Costa José Jackson do Nascimento, de Souza Glaucinete Borges, Bernardo Joyla Maria Pires, Ribeiro Regislane Pinto, Passos José Renato de Souza, Bezerra Francisco Taiã Gomes, Saraiva Márcia Viviane Alves, Silva José Roberto Viana
Biotechnology Nucleus of Sobral - NUBIS,Federal University of Ceara,CEP 62042-280,Sobral,CE,Brazil.
Biotechnology Nucleus of Sobral - NUBIS,Federal University of Ceara,Av. Comandante Maurocélio Rocha Ponte 100,CEP: 62.041-040,Sobral,CE,Brazil.
Zygote. 2017 Jun;25(3):341-357. doi: 10.1017/S0967199417000211. Epub 2017 Jul 3.
This study aims to investigate the effect 5-azacytidine (5-Aza) during induction of pluripotency in bovine fibroblasts and to evaluate the effects of BMP2, BMP4 or follicular fluid in the differentiation of reprogrammed fibroblasts in primordial germ cells and oocytes. It also analysis the mRNA levels for OCT4, NANOG, REX, SOX2, VASA, DAZL, cKIT, SCP3, ZPA and GDF9 after culturing 5-Aza treated fibroblasts in the different tested medium. Dermal fibroblasts were cultured and exposed to 0.5, 1.0 or 2.0 μM of 5-Aza for 18 h, 36 h or 72 h. Then, the cells were cultured in DMEM/F12 supplemented with 10 ng/ml BMP2, 10 ng/ml BMP4 or 5% follicular fluid. After culture, morphological characteristics, viability and gene expression were evaluated by qPCR. Treatment of skin fibroblasts with 2.0 μM 5-Aza for 72 h significantly increased expression of mRNAs for SOX2, OCT4, NANOG and REX. The culture in medium supplemented with BMP2, BMP4 or follicular fluid for 7 or 14 days induced formation of oocyte-like cells, as well as the expression of markers for germ cells and oocyte. In conclusion, treatment of bovine skin-derived fibroblasts with 2.0 μM 5-Aza for 72 h induces the expression of pluripotency factors. Culturing these cells in differentiation medium supplemented with BMP2, BMP4 or follicular fluid induces morphological changes and promotes expression of markers for germ cells, meiosis and oocyte.
本研究旨在探究5-氮杂胞苷（5-Aza）在牛成纤维细胞重编程诱导过程中的作用，并评估骨形态发生蛋白2（BMP2）、骨形态发生蛋白4（BMP4）或卵泡液对重编程后的成纤维细胞向原始生殖细胞和卵母细胞分化的影响。研究还分析了在不同测试培养基中培养经5-Aza处理的成纤维细胞后，八聚体结合转录因子4（OCT4）、 Nanog同源蛋白（NANOG）、REX1转录因子（REX）、性别决定区Y盒2（SOX2）、血管生成素（VASA）、无精症缺失基因（DAZL）、干细胞生长因子受体（cKIT）、联会复合体蛋白3（SCP3）、锌指蛋白基因（ZPA）和生长分化因子9（GDF9）的mRNA水平。培养真皮成纤维细胞，并将其暴露于0.5、1.0或2.0 μM的5-Aza中18小时、36小时或72小时。然后，将细胞培养在添加有10 ng/ml BMP2、10 ng/ml BMP4或5%卵泡液的DMEM/F12培养基中。培养后，通过实时定量聚合酶链反应（qPCR）评估细胞的形态特征、活力和基因表达。用2.0 μM 5-Aza处理皮肤成纤维细胞72小时可显著增加SOX2、OCT4、NANOG和REX的mRNA表达。在添加BMP2、BMP4或卵泡液的培养基中培养7天或14天可诱导形成类卵母细胞，并促进生殖细胞和卵母细胞标志物的表达。总之，用2.0 μM 5-Aza处理牛皮肤来源的成纤维细胞72小时可诱导多能性因子的表达。在添加BMP2、BMP4或卵泡液的分化培养基中培养这些细胞可诱导形态变化，并促进生殖细胞、减数分裂和卵母细胞标志物的表达。
