TGF-β_1联合内脏内胚层END-2细胞诱导胚胎干细胞分化为心肌细胞及与凋亡的关系

Relationship between apoptosis and differentiation from ESCs into cardiomyocytes induced by TGF-β_1 and co-culture with visceral endoderm like END-2 cells

目的:探讨体外诱导胚胎干细胞(ESCs)向心肌细胞分化过程中分化与凋亡的关系,为优化组织工程的种子细胞提供依据。方法:先将ESCs悬浮培养形成2-3 d拟胚体(embryoid bodies,EBs),再向培养液中添加转化生长因子-β1(TGF-β1),以及与内脏内胚层样END-2细胞条件培养液共培养联合诱导ESCs向心肌细胞分化。以自然分化为对照组。激光共聚焦显微技术检测心肌细胞特异性肌钙蛋白T(TnT)的表达,透射电镜观察分化心肌细胞的超微结构。用AnnexinⅤ/PI双染后,在流式细胞仪上检测ESCs分化过程中的凋亡情况。结果:EBs经TGF-β1诱导后,有(43.00±2.08)%的拟胚体出现自发节律性收缩,表达心肌细胞特异性蛋白TnT,以及观察到心肌样超微结构,尤其是联合TGF-β1和END-2细胞条件培养液诱导,自发性收缩的拟胚体高达(88.00±1.42)%(P<0.01),收缩区域较大且拥有较成熟的心肌样结构。然而自然分化组发生自发节律性收缩的拟胚体只有(12.00±1.53)%。经2种诱导条件诱导后,检测两者的凋亡率分别为(5.58±0.65)%,(9.60±0.75)%(P<0.05)。结论:联合TGF-β1和内脏内胚层样END-2细胞条件培养液对心肌细胞分化有较高的诱导率,两者发挥协同诱导作用,可能机制是直接诱导ESCs分化或促进不向心肌细胞分化的ESCs凋亡。

AIM ： To provide the reference for optimizing the seed cells for tissue engineering, the relationship between apoptosis and differentiation in the process of induction on embryonic stem cells （ESCs） was investigated. METH- ODS： Day 2 -3 embryoid bodys （EBs） were derived from ESCs, and then tissue growth factor - β1 （TGF - β1 ） was added, and co - cultured with visceral endoderm like END - 2 cell conditioned medium. The induced cells were evaluated by using immunofluoresence and transmission electron micrography. Cell apoptosis was analyzed using flow cytometry. RE- SULTS： The total percentage of beating EBs treated with TGF - β1 was 43%. All the beating cardiomyocytes derived from ESCs expressed cardiac - specific proteins for TnT, and were observed the cardiac - specific uhrastructure. Interestingly, the total percentage of beating EBs treated with TGF - β1 combined with END - 2 cell conditioned medium was 88% （P 〈0.01 ）, and the beating areas were bigger. After 3 days of induction with different conditions, the apoptotic levels were （5. 58% ± 0. 65% and 9. 60% ± 0. 75% , P 〈 0. 05 ）, respectively. CONCLUSION： combination of TGF - β1 with co - cultured visceral endoderm like END - 2 cell conditioned medium could get a higher induction efficiency on the differentiation of ESCs into the cardiomyocytes. The partly inducible effect mechanisms may induce EDCs differentiation toward a cardiac phenotype and enhance apoptosis in cells not committed to cardiac differentiation.