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目的 :5 -氮胞苷作为分化诱导剂 ,初步探讨其单独或联合全反式维甲酸应用时对小鼠胚胎干细胞(mESC)分化为心肌细胞的影响 ,旨在建立一种体外诱导mESC分化为心肌细胞的实验方法。方法 :采用MTT法确定 5 -氮胞苷的非细胞毒性参考剂量。设计不同条件培养基 (5 -氮胞苷单独或配伍全反式维甲酸应用 )对mESC进行诱导分化 ,并通过免疫组化技术及RT -PCR方法等对分化细胞进行鉴定。结果 :5 -氮胞苷的非细胞毒性参考剂量为 8μmol/L ,能够诱导mESC分化为心肌合胞体 (与阴性对照组比较 ,P <0 0 1) ,诱导分化率可达 5 0 %。配伍全反式维甲酸持续诱导的结果等同于单独应用全反式维甲酸的作用效果 (P >0 0 5 ) :即对ESC向心肌细胞的诱导分化没有促进作用。结论 :5 -氮胞苷能够诱导mESC分化为心肌合胞体 ,从而得以建立一种体外诱导mESC分化为心肌细胞的方法。
AIM: To investigate the effect of 5 - azacytidine on the differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes when used alone or in combination with all - trans retinoic acid, in order to establish an in vitro induction of mESC differentiation into Experimental method of cardiomyocytes. Methods: The non - cytotoxic reference dose of 5 - azacytidine was determined by MTT assay. Design different conditions of medium (5 - azacytidine alone or in combination with all-trans retinoic acid application) mESC induced differentiation, and by immunohistochemical techniques and RT-PCR method for identification of differentiated cells. Results: The noncytotoxic reference dose of 5 - azacytidine was 8 μmol / L, which could induce the differentiation of mESC into cardiomyocyte syncytiotrophoblast (P <0.01). The rate of induced differentiation was 50%. The result of continuous induction of all-trans retinoic acid was equivalent to the effect of all-trans retinoic acid alone (P> 0 05): it did not promote the differentiation of ESC into cardiomyocytes. CONCLUSION: 5 - Azacytidine can induce the differentiation of mESC into cardiomyocyte syncytiotrophoblast, so that a method of inducing mESC differentiation into cardiomyocytes can be established in vitro.