【作者】 武艳群；

【导师】 张才乔；

【作者基本信息】 浙江大学 ， 动物遗传与繁殖， 2009， 博士

【摘要】 与哺乳动物相比,鸡胚具有易于获取和操作、结合病毒转染和电转移可进行转基因操作等优势,因而常被作为发育生物学和比较医学研究的模型动物。尤其是近年来有关鸡胚各种组织器官发育分子机理的研究进展较快,这为鸡胚更好的作为模型动物奠定了一定的基础。为了更深层次揭示细胞发育和分化的机理,在内源性调节的基础上力图通过外源性调控,在体外获得干细胞来源、可用于组织功能修复的组织和器官,本实验以艾维因鸡胚为材料,分离了胚胎期原始生殖细胞(primordial germ cell, PGC)并通过传代培养获得了大量胚胎生殖(embryonic germ cell, EG)细胞,建立了EG细胞体内形成畸胎瘤及体外形成类胚体(embryoid body, EB)的模型,探讨了多种因素对EG细胞体外多向分化的调控,研究了EG细胞体外向成熟神经细胞、脂肪细胞、肝细胞和心肌细胞分化的潜能。1.鸡胚胎生殖细胞分离和培养模型的建立在体视显微镜下用玻璃细针分离3.5-4日胚龄鸡胚生殖嵴部位,用胰蛋白酶-EDTA消化分散组织,然后将分散的细胞进行培养以建立PGC的原代培养模型。所用的高糖DMEM培养液中添加5%胎牛血清(FCS)、10ng/ml白血病抑制因子(LIF)、10ng/ml碱性成纤维生长因子(bFGF)、10ng/ml干细胞生长因子(SCF), 0.1mmol/L MEM非必需氨基酸、0.1mmol/Lβ-巯基乙醇、2mmol/L L-谷氨酰胺(Gln)、100IU/ml青霉素和100μg/ml链霉素(完全培养液)。与体细胞共培养5d后,利用RT-PCR方法检测PGC特异性基因Cvh、Dazl和DEH在原代培养形成的PGC集落中的表达。将表达PGC特异基因的集落进行传代培养,然后用过碘酸雪夫氏反应(PAS)、SSEA-1和SSEA-3及SSEA-4免疫组化、和干细胞多能性相关基因PouV、Nanog和Sox2 RT-PCR检测均证实传代后形成的集落为EG阳性克隆。上述结果表明,PGC-体细胞原代及传代共培养并添加多种因子的条件下,PGC在体外可转变为EG细胞,这为研究生殖干细胞的分化机制提供了细胞来源。2.鸡胚胎生殖细胞体内形成畸胎瘤通过灌饲具有免疫抑制作用的药物环孢素A (CsA,5mg/kg)制作免疫抑制雏鸡,将含有1.0×106个鸡EG细胞的PBS注射到受到免疫抑制的雏鸡皮下,观察其分化情况。5周后取皮下形成的组织瘤进行切片和HE染色及免疫组化染色鉴定。结果表明,EG细胞在免疫缺陷的雏鸡皮下可自发分化为含有神经细胞、软骨细胞、脂肪细胞、血管、骨细胞、平滑肌、横纹肌和腺上皮细胞等三个胚层来源的多种细胞类型的畸胎瘤。而注射同样数量EG细胞到未饲喂环孢素A组(模型对照组)和注射不含有EG细胞的免疫抑制雏鸡组(细胞对照组)均未观察到组织瘤的形成。上述结果表明通过饲喂免疫抑制作用的药物环孢素A可制作免疫抑制雏鸡模型,本实验利用此模型证明了鸡EG细胞在体内具有多向分化为含有三个胚层来源细胞畸胎瘤的潜能,这为开展生殖干细胞体内分化潜能的研究提供了实验平台。3.鸡胚胎生殖细胞体外形成EB模型的建立与优化将EG克隆与体细胞进行胰酶消化,并利用差速贴壁方法富集EG细胞。然后无饲养层、无LIF和β-巯基乙醇条件下将鸡EG细胞接种到涂有1%琼脂的培养板中悬浮培养形成EB。形成的EB进行丫啶橙(AO)染色后在荧光共聚焦显微镜下观察其内部结构。对形成7d和14d的类胚体分别进行切片和HE组织学染色,并通过RT-PCR检测内胚层标志基因AFP、外胚层标志基因Sox3、中胚层标志基因GATA6和滋养层标志基因Cdx2在其中的表达。结果表明：将富集的鸡EG单细胞采用不含LIF的培养液进行悬浮培养4-7d可形成简单EB,继续悬浮培养则其内部形态也发生改变,最终形成囊状EB。不同时间点的EB切片显示随着时间的变化,EB中出现含有神经细胞、脂肪细胞和胰腺细胞等三个胚层的细胞类型。此外,在体外悬浮培养时比较了FCS和Gln对EB形成的影响。统计显示,随着悬浮培养时间的延长,EB的面积不断增大,FCS浓度和Gln添加量对EB的形成产生影响显著,其中以含15%FCS、2 mmol/L Gln的培养液中EB的形成数量和状态较好。同时,在EG细胞体外分化形成EB的第0d、3d、5d和10d分别检测多能性相关基因转录调控因子如PouV、Nanog和Sox2 mRNA的表达。结果显示：在EB体外分化过程中,调控干细胞多能性和自我更新的三个重要转录调控因子PouV、Nanog和Sox2表达量均呈明显下调趋势,提示PouV、Nanog和Sox2在维持鸡EG细胞多潜能性中起着重要作用。上述结果表明,鸡胚EG细胞通过悬浮培养方法可制备具有向三个胚层分化能力的EB,此结果为研究EG细胞体外定向诱导分化为特定的细胞类型奠定了实验基础。4.鸡胚EG细胞体外多向分化调控的研究在贴壁培养条件下,利用含10-6 mol/L RA的ITS诱导液可将EG细胞定向诱导分化为表达成熟神经元、星型胶质细胞和小胶质细胞特异蛋白及基因的神经细胞。部分神经元经免疫组化及RT-PCR方法鉴定,表明其具有合成多巴胺和胆碱能神经递质的功能。利用10μg/ml胰岛素、1μmol/L地塞米松和0.2 mmol/L吲哚美辛联合处理,可诱导EG细胞定向分化为含有大量脂滴并表达脂肪细胞特异性基因PPARγ, GLUT1和LPL的成熟脂肪细胞。利用与肝细胞条件培养联合培养的方式可定向诱导EG细胞分化为肝细胞,利用PAS染色、免疫细胞化学、RT-PCR方法鉴定EG诱导来源的肝细胞,提示EG细胞具有分化为含有糖原并分泌白蛋白功能的成熟的肝细胞。利用添加去甲基化试剂5-aza的培养液悬浮培养,可将EG细胞诱导分化为心肌细胞,表明5-aza具有显著提高形成规律性收缩EB比例的作用,其中以添加5μmol／L 5-aza组形成的比例最高。