發(fā)布時(shí)間：2018-06-20 06:08

  本文選題：阿爾巴斯絨山羊 + 毛囊干細(xì)胞��； 參考：《內(nèi)蒙古大學(xué)》2016年博士論文

【摘要】：毛囊是一個(gè)動(dòng)態(tài)的微小器官。它不僅具有預(yù)防皮膚組織受損、感覺和免疫防護(hù)等重要功能,且獲取方便。毛囊組織中含有與自我更新、分化、調(diào)控毛發(fā)生長(zhǎng)相關(guān)的多種干細(xì)胞,維持著皮膚的體內(nèi)平衡。而毛囊的發(fā)生和再生都離不開毛囊干細(xì)胞。毛囊干細(xì)胞的生物學(xué)研究日漸成為毛囊領(lǐng)域、皮膚學(xué)領(lǐng)域、再生醫(yī)學(xué)領(lǐng)域的前沿。轉(zhuǎn)錄因子Sox9,在胚胎發(fā)育、三胚層分化、胚胎干細(xì)胞和多種成體干細(xì)胞的維持、先天性疾病等重要的生物學(xué)過(guò)程中具有重要的功能。近幾年發(fā)現(xiàn),在早期胚胎生發(fā)板形成和毛囊發(fā)生中,Sox9有著至關(guān)重要的作用。目前,國(guó)際上對(duì)小鼠和人的毛囊干細(xì)胞研究較多,對(duì)家畜動(dòng)物毛囊干細(xì)胞的研究甚少。在本研究中,我們首次在體外分離鑒定了阿爾巴斯絨山羊毛囊干細(xì)胞(gHFSCs),并對(duì)其進(jìn)行了多能性分析；初步驗(yàn)證了Sox9在gHFSCs中的功能；并且進(jìn)行了Sox9染色質(zhì)共沉淀(ChIP),以期研究其在gHFSCs中的作用機(jī)制,為今后絨山羊毛囊生長(zhǎng)及其調(diào)控機(jī)理的研究提供實(shí)驗(yàn)材料和依據(jù)。一、阿爾巴斯絨山羊毛囊干細(xì)胞(gHFSCs)的體外分離培養(yǎng)及鑒定我們利用組織貼壁培養(yǎng)法在體外分離gHFSCs,對(duì)其進(jìn)行純化。從細(xì)胞形態(tài)、特異標(biāo)記、增殖能力、體外分化能力等方面對(duì)其進(jìn)行了鑒定。結(jié)果顯示：實(shí)驗(yàn)所得到的細(xì)胞較小,呈典型的鵝卵石樣,折光度高且貼附能力強(qiáng),符合毛囊干細(xì)胞的形態(tài)特征,且在體外培養(yǎng)至20代未出現(xiàn)明顯形態(tài)變化。免疫組化染色顯示,gHFSCs陽(yáng)性表達(dá)毛囊干細(xì)胞標(biāo)記分子Krt15、Krt19、CD34、Itgβ1和Krt14。CD34在gHFSCs中FACS(fluorescence-activated cell sorting)陽(yáng)性率為99.8%。從細(xì)胞生長(zhǎng)曲線可以看出,分離得到的gHFSCs具有較強(qiáng)的增殖能力。在轉(zhuǎn)錄水平上Krtl4和CD34高表達(dá)(p0.01),分別為絨山羊角質(zhì)細(xì)胞的39.68倍和24.37倍,Krtl5的表達(dá)量為5.62倍,Itg β1表達(dá)量低(p0.01),為1.81倍。同樣Western blot檢測(cè)到了以上特異標(biāo)記蛋白在gHFSCs中表達(dá)。體外成骨誘導(dǎo)后,Von Kossa法染色呈陽(yáng)性；成軟骨誘導(dǎo)阿爾新藍(lán)染色為陽(yáng)性；成肌誘導(dǎo)后Hoechst33342染色觀察到細(xì)胞融合現(xiàn)象,免疫組化染色檢測(cè)到成績(jī)誘導(dǎo)細(xì)胞MyoG表達(dá)呈陽(yáng)性。二、阿爾巴斯絨山羊毛囊干細(xì)胞(gHFSCs)的多能性分析我們對(duì)gHFSCs中與干細(xì)胞多能性建立和維持及自我更新能力相關(guān)的幾個(gè)因子Oct4、Nanog、Sox2、AKP和TERT等,通過(guò)細(xì)胞免疫組化、FACS、Q-PCR和Western blot的方法進(jìn)行了的檢測(cè)。結(jié)果表明：細(xì)胞免疫組化染色顯示Oct4、Nanog、Sox2、AKP和TERT等五個(gè)因子均在gHFSCs中陽(yáng)性表達(dá)；Oct4、Nanog和Sox2在gHFSCs中FACS陽(yáng)性率在99.9%以上；與阿爾巴斯絨山羊脂肪間充質(zhì)干細(xì)胞(gADSCs)對(duì)比,在轉(zhuǎn)錄水平上,gHFSCs中Oct4高表達(dá),為對(duì)照細(xì)胞的41.36倍；Nanog、AKP、TERT中等表達(dá),分別為對(duì)照組的5.61倍、2.74倍和2.10倍(p0.01)；在蛋白水平,Oct4、Nanog、AKP和TERT的相對(duì)表達(dá)量,分別為對(duì)照組的5.94倍、10.78倍、1.33倍和1.39倍。Sox2在gHFSCs中mRNA和蛋白水平均表達(dá),而在gADSCs中均不表達(dá)。三、Sox9在阿爾巴斯絨山羊毛囊干細(xì)胞(gHFSCs)中的功能驗(yàn)證通過(guò)毛囊(gHF)冰凍切片和細(xì)胞免疫組化、Q-PCR. Western blot檢測(cè)Sox9在gHF和gHFSCs中的表達(dá)；利用shRNA載體,干擾gHFSCs中Sox9的表達(dá),對(duì)與細(xì)胞增殖分化相關(guān)基因進(jìn)行轉(zhuǎn)錄水平和蛋白水平表達(dá)變化的檢測(cè)；對(duì)gHFSCs特異標(biāo)記和干細(xì)胞多能因子在轉(zhuǎn)錄水平和蛋白水平的表達(dá)變化進(jìn)行檢測(cè)；鈣誘導(dǎo)培養(yǎng)后,對(duì)gHFSCs中Sox9和Loricrin(Lor)的表達(dá)變化進(jìn)行檢測(cè)。結(jié)果顯示：Sox9在gHF整個(gè)外根鞘部位都有表達(dá),在隆突部表達(dá)比其他部位強(qiáng),在毛母質(zhì)中也有少量表達(dá),既Sox9主要在隆突部集中表達(dá)；在體外培養(yǎng)的gHFSCs呈Sox9陽(yáng)性,在細(xì)胞質(zhì)和細(xì)胞核內(nèi)都有不同程度的表達(dá)；Q-PCR和Western blot進(jìn)一步驗(yàn)證了Sox9在gHFSCs中的轉(zhuǎn)錄水平和蛋白水平的表達(dá)。