罗氏易位伴少弱精子症来源的胚胎干细胞向生殖细胞诱导分化的研究

Differentiation of embryonic stem cells from the embryos of the couples with male asthenozoospermia and Robertsonian translocation into germ cells

目的:建立罗氏易位伴少弱精子症来源的人胚胎干细胞(ESC),利用体外诱导ESC向生殖细胞分化作为模型,评估辅助生殖技术子代的潜在风险。方法:利用罗氏易位伴少弱精子症患者夫妇捐赠的胚胎,通过分离囊胚内细胞团,培养、传代、扩增,建立ESC CCRM16,其核型为46,XY,+14,rob(13;14)(q10;q10);添加2 mol/L维甲酸体外诱导分化,分析其向生殖细胞分化过程及其相关基因的表达,并与遗传背景完全正常的ESC CCRM23相比较。结果:CCRM16表达OCT4,TRA-1-81,NANOG及SSEA4多能性标记基因,体外能形成拟胚体(EB),体内、体外都能向3个胚层分化。添加维甲酸可直接诱导ESC向生殖细胞分化。原始生殖细胞标志基因DAZL和减数分裂标记基因SCP3的表达水平在CCRM16中较正常夫妇捐赠胚胎建立的ESC CCRM23明显减少。结论:核型为46,XY,+14,rob(13;14)(q10;q10)CCRM16具有ESC典型特征,但其向早期精子分化的过程异常。体外诱导CCRM16向精子分化作为研究模型,可应用于评估少弱精子症患者辅助生殖出生子代的健康风险。

Objective： To assess the risk of male infertility in the offspring conceived through assisted reproductive technology （ART） byin vitroinductionof the differentiation of embryonic stem cells （ESCs） derived from the embryos of the couples with male as- thenozoospermia and Robertsonian translocation （RT） into germ cells. Methods： We established a CCRM16ESC line with the karyo- type of 46, XY, ＋ 14, rob（ 13; 14） （ql0; ql0） from the embryo donated by a patientwithasthenozoospermiaand RT and his wife by isolation of the inner cell mass of blastula, culturing, passaging, and amplification, followed by in vitro induction and differentiationof the ESCs into germ cells with ratinoic acid（RA） at 2 mol/L. Then, we analyzed the process of differentiation and the expressions of its related genes and compared them with those in the normal CCRM23ESCs. Results：CCRM16 showed the typical characteristics of ESCs, expressing the pluripotency makers of NANOG, OCT4, TRA-l-181 and SSEA4, forming embryoid bodies, and differentiating in-to three germlayer tissues in vitro and in vivo. Intervention with 2 mol/LRAinduced direct differentiation of the ESCs into germ cells. The expressions of the primordial germ cell marker geneDAZLand the meiosis marker geneSCP3were markedly decreased in the CCRM16 as compared with those in the normal CCRM23 ESCs. Conclusion： The CCRM16ESC linewith the karyotype of 46, XY, ＋ 14, rob（ 13; 14） （ql0; ql0） has thetypical characteristics of ESCs but an abnormal process of differentiation into germ cells in the early stage. In vitroinductionof the differentiation of ESCs into germ cells can be used for assessing the risk of male infertility in the off- spring conceived through ART for asthenozoospermia patients.