阿尔巴斯绒山羊毛囊干细胞的分离鉴定及转录因子Sox9对其作用机理的研究

发布时间：2018-06-20 06:08

  本文选题：阿尔巴斯绒山羊 + 毛囊干细胞　； 参考：《内蒙古大学》2016年博士论文

【摘要】：毛囊是一个动态的微小器官。它不仅具有预防皮肤组织受损、感觉和免疫防护等重要功能,且获取方便。毛囊组织中含有与自我更新、分化、调控毛发生长相关的多种干细胞,维持着皮肤的体内平衡。而毛囊的发生和再生都离不开毛囊干细胞。毛囊干细胞的生物学研究日渐成为毛囊领域、皮肤学领域、再生医学领域的前沿。转录因子Sox9,在胚胎发育、三胚层分化、胚胎干细胞和多种成体干细胞的维持、先天性疾病等重要的生物学过程中具有重要的功能。近几年发现,在早期胚胎生发板形成和毛囊发生中,Sox9有着至关重要的作用。目前,国际上对小鼠和人的毛囊干细胞研究较多,对家畜动物毛囊干细胞的研究甚少。在本研究中,我们首次在体外分离鉴定了阿尔巴斯绒山羊毛囊干细胞(gHFSCs),并对其进行了多能性分析；初步验证了Sox9在gHFSCs中的功能；并且进行了Sox9染色质共沉淀(ChIP),以期研究其在gHFSCs中的作用机制,为今后绒山羊毛囊生长及其调控机理的研究提供实验材料和依据。一、阿尔巴斯绒山羊毛囊干细胞(gHFSCs)的体外分离培养及鉴定我们利用组织贴壁培养法在体外分离gHFSCs,对其进行纯化。从细胞形态、特异标记、增殖能力、体外分化能力等方面对其进行了鉴定。结果显示：实验所得到的细胞较小,呈典型的鹅卵石样,折光度高且贴附能力强,符合毛囊干细胞的形态特征,且在体外培养至20代未出现明显形态变化。免疫组化染色显示,gHFSCs阳性表达毛囊干细胞标记分子Krt15、Krt19、CD34、Itgβ1和Krt14。CD34在gHFSCs中FACS(fluorescence-activated cell sorting)阳性率为99.8%。从细胞生长曲线可以看出,分离得到的gHFSCs具有较强的增殖能力。在转录水平上Krtl4和CD34高表达(p0.01),分别为绒山羊角质细胞的39.68倍和24.37倍,Krtl5的表达量为5.62倍,Itg β1表达量低(p0.01),为1.81倍。同样Western blot检测到了以上特异标记蛋白在gHFSCs中表达。体外成骨诱导后,Von Kossa法染色呈阳性；成软骨诱导阿尔新蓝染色为阳性；成肌诱导后Hoechst33342染色观察到细胞融合现象,免疫组化染色检测到成绩诱导细胞MyoG表达呈阳性。二、阿尔巴斯绒山羊毛囊干细胞(gHFSCs)的多能性分析我们对gHFSCs中与干细胞多能性建立和维持及自我更新能力相关的几个因子Oct4、Nanog、Sox2、AKP和TERT等,通过细胞免疫组化、FACS、Q-PCR和Western blot的方法进行了的检测。结果表明：细胞免疫组化染色显示Oct4、Nanog、Sox2、AKP和TERT等五个因子均在gHFSCs中阳性表达；Oct4、Nanog和Sox2在gHFSCs中FACS阳性率在99.9%以上；与阿尔巴斯绒山羊脂肪间充质干细胞(gADSCs)对比,在转录水平上,gHFSCs中Oct4高表达,为对照细胞的41.36倍；Nanog、AKP、TERT中等表达,分别为对照组的5.61倍、2.74倍和2.10倍(p0.01)；在蛋白水平,Oct4、Nanog、AKP和TERT的相对表达量,分别为对照组的5.94倍、10.78倍、1.33倍和1.39倍。Sox2在gHFSCs中mRNA和蛋白水平均表达,而在gADSCs中均不表达。三、Sox9在阿尔巴斯绒山羊毛囊干细胞(gHFSCs)中的功能验证通过毛囊(gHF)冰冻切片和细胞免疫组化、Q-PCR. Western blot检测Sox9在gHF和gHFSCs中的表达；利用shRNA载体,干扰gHFSCs中Sox9的表达,对与细胞增殖分化相关基因进行转录水平和蛋白水平表达变化的检测；对gHFSCs特异标记和干细胞多能因子在转录水平和蛋白水平的表达变化进行检测；钙诱导培养后,对gHFSCs中Sox9和Loricrin(Lor)的表达变化进行检测。