Sox9-shRNA的干擾效率達(dá)到了53.58%。干擾Sox9表達(dá)之后：細(xì)胞增殖緩慢,無(wú)法進(jìn)入對(duì)數(shù)生長(zhǎng)期,隨著生長(zhǎng)周期,凋亡細(xì)胞增多；流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期發(fā)現(xiàn),干擾Sox9表達(dá)后的gHFSCs無(wú)法完成從S期到G2/M期的過(guò)渡；Q-PCR檢測(cè)與對(duì)照組相比發(fā)現(xiàn),p21和細(xì)胞核抗原PCNA的表達(dá)量顯著減少(p0.01),p53表達(dá)無(wú)顯著差異(p0.05),皮膚細(xì)胞終末分化標(biāo)記Lor表達(dá)量顯著增加(p0.01); gHFSCs不僅失去了毛囊干細(xì)胞形態(tài)特征,其特異標(biāo)記Krt15、 Krt19、Krt14、CD34和Itgβ1在mRNA水平和蛋白水平的表達(dá)量均大幅降低(p0.01)；與干細(xì)胞多能性和自我更新能力相關(guān)的因子Oct4、Nanog、Sox2、 AKP和TERT的表達(dá)量在轉(zhuǎn)錄水平和蛋白水平表達(dá)量均急劇減少(p0.01)；低鈣培養(yǎng)的細(xì)胞形態(tài)無(wú)明顯變化,而高鈣誘導(dǎo)培養(yǎng)的細(xì)胞出現(xiàn)形態(tài)變化,呈長(zhǎng)梭形。Western blot蛋白條帶可以看出,高鈣誘導(dǎo)分化后的細(xì)胞中Sox9表達(dá)量減少,而低鈣和正常培養(yǎng)的細(xì)胞中無(wú)明顯變化；高鈣誘導(dǎo)分化后的細(xì)胞中Lor表達(dá)量增多,在低鈣和正常培養(yǎng)的細(xì)胞中表達(dá)量很少。四、ChIP結(jié)合ChIP-seq尋找Sox9結(jié)合的基因ChIP是目前研究體內(nèi)蛋白與DNA相互作用的唯一手段。我們?cè)谇叭聝?nèi)容的基礎(chǔ)上,對(duì)gHFSCs進(jìn)行轉(zhuǎn)錄因子Sox9-ChIP得到蛋白-DNA復(fù)合物,利用ChIP-seq進(jìn)行測(cè)序,獲得與Sox9互作的基因信息。結(jié)果顯示：對(duì)超聲破碎效果良好的蛋白-DNA復(fù)合物進(jìn)行純化測(cè)序,ChIP-seq測(cè)序后得到了共10736個(gè)MACS-peaks和峰的位置信息。其中位于外顯子區(qū)(exonic)的有2008個(gè),內(nèi)含子(intronic)區(qū)有2827個(gè),基因間(intergenic)區(qū)有5433個(gè),下游(downstream)區(qū)域有48個(gè),上游(upstream)區(qū)域有61個(gè),3、端非翻譯區(qū)(UTR3)有292個(gè),5、端非翻譯區(qū)(UTR5)有28個(gè),剪接(splicing)區(qū)有39個(gè)；利用de novo finding找到了36個(gè)未知的motif信息,通過(guò)轉(zhuǎn)錄因子motif數(shù)據(jù)和GO數(shù)據(jù)庫(kù)比對(duì)得出96個(gè)已知的motif。Top motifs中排列第一的基序與Sox9 in vitro motif匹配。已知motif比對(duì)中包括明確的胚胎發(fā)育相關(guān)轉(zhuǎn)錄因子Nanog、 Oct4、Sox2、Tcf;對(duì)測(cè)序得到的所有基因進(jìn)行KEGG-pathway分析,跟據(jù)GO和KEGG中已有的信息共得到278條通路。其中p值小于0.05p0.05)的通路有FoxO信號(hào)通路、MAPK信號(hào)通路、cAMP信號(hào)通路、干細(xì)胞多能性調(diào)控通路和細(xì)胞骨架肌動(dòng)蛋白調(diào)控通路等24條通路；p值小于0.01(p0.01)的通路有黏著斑(Focal adhesion)、粘著連接(Adherens junction)、Rap1信號(hào)通路、Wnt信號(hào)通路、Hippo信號(hào)通路等9條通路。
[Abstract]:Hair follicle is a dynamic and tiny organ. It not only has the important functions of preventing the damage of skin tissue, feeling and immune protection, but also is convenient to obtain. The hair follicle contains a variety of stem cells related to self renewal, differentiation, regulation of hair growth and the balance of the skin. The occurrence and regeneration of hair follicles can not be separated from the hair follicle dry fine. The biological research of hair follicle stem cells has increasingly become the field of hair follicle, dermatology, and the frontier of regenerative medicine. The transcription factor Sox9 has important functions in the important biological processes such as embryonic development, three embryo layer differentiation, embryonic stem cells and the