生长分化因子-11促进小鼠诱导性多能干细胞向心肌细胞定向分化的研究

目的 明确生长分化因子（GDF）-11对小鼠诱导多能干细胞（miPSCs）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miPSCs,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cTnT的表达变化。用免疫荧光染色观察心肌结构蛋白cTnI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）；cTnT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cTnI阳性率要显著高于对照组（P<0.01）。结论 GDF-1能够显著促进miPSCs的心肌定向分化。

Growth differentiation factor 11 promotes cardiac differentiation of mouse-induced pluripotent stem cells

AIM To study the effect of growth differentiation factor 11 (GDF-11) on the cardiac differentiation of mouse-induced pluripotent stem cells (miPSCs) and to provide promising cells and an experimental basis for the cellular biological therapy of cardiomyocyte regeneration. METHODS MiPSCs were cultured using conventional methods and were divided into GDF-11 group and control group. The differentiation medium of the GDF-11 group contained 10 ng/ml GDF-11. Embryoid bodies (EBs) were formed using the hanging drop method and the number of beating EBs was calculated daily. Expression of pluripotent stem cell marker Oct-4, cardiac mesoderm marker Flk-1, cardiac progenitor marker Nkx2.5 and cardiac specific marker cTnT were detected using qRT-PCR. The expression of cTnI was observed using immunoflourensence staining. RESULTS Expression levels of Oct-4 in GDF-11 group at day 3 and day 7 were (0.55±0.31)-fold (P<0.01) and (0.41±0.57)-fold (P<0.05) of those in the control group. The expressions of flk-1 and Nkx2.5 in GDF-11 group at day 10 were higher (2.09±0.8)-fold (P<0.05) and (2.47±0.22)-fold (P<0.01) than the control group. In addition, GDF-11 group at days 10 and 14 had a (1.81±0.19)-fold change (P<0.01) and a (1.61±0.20)-fold change (P<0.01) of cTnT expression compared with those in the control group. Beating areas were found in both groups, but GDF-11 group had a higher percentage of beating EBs. Immunoflourensence staining showed more cTnI positive cells in the GDF-11 group (P<0.01). CONCLUSION GDF-11 significantly enhances cardiac differentiation of miPSCs.