形成的EB经贴壁分化培养后,采用透射电镜观察、荧光免疫组化及RT-PCR方法鉴定EG细胞来源的心肌细胞,结果显示5-aza可诱导EG细胞分化为含有肌小结和缝隙连接、表达心肌细胞特异性蛋白和标志基因的心肌细胞。以上实验结果表明：从鸡胚生殖嵴分离的PGC与体细胞原代共培养并经传代所建立的培养模型可用于EG细胞制备和分化调控的研究,经PAS组化、SSEA-1和SSEA-3及SSEA-4免疫细胞化学染色、干细胞多能性相关基因转录调控因子PouV、Nanog和Sox2的RT-PCR法检测、在免疫抑制雏鸡体内形成含有三个胚层来源细胞的畸胎瘤等多种方法均证实了EG细胞的干细胞特性。将富集的鸡EG细胞悬浮培养形成简单EB和囊状EB,培养液中添加15%FCS和2mmol／L Gln可促进EB的形成。在EB体外分化过程中,PouV Nanog和Sox2表达量均呈明显下调趋势,而三个胚层的标志基因AFP.Sox3和GATA6及滋养层标志基因Cdx2出现表达,表明EG细胞通过悬浮培养方法可制备具有向三个胚层分化能力的EB。贴壁培养的EB经不同的诱导剂处理后,经细胞特异性的标志基因或蛋白的表达鉴定,EB可定向分化成有功能的神经细胞、脂肪细胞、肝细胞及心肌细胞。以上结果提示利用所建立的EG／EB体外制备和定向诱导分化模型可用于生殖干细胞发育调控机制的研究,EG细胞可诱导分化成三个胚层的细胞,这为禽类干细胞发育生物学和比较医学的研究提供了实验平台和理论基础。更多还原

【Abstract】 Although the chick embryo has long been used as a model for developmental biology, its potential use as an experiment model for the repair and regeneration of adult tissues is often overlooked. This model has several advantages over mammalian systems for in vivo studies, as it is cost-effective, easily manipulated, and can be used for transgenesis in conjunction with viral vectors or electroporation. The molecular mechanisms underlying the development of nearly all major organ systems have been elucidated in the chick, making data interpretation more complete within this knowledge landscape. In this study, embryonic germ (EG) cells were derived from primordial germ cells (PGCs) of genital ridges of 3.5-4-days-old chicken embryos. These cells satisfied the criteria previously used for defining chicken EG cells by using the expression of markers characteristic to ES cells. When injected subcutaneously, chicken EG cells could form teratoma that enable differentiation into a wide range of tissue types of all three primary cell lineages including neural cells, cartilage, forming bone, adipocytes, blood vessels, smooth muscle, striated muscle and secretory epithelia in recipient. Furthermore, cells in embryoid bodies (EBs) expressed lineage-specific markers of three germ layers and could be induced to differentiate into more advanced stages of various committed cell types, including dopamine and cholinergic neurons, astrocytes, oligodendrocytes, adipocytes, cardiomyocytes and hepatocytes, that were demonstrated by immunocytochemical staining or RT-PCR analysis. These findings support the multiple differentiation capability of chicken pluripotent EG cells, thus confirming the presumption that chicken embryos may used as a potential model for better understanding the mechanisms of tissue-specific differentiation and regeneration, that will help to devise strategies based on the