maintenance of multiple adult stem cells, congenital diseases and other important biological processes. In recent years, the early embryo was found in the early embryo. Sox9 plays an important role in the formation of viviparous hair plate and hair follicle. At present, there are many studies on the hair follicle stem cells of mice and human beings and few studies on the hair follicle stem cells of domestic animals. In this study, we first isolated and identified the gHFSCs in vitro. The function of Sox9 in gHFSCs was preliminarily verified and the Sox9 chromatin coprecipitation (ChIP) was carried out in order to study the mechanism of its action in gHFSCs and to provide experimental materials and basis for the study of the growth and regulation mechanism of wool sac in the future. 1. In vitro isolation and culture of the wool sac stem cells (gHFSCs) in the Mashan mountains and the isolation and culture of the hair follicle stem cells (gHFSCs). GHFSCs was isolated and purified by tissue adherence culture in vitro. It was identified from cell morphology, specific labeling, proliferation ability and ability to differentiate in vitro. The results showed that the cells obtained in the experiment were smaller, with a typical cobblestone like, high refractive index and strong attachment ability, conforming to the hair follicle stem cells. Morphological characteristics, and no obvious morphological changes were found in the 20 generation in vitro. Immunohistochemical staining showed that gHFSCs positive expression of hair follicle stem cell marker molecules Krt15, Krt19, CD34, Itg beta 1 and Krt14.CD34 in gHFSCs FACS (fluorescence-activated cell sorting) positive rate was seen from the cell growth curve. The high expression of Krtl4 and CD34 (P0.01) at the transcriptional level (P0.01) was 39.68 times and 24.37 times of the keratinocytes of cashmere goats, respectively, the expression of Krtl5 was 5.62 times, the expression of Itg beta 1 was low (P0.01), 1.81 times. The same Western blot detected the expression of the specific marker protein in gHFSCs. After induction of osteogenesis in vitro, Von Kossa The method of staining was positive; the chondrogenic induced alanine blue staining was positive; the cell fusion was observed by Hoechst33342 staining after induction of myoblast. Immunohistochemical staining was used to detect the positive expression of MyoG. Two, the pluripotent analysis of Alba cashmere wool sac stem cells (gHFSCs) in the gHFSCs and the pluripotent cells in the stem cells Several factors, such as Oct4, Nanog, Sox2, AKP and TERT, were established to establish and maintain the capacity of self renewal, such as cell immunization, FACS, Q-PCR and Western