结果显示：Sox9在gHF整个外根鞘部位都有表达,在隆突部表达比其他部位强,在毛母质中也有少量表达,既Sox9主要在隆突部集中表达；在体外培养的gHFSCs呈Sox9阳性,在细胞质和细胞核内都有不同程度的表达；Q-PCR和Western blot进一步验证了Sox9在gHFSCs中的转录水平和蛋白水平的表达。Sox9-shRNA的干扰效率达到了53.58%。干扰Sox9表达之后：细胞增殖缓慢,无法进入对数生长期,随着生长周期,凋亡细胞增多；流式细胞术检测细胞周期发现,干扰Sox9表达后的gHFSCs无法完成从S期到G2/M期的过渡；Q-PCR检测与对照组相比发现,p21和细胞核抗原PCNA的表达量显著减少(p0.01),p53表达无显著差异(p0.05),皮肤细胞终末分化标记Lor表达量显著增加(p0.01); gHFSCs不仅失去了毛囊干细胞形态特征,其特异标记Krt15、 Krt19、Krt14、CD34和Itgβ1在mRNA水平和蛋白水平的表达量均大幅降低(p0.01)；与干细胞多能性和自我更新能力相关的因子Oct4、Nanog、Sox2、 AKP和TERT的表达量在转录水平和蛋白水平表达量均急剧减少(p0.01)；低钙培养的细胞形态无明显变化,而高钙诱导培养的细胞出现形态变化,呈长梭形。Western blot蛋白条带可以看出,高钙诱导分化后的细胞中Sox9表达量减少,而低钙和正常培养的细胞中无明显变化；高钙诱导分化后的细胞中Lor表达量增多,在低钙和正常培养的细胞中表达量很少。四、ChIP结合ChIP-seq寻找Sox9结合的基因ChIP是目前研究体内蛋白与DNA相互作用的唯一手段。我们在前三章内容的基础上,对gHFSCs进行转录因子Sox9-ChIP得到蛋白-DNA复合物,利用ChIP-seq进行测序,获得与Sox9互作的基因信息。结果显示：对超声破碎效果良好的蛋白-DNA复合物进行纯化测序,ChIP-seq测序后得到了共10736个MACS-peaks和峰的位置信息。其中位于外显子区(exonic)的有2008个,内含子(intronic)区有2827个,基因间(intergenic)区有5433个,下游(downstream)区域有48个,上游(upstream)区域有61个,3、端非翻译区(UTR3)有292个,5、端非翻译区(UTR5)有28个,剪接(splicing)区有39个；利用de novo finding找到了36个未知的motif信息,通过转录因子motif数据和GO数据库比对得出96个已知的motif。Top motifs中排列第一的基序与Sox9 in vitro motif匹配。已知motif比对中包括明确的胚胎发育相关转录因子Nanog、 Oct4、Sox2、Tcf;对测序得到的所有基因进行KEGG-pathway分析,跟据GO和KEGG中已有的信息共得到278条通路。其中p值小于0.05p0.05)的通路有FoxO信号通路、MAPK信号通路、cAMP信号通路、干细胞多能性调控通路和细胞骨架肌动蛋白调控通路等24条通路；p值小于0.01(p0.01)的通路有黏着斑(Focal adhesion)、粘着连接(Adherens junction)、Rap1信号通路、Wnt信号通路、Hippo信号通路等9条通路。
[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.
【学位授予单位】：内蒙古大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S827

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2 ;Stem cell pluripotency and transcription factor Oct4[J];Cell Research;2002年Z2期

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