cells and mouse embryonic stem cells in vitro so as to provide an experimental basis for further study of inducing the differentiation of embryonic stem cells into cardiomyocytes in vitro.Methods Human embryonic stem cells are induced into cardiomyocytes by suspension method which dosen't using inducersand adherence method which using inducers respectively.Mouse embryonic stem cells are induced into cardiomyocytes by hanging drop method which dosent using inducers and using inducer 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 24 h.Results The group of hESC without inducers results in that the mean time days of appearing beating cardiomyocytes is (13.9 ± 0.9) days,the percentage is 20.8 ％ and the average frequency of beating is (63.8 ± 5.6) times/min.The group of hESC with inducers results in that the mean time days of appearing beating cardiomyocytes is (13.0 ± 1.1) days,the percentage is 66.7％ and the average frequency of beating is (63.0 ± 7.0) times/min.The group of mESC without inducers results in that the mean time days of appearing beating cardiomyocytes is (14.3 ± 1.0) days,the percentage is 12.5％ and the average frequency of beating is (80.2 ± 3.9) times/min.The group of mESC with inducers results in that the mean time days of appearing beating cardiomyocytes is (12.2 ± 1.2) days,the percentage is 81.3％ and the average frequency of beating is (79.9 ±7.7) times/min.Beating cardiomyocytes of each group are positive to cTnT staining.Different apoptosis ratio are detected of beating cardiomyocytes of each group.Conclusion The four methods can all successfully induce the differentiation of embryonic stem cells into myocardiocytes,and the adherent method of hESC induced with activin A + BMP4 is the first successful induction in China.The groups adding inducers improve the differentiation efficiency more significantly than the groups without adding inducers.Inducing mouse embryonic stem cells into cardiomyocytes is more simple and efficient compared with human embryonic stem cells.Without the presence of other protective factor,anti-hypoxia ability of cardiomyocytes induced from human embryonic stem cells is stronger and the beating time are longer in vitro compared with mouse embryonic stem cells.
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