blot. The results showed that the cytochemical staining showed that five factors such as Oct4, Nanog, Sox2, AKP and Western were all positive. The positive rate of FACS in gHFSCs was more than 99.9%; compared with the adipose mesenchymal stem cells (gADSCs) of the alpha cashmere goat (gADSCs), at the transcriptional level, the expression of Oct4 was 41.36 times as high as that of the control cells; Nanog, AKP, and TERT were moderately expressed, respectively, 5.61 times, 2.74 times and 2.10 times (P0.01), respectively, at the protein level, Oct4, Nanog, AKP, and societies. The relative expression was 5.94 times, 10.78 times, 1.33 times and 1.39 times.Sox2, respectively, in gHFSCs, mRNA and protein levels were all expressed, but not in gADSCs. Three, Sox9 in the wool sac stem cells (gHFSCs) in the Mashan (gHFSCs) was validated through the follicle (gHF) frozen section and cell immunohistochemistry, Q-PCR. Western blot detection Sox9 Expression in gHF and gHFSCs; using shRNA vector to interfere with the expression of Sox9 in gHFSCs, detection of transcriptional level and protein level expression with cell proliferation and differentiation related genes; detection of gHFSCs specific markers and stem cell pluripotent factors at transcriptional level and protein level; calcium induced culture, gH The expression changes of Sox9 and Loricrin (Lor) in FSCs were detected. The results showed that Sox9 was expressed in the whole outer root sheath of gHF, expressed in the protuberance and a small amount of expression in the hair mother, not only the Sox9 was mainly expressed in the protuberance, but the gHFSCs in vitro was positive in Sox9, but not in the cytoplasm and nucleus. Q-PCR and Western blot further verified that Sox9's transcription level and protein level in gHFSCs expressed.Sox9-shRNA interference efficiency after 53.58%. interference Sox9 expression: the cell proliferation was slow, the logarithmic growth period was unable to enter, and the apoptotic cells increased with the growth cycle; flow cytometry was used to detect the cell cycle. It was found that the gHFSCs after interference of Sox9 could not complete the transition from S to G2/M. Compared with the control group, Q-PCR detected a significant decrease in the expression of p21 and nuclear antigen PCNA (P0.01), and there was no significant difference in p53 expression (P0.05), and the Lor expression of the terminal differentiation marker of the skin cells was significantly increased (P0.01), and not only lost Mao Nanggan. Cell morphological characteristics, its specific markers Krt15, Krt19, Krt14, CD34 and Itg beta 1 were significantly reduced at mRNA levels and protein levels (P0.01); the expression of