梗阻性和非梗阻性无精子症患者睾丸组织来源iPSCs系的建立

Generation of testis-derived iPSCs from obstructive azoospermia and non-obstructive azoospermia

目的利用梗阻性无精子症(OA)和非梗阻性无精子症(NOA)患者的睾丸组织建立诱导性多能干细胞(iPSCs)系,比较两者建系过程有无差别。方法分别取3例OA患者和特发性NOA患者5～6 mm^3的睾丸组织,组织块培养方法进行原代培养,传至3～4代转染仙台病毒,待细胞增殖满后转移至饲养层细胞上继续培养,第21～22天挑取克隆,建立iPSCs系并进行多能性和分化能力的鉴定。结果两株iPSCs系均成功建立,建系过程无明显差异,碱性磷酸酶(AP)染色均为阳性;八聚体结合转录因子4(OCT4)、性别决定基因相关蛋白2(SOX2)、阶段特异性胚胎抗原-4(SSEA-4)、肿瘤排斥抗原-1-60(TRA-1-60)的免疫荧光染色均为阳性;小鼠体内的畸胎瘤实验也表明获得的两株iPSCs系具有向3个胚层分化的潜能。结论利用仙台病毒非基因整合方法,OA患者和特发性NOA患者的睾丸组织均能建立iPSCs系。

Objective To establish testis-derived induced pluripotent stem cell(iPSCs)from patients with obstructive azoospermia(OA)and non-obstructive azoospermia(NOA),and to find the difference in the two lines process.Methods 5 to 6 mm^3 testicular tissue of 3 OA and idiopathic NOA patients were excised for primary culture,transfected with Sendai virus in 3rd or 4th passages,and transferred cells were cultured on feeder after they were dense,then picked the iPS clone on the 21st to the 22nd day,established iPSCs line and to identify iPSCs pluripotency and differentiation potential.Results Two iPSCs lines were successfully established,there was no significant difference in the two lines.Both AP stains were positive,Octamer-binding transcription factor 4(OCT4),SRY-related-box protein-2(SOX2),and stage-specific embryonic antigen-4(SSEA-4)and tumor rejection antigen-1-60(TRA-1-60)were all positive for immunofluorescence staining;teratoma test in mice also showed that the obtained two iPSCs differentiated to three-dimensional germ layers.Conclusions Using the Sendai virus non-genomic integration transfection approach,testis-derived iPSCs can be established from OA and idiopathic NOA patients.