本文選題：豬 + 睪丸間質(zhì)干細(xì)胞(SLCs)　； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：睪丸間質(zhì)干細(xì)胞(stem Leydig cells,SLCs)是雄性動(dòng)物睪丸中的一類成體干細(xì)胞,可定向分化為成熟睪丸間質(zhì)細(xì)胞(adult Leydig cells,ALCs),調(diào)控精子發(fā)生。豬作為重要的經(jīng)濟(jì)動(dòng)物和理想的哺乳動(dòng)物模型,可代替小鼠等模式動(dòng)物在輔助生殖領(lǐng)域發(fā)揮重要作用,然而此前并無(wú)豬SLCs的相關(guān)研究報(bào)道。因此,本研究以不同時(shí)期豬睪丸為研究對(duì)象,利用HE染色、免疫組織化學(xué)和qRT-PCR等生物技術(shù)手段,旨在鑒定并分離豬SLCs,并探究其體外培養(yǎng)體系,為后續(xù)進(jìn)一步研究其增殖分化調(diào)控的機(jī)制提供基礎(chǔ)。本研究獲得如下重要研究結(jié)果:1.豬SLCs的組織學(xué)鑒定PDGFRα+細(xì)胞主要分布在豬睪丸曲細(xì)精管管周和間質(zhì)部分,部分為紡錘形,證明SLCs在豬睪丸中是存在的。PDGFRα+細(xì)胞在7 d睪丸間質(zhì)中所占比例顯著高于2 m睪丸間質(zhì)中。同時(shí),Nestin在7 d豬睪丸的mRNA表達(dá)量高于2 m豬的表達(dá)量,而CYP17A1在2 m豬睪丸的表達(dá)量最高,表明:相比于2 m與成年豬睪丸,7 d豬是分離SLCs最適宜的采樣時(shí)間點(diǎn)。2.豬原代SLCs的分離與鑒定本研究采用膠原酶消化法分離得到7 d豬睪丸間質(zhì)中的原代細(xì)胞。發(fā)現(xiàn)該原代細(xì)胞表達(dá)SLCs和干細(xì)胞的分子標(biāo)記(Nestin、PDGFRα、Oct4和LIFR),但不表達(dá)精原干細(xì)胞的分子標(biāo)記(PLZF)和支持細(xì)胞的分子標(biāo)記(SOX9),提示本研究采用的分離方法可有效除去曲細(xì)精管內(nèi)細(xì)胞。隨后,LIFR和PDGFRα在原代細(xì)胞中的表達(dá)量顯著高于在7 d豬睪丸組織的表達(dá)量,表明本研究可分離和富集豬SLCs。1.00 mg/mL EDS處理發(fā)現(xiàn)該原代細(xì)胞含約23.3%的已分化LCs。3.豬原代SLCs的體外短期培養(yǎng)本研究將豬睪丸組織液(porcine testicular fluid,pTF)添加在培養(yǎng)基中,以期實(shí)現(xiàn)對(duì)豬SLCs的體外培養(yǎng)。結(jié)果發(fā)現(xiàn),在添加pTF的情況下,原代SLCs培養(yǎng)1周后,有細(xì)胞克隆形成,且形成克隆的細(xì)胞表達(dá)PDGFRα。而在不添加pTF的情況下,無(wú)細(xì)胞克隆形成,且這些細(xì)胞表達(dá)CY17A1,提示SLCs在普通培養(yǎng)條件下已自發(fā)分化成LCs。以上結(jié)果說(shuō)明,添加pTF有助于SLCs的干細(xì)胞特性的維持,并可在體外進(jìn)行短期培養(yǎng)。綜上,本研究成功鑒定并分離出了豬SLCs,且pTF對(duì)豬SLCs干細(xì)胞特性的維持有支持作用,并以此為基礎(chǔ),建立了豬SLCs的體外短期培養(yǎng)體系。這些研究結(jié)果為進(jìn)一步研究豬SLCs的增殖分化調(diào)控機(jī)制奠定了基礎(chǔ),為人SLCs的分離與培養(yǎng)研究積累了科學(xué)資料。
[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 偽 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 偽 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 偽 -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 偽 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 偽. 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.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S828

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