Nanog, Sox2, AKP, and TERT were decreased sharply in the transcription and protein levels of the stem cell pluripotent and self renewal capacity, and the expression of Nanog, Sox2, AKP and TERT, and low calcium levels; There was no obvious change in the cell morphology of the cultured cells, while the cells in the high calcium induced cells showed morphological changes. The length of the long shuttle shaped.Western blot protein strip showed that the expression of Sox9 decreased in the cells with high calcium induced differentiation, but there was no obvious change in the low calcium and normal cultured cells, and the increased expression of Lor in the cells with high calcium induced differentiation was increased. Low calcium and normal cultured cells have little expression. Four, ChIP combined with ChIP-seq to find Sox9 binding gene ChIP is the only means to study the interaction of protein and DNA in the body. On the basis of the first three chapters, we use the transcription factor Sox9-ChIP to obtain the protein -DNA complex, and the gHFSCs is sequenced by ChIP-seq, and Sox is obtained and Sox. 9 mutual gene information. The results showed that the protein -DNA complex with good ultrasonic fragmentation was purified and sequenced. After ChIP-seq sequencing, a total of 10736 MACS-peaks and peak positions were obtained. Among them, there were 2008 exons (exonic), 2827 in Chi Ko (intronic), 5433 in intergenic region, and downstream (Dow). Nstream) there are 48 regions in the region, 61 in the upstream (upstream) area, 3 in the end non translation area (UTR3), 5, 28 in the end non translation area (UTR5) and 39 in the splice (splicing) area; and 36 unknown motif information is found by using de novo finding, and 96 known motif.Top schemes are arranged through the transcription factor motif data and GO database alignment. The first sequence is matched with the Sox9 in vitro motif. The known motif alignment includes a clear fetal development related transcription factor Nanog, Oct4, Sox2, Tcf; KEGG-pathway analysis of all the genes that are sequenced, and a total of 278 pathways according to the information existing in GO and KEGG. Signal pathway, cAMP signaling pathway, stem cell pluripotent regulation pathway and cytoskeleton actin regulation pathway, and the p value less than 0.01 (P0.01) pathways are adherent plaque (Focal adhesion), adhesion junction (Adherens junction), Rap1 signaling pathway, Wnt signaling pathway, Hippo signaling pathway, and other 9 pathways.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S827

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相關(guān)期刊論文 前2條

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本文編號(hào)：2043255