猪睾丸间质干细胞的分离、鉴定及其短期培养体系的建立

发布时间：2018-07-10 20:06

  本文选题：猪 + 睾丸间质干细胞(SLCs)　； 参考：《西北农林科技大学》2017年硕士论文

【摘要】：睾丸间质干细胞(stem Leydig cells,SLCs)是雄性动物睾丸中的一类成体干细胞,可定向分化为成熟睾丸间质细胞(adult Leydig cells,ALCs),调控精子发生。猪作为重要的经济动物和理想的哺乳动物模型,可代替小鼠等模式动物在辅助生殖领域发挥重要作用,然而此前并无猪SLCs的相关研究报道。因此,本研究以不同时期猪睾丸为研究对象,利用HE染色、免疫组织化学和qRT-PCR等生物技术手段,旨在鉴定并分离猪SLCs,并探究其体外培养体系,为后续进一步研究其增殖分化调控的机制提供基础。本研究获得如下重要研究结果:1.猪SLCs的组织学鉴定PDGFRα+细胞主要分布在猪睾丸曲细精管管周和间质部分,部分为纺锤形,证明SLCs在猪睾丸中是存在的。PDGFRα+细胞在7 d睾丸间质中所占比例显著高于2 m睾丸间质中。同时,Nestin在7 d猪睾丸的mRNA表达量高于2 m猪的表达量,而CYP17A1在2 m猪睾丸的表达量最高,表明:相比于2 m与成年猪睾丸,7 d猪是分离SLCs最适宜的采样时间点。2.猪原代SLCs的分离与鉴定本研究采用胶原酶消化法分离得到7 d猪睾丸间质中的原代细胞。发现该原代细胞表达SLCs和干细胞的分子标记(Nestin、PDGFRα、Oct4和LIFR),但不表达精原干细胞的分子标记(PLZF)和支持细胞的分子标记(SOX9),提示本研究采用的分离方法可有效除去曲细精管内细胞。随后,LIFR和PDGFRα在原代细胞中的表达量显著高于在7 d猪睾丸组织的表达量,表明本研究可分离和富集猪SLCs。1.00 mg/mL EDS处理发现该原代细胞含约23.3%的已分化LCs。3.猪原代SLCs的体外短期培养本研究将猪睾丸组织液(porcine testicular fluid,pTF)添加在培养基中,以期实现对猪SLCs的体外培养。结果发现,在添加pTF的情况下,原代SLCs培养1周后,有细胞克隆形成,且形成克隆的细胞表达PDGFRα。而在不添加pTF的情况下,无细胞克隆形成,且这些细胞表达CY17A1,提示SLCs在普通培养条件下已自发分化成LCs。以上结果说明,添加pTF有助于SLCs的干细胞特性的维持,并可在体外进行短期培养。综上,本研究成功鉴定并分离出了猪SLCs,且pTF对猪SLCs干细胞特性的维持有支持作用,并以此为基础,建立了猪SLCs的体外短期培养体系。这些研究结果为进一步研究猪SLCs的增殖分化调控机制奠定了基础,为人SLCs的分离与培养研究积累了科学资料。
[Abstract]:Stem Leydig cells (SLCs) are one of the adult stem cells in the testis of male animals. They can differentiate into adult Leydig cells and regulate spermatogenesis. As an important economic animal and an ideal mammalian model, pigs can play an important role in the assisted reproduction field instead of mice and other model animals. However, there have been no previous studies on porcine SLCs. Therefore, the purpose of this study was to identify and isolate porcine SLCsby HE staining, immunohistochemistry and qRT-PCR, and to explore its culture system in vitro. It provides a basis for further study on the mechanism of proliferation and differentiation regulation. The results of this study are as follows: 1. 1. The histology of porcine SLCs revealed that PDGFR 伪 cells were mainly distributed around the tubules and the interstitial parts of the testis, and some of them were fusiform. It was proved that the percentage of PDGFR 伪 cells in the testis of pigs was significantly higher than that in the stroma of 2 m testis at 7 days. At the same time, the mRNA expression of nestin in the testis was higher than that in the testis of 2 m pigs, while CYP17A1 was the highest in the testis of 2 m pigs. Isolation and Identification of Porcine Primary SLCs in this study primary cells were isolated from the interstitial cells of pig testis for 7 days by collagenase digestion. It was found that the primary cells expressed SLCs and molecular markers of stem cells (Nestinine PDGFR 伪 -Oct4 and LIFR), but did not express the molecular markers of spermatogonial stem cells (PLZF) and Sertoli cells (SOX9). Subsequently, the expression of LIFR and PDGFR 伪 in primary cells was significantly higher than that in testicular tissues on day 7, indicating that this study could isolate and enrich porcine SLCs.1.00 mg / mL EDS. It was found that the primary cells contained 23.3% of differentiated LCs.3. In this study, porcine testicular tissue fluid (porcine testicular fluidd) was added to the culture medium in order to achieve the in vitro culture of porcine SLCs. The results showed that after the primary SLCs were cultured for 1 week, the clones were formed and the cloned cells expressed PDGFR 伪. However, without the addition of PTFs, no cell clones were formed, and these cells expressed CY17A1, suggesting that SLCs had spontaneously differentiated into LCsunder normal culture conditions. These results suggest that the addition of PTFs is helpful to maintain the stem cell characteristics of SLCs and can be cultured in vitro for a short time. In conclusion, porcine SLCs were successfully identified and isolated in this study, and PTFs could support the maintenance of the characteristics of porcine SLCs stem cells. Based on this, a short-term culture system of porcine SLCs was established in vitro. These results laid a foundation for further study on the regulation mechanism of porcine SLCs proliferation and differentiation, and accumulated scientific data for the isolation and culture of human SLCs.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S828

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