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目的明确生长分化因子(GDF)-11对小鼠诱导多能干细胞(mi PSCs)向心肌细胞定向分化的促进作用,为心肌再生的细胞生物学治疗提供种子细胞和实验依据。方法常规培养小鼠mi PSCs,分为对照组和GDF-11组。GDF-11组在普通分化培养基中加用10 ng/ml的GDF-11。悬滴法诱导培养形成拟胚体(EBs),每日观察搏动拟胚体数目。采用实时荧光定量PCR检测多能干细胞标志物Oct-4、心脏中胚层标志物Flk-1、心脏祖细胞标志物Nkx2.5、和心肌特异性标志物c Tn T的表达变化。用免疫荧光染色观察心肌结构蛋白c Tn I的表达水平。结果GDF-11组的Oct-4表达水平在诱导分化的第3、7天分别为同期对照组的(0.55±0.31)倍(P<0.01)和(0.41±0.57)倍(P<0.05);诱导分化的第10天,GDF-11组的Flk-1和Nkx2.5表达相分别为对照组的(2.09±0.8)倍(P<0.05)和(2.47±0.22)倍(P<0.01);c Tn T的表达在分化后第10天和第14天分别为对照组的(1.81±0.19)倍(P<0.01)和(1.61±0.20)倍(P<0.01);两组均出现了心肌样搏动细胞团,GDF-11组搏动EBs百分比要显著高于对照组(P<0.01)。免疫荧光染色显示,GDF-11组的c Tn I阳性率要显著高于对照组(P<0.01)。结论 GDF-1能够显著促进mi PSCs的心肌定向分化。
Objective To investigate the effect of growth factor (GDF) -11 on the directional differentiation of mouse induced pluripotent stem cells (mi PSCs) into cardiomyocytes and to provide seed cells and experimental evidences for cell biology treatment of myocardial regeneration. Methods Mouse mi PSCs were cultured routinely and divided into control group and GDF-11 group. GDF-11 group added 10 ng / ml GDF-11 to ordinary differentiation medium. The hanging drop method induced the formation of embryoid bodies (EBs) and observed the number of beating embryoid bodies daily. Real-time quantitative PCR was used to detect the expression changes of pluripotent stem cell marker Oct-4, cardiac mesoderm marker Flk-1, cardiac progenitor cell marker Nkx2.5, and cardiac specific marker cTnT. Immunofluorescence staining was used to observe the expression of cTn I. Results The expression level of Oct-4 in GDF-11 group was (0.55 ± 0.31) times (0.41 ± 0.57) times (0.41 ± 0.57) times higher than that in the control group on the 3rd and 7th day respectively (P <0.05) On day 10, the expression of Flk-1 and Nkx2.5 in GDF-11 group was (2.09 ± 0.8) times (2.47 ± 0.22) times (2.47 ± 0.22) fold higher than that in control group (P <0.01) The expression of cTn T was (1.81 ± 0.19) times (1.81 ± 0.19) times and (1.61 ± 0.20) times higher than that of the control group (P <0.01) on the 10th and 14th day after differentiation respectively Like beating cell mass, the percentage of beating EBs in GDF-11 group was significantly higher than that in control group (P <0.01). Immunofluorescence staining showed that the positive rate of cTn I in GDF-11 group was significantly higher than that in control group (P <0.01). Conclusion GDF-1 can significantly promote the directional differentiation of mi PSCs.