无精症患者诱导多能干细胞系的建立及其在体外分化成原始生殖样细胞的研究

发布时间：2018-06-04 13:55

本文选题：无精症 + 外周血单个核细胞　；参考：《广州医科大学》2017年硕士论文

【摘要】：第一部分:建立无精症患者诱导多能干细胞系目的:建立无精症患者诱导多能干细胞系,为进一步研究该疾病的发病机制和治疗提供合适的细胞模型。方法:利用仙台病毒非基因整合的方法,我们建立了无精症患者外周血单个核细胞来源的诱导多能干细胞系。采用无血清完全培养基(Te SR?2)和胚胎干细胞培养基(Stem Adhere?Defined Matrix)限定培养体系进行培养,应用免疫荧光染色、染色体核型分析、拟胚体和畸胎瘤实验对iPSCs系的干性、多能性、体内外分化能力进行验证。结果:我们建立了具有人胚胎干细胞特征的无精症患者来源的iPSCs系。免疫荧光染色显示,这些细胞均表达SOX2和OCT4以及TRA-1-60和SSEA-4干细胞多能性基因;染色体核型分析显示正常的核型;拟胚体和畸胎瘤实验表明其在体内、体外具有向三个胚层分化的能力。结论:利用非基因整合方法成功构建了无精症患者外周血来源的诱导多能干细胞系,为无精症发病机制的研究及治疗提供了细胞模型。第二部分:无精症患者iPSCs在体外分化成原始生殖样细胞的研究目的:将建立的无精症患者诱导多能干细胞(Patients with azoospermia induce pluripotent stem cells,pa-iPSCs)在体外定向诱导分化为原始生殖样细胞(primordial germ cell-like cells,PGCLCs)。方法:我们利用“4i”(PD0325901+CHIR99021+SB203580+SP600125)的培养体系将无精症患者来源iPSCs转化成naive iPSCs,然后将naive iPSCs在体外在含有b FGF、TGF-β1和1%KSR的预诱导培养基中培养2天,然后以2000-4000个细胞/孔传到低吸附的96孔板中,细胞培养在含BMP4、LIF、SCF、EGF、和ROCK inhibitor PGCLCs的分化培养基中。并用免疫荧光染色的方法对PGCLCs进行了特异标记鉴定。结果:我们成功将iPSCs转变成了na?ve态i PS细胞,然后在体外成功诱导出PGCLCs,免疫荧光染色显示PGCLCs表达PGC的特异标记OCT4,SOX17。结论:通过“4i”体系将pa-iPSCs转变成naive状态的干细胞,并将其在体外诱导分化成了PGCLCs,PGCLCs表达PGC的特异标记SOX17和OCT4,为体外人造精子治疗无精症患者提供了理论基础。
[Abstract]:The first part: to establish a cell line induced by azoospermia. Objective: to establish a cell line induced by azoospermia and to provide a suitable cell model for further study of the pathogenesis and treatment of azoospermia. Methods: by using Sendai virus non-gene integration method, we established an induced pluripotent cell line derived from peripheral blood mononuclear cells (PBMC) in patients with azoospermia. Serum-free complete culture medium (te SR2) and embryonic stem cell medium (Stem Adhere?Defined Matrix) were used to culture. The dry and pluripotent properties of iPSCs lines were studied by immunofluorescence staining, chromosome karyotype analysis, embryoid body and teratoma test. The ability of differentiation in vivo and in vitro was verified. Results: we established iPSCs lines from azoospermia patients with human embryonic stem cells. Immunofluorescence staining showed that these cells expressed SOX2 and OCT4 and TRA-1-60 and SSEA-4 stem cell pluripotent genes; chromosome karyotype analysis showed normal karyotype; embryoid and teratoma experiments showed that they were in vivo. In vitro, there is the ability to differentiate into three embryo layers. Conclusion: the induced multipotent cell lines from peripheral blood of patients with azoospermia were successfully constructed by using non-gene integration method, which provided a cell model for the study and treatment of the pathogenesis of azoospermia. Part 2: study on the differentiation of iPSCs from azoospermia into primordial reproductive cells in vitro objective: to induce the differentiation of pluripotent with azoospermia induce pluripotent stem cells into primordial germ cell-like cells of primordial germ cell-like cells (PGCLCsN) in vitro in azoospermia patients. Methods: we transformed iPSCs from azoospermia patients into naive iPSCsusing the culture system of "4i" PD0325901 CHIR99021 SB203580 SP600125), and then cultured naive iPSCs in pre-induction medium containing bFGF- 尾 1 and 1%KSR for 2 days. Then, 2000-4000 cells / well were transferred to the low-adsorbed 96-well plate, and the cells were cultured in the differentiation medium containing BMP4LIF-SCF and ROCK inhibitor PGCLCs. PGCLCs was identified by immunofluorescence staining. Results: we successfully transformed iPSCs into na?ve iPS cells, and then induced PGCLCs in vitro. Immunofluorescence staining showed that PGCLCs expressed PGC specifically, OCT4 and SOX17. Conclusion: the stem cells transformed from pa-iPSCs to naive by "4i" system can be induced to differentiate into SOX17 and OCT4, which express PGC in vitro, which provides a theoretical basis for the treatment of azoospermia with artificial spermatozoa in vitro.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R698.2

【相似文献】