本文選題：無(wú)精癥 + 外周血單個(gè)核細(xì)胞　； 參考：《廣州醫(yī)科大學(xué)》2017年碩士論文

【摘要】：第一部分:建立無(wú)精癥患者誘導(dǎo)多能干細(xì)胞系目的:建立無(wú)精癥患者誘導(dǎo)多能干細(xì)胞系,為進(jìn)一步研究該疾病的發(fā)病機(jī)制和治療提供合適的細(xì)胞模型。方法:利用仙臺(tái)病毒非基因整合的方法,我們建立了無(wú)精癥患者外周血單個(gè)核細(xì)胞來(lái)源的誘導(dǎo)多能干細(xì)胞系。采用無(wú)血清完全培養(yǎng)基(Te SR?2)和胚胎干細(xì)胞培養(yǎng)基(Stem Adhere?Defined Matrix)限定培養(yǎng)體系進(jìn)行培養(yǎng),應(yīng)用免疫熒光染色、染色體核型分析、擬胚體和畸胎瘤實(shí)驗(yàn)對(duì)iPSCs系的干性、多能性、體內(nèi)外分化能力進(jìn)行驗(yàn)證。結(jié)果:我們建立了具有人胚胎干細(xì)胞特征的無(wú)精癥患者來(lái)源的iPSCs系。免疫熒光染色顯示,這些細(xì)胞均表達(dá)SOX2和OCT4以及TRA-1-60和SSEA-4干細(xì)胞多能性基因;染色體核型分析顯示正常的核型;擬胚體和畸胎瘤實(shí)驗(yàn)表明其在體內(nèi)、體外具有向三個(gè)胚層分化的能力。結(jié)論:利用非基因整合方法成功構(gòu)建了無(wú)精癥患者外周血來(lái)源的誘導(dǎo)多能干細(xì)胞系,為無(wú)精癥發(fā)病機(jī)制的研究及治療提供了細(xì)胞模型。第二部分:無(wú)精癥患者iPSCs在體外分化成原始生殖樣細(xì)胞的研究目的:將建立的無(wú)精癥患者誘導(dǎo)多能干細(xì)胞(Patients with azoospermia induce pluripotent stem cells,pa-iPSCs)在體外定向誘導(dǎo)分化為原始生殖樣細(xì)胞(primordial germ cell-like cells,PGCLCs)。方法:我們利用“4i”(PD0325901+CHIR99021+SB203580+SP600125)的培養(yǎng)體系將無(wú)精癥患者來(lái)源iPSCs轉(zhuǎn)化成naive iPSCs,然后將naive iPSCs在體外在含有b FGF、TGF-β1和1%KSR的預(yù)誘導(dǎo)培養(yǎng)基中培養(yǎng)2天,然后以2000-4000個(gè)細(xì)胞/孔傳到低吸附的96孔板中,細(xì)胞培養(yǎng)在含BMP4、LIF、SCF、EGF、和ROCK inhibitor PGCLCs的分化培養(yǎng)基中。并用免疫熒光染色的方法對(duì)PGCLCs進(jìn)行了特異標(biāo)記鑒定。結(jié)果:我們成功將iPSCs轉(zhuǎn)變成了na?ve態(tài)i PS細(xì)胞,然后在體外成功誘導(dǎo)出PGCLCs,免疫熒光染色顯示PGCLCs表達(dá)PGC的特異標(biāo)記OCT4,SOX17。結(jié)論:通過(guò)“4i”體系將pa-iPSCs轉(zhuǎn)變成naive狀態(tài)的干細(xì)胞,并將其在體外誘導(dǎo)分化成了PGCLCs,PGCLCs表達(dá)PGC的特異標(biāo)記SOX17和OCT4,為體外人造精子治療無(wú)精癥患者提供了理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣州醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R698.2

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