transplantation of stem cell-derived tissues for restoring function to damaged or diseased tissues.1. Preparation of of chicken EG cells and establishment of the culture modelsGenital ridges were collected by dissection of chicken embryos at 3.5-4 days with a fine glass needle under a microsurgery scope. For primary culture, cell suspension containing both PGCs and somatic cells was seeded onto gelatin-treated 35 mm culture plates at a density of 1×106/well in DMEM supplemented with 5% fetal calf serum (FCS), 10ng/ml leukemia inhibitory factor (LIF), 10ng/ml human basic fibroblast growth factor (bFGF), 0.1mmol/L MEM nonessential amino acids, O.lmmol/L 2-mercaptoethanol,2mmol/L L-glutamine (Gln), 100U/ml penicillin and 100μg/ml streptomycin. The seeded cells were then maintained at 38.5℃in 5% CO2/95% air with 60%-70% relative humidity until the PGCs colonized as a primary culture. To trace the origin of the colonies, the primary formed colonies were picked up with a fine glass needle, dissociated with 0.25% trypsin-EDTA 5 days after plating and then subjected to RT-PCR analysis for expression of PGC-specific markers. For further subculture, colonies that were positive for PGC markers were picked up and treated with 0.25% trypsin-EDTA to achieve single cell suspension and reseeded onto 6-well dishes. After three passages, staining of periodic acid-Schiff regent (PAS), stage-specific embryonic antigens (SSEA-1, SSEA-3 and SSEA-4) immunocytochemistry, and the expression of the pluripotency-associated genes cPouV, cNanog and Sox2 analysis all confirmed the characteristics of cultured EG cells. The above results indicated that the primary and subculture models of PGCs could be used for obtaining EG cells which represents a source of EG cells for studies about differentiation of germline stem cells (GSC).2. Teratoma formation of chicken EG cells in vivoSeven-day-old chicks were obtained from a commercial hatchery and fed with cyclosporine (5 mg/kg BW) known as an immunosuppressive agent to dampen down the body’s immune reaction. EG colonies were collected and treated with 0.25% trypsin-EDTA to achieve single cell suspension. About 1×106 cells in 200μl PBS were injected subcutaneously into axilla region of the chicks. After 5 weeks, chicks were sacrificed and emerging tissue materials were dissected. Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5μm) were placed on slides for HE and irnmunohistochemical staining. Results showed that chicken EG cells were capable of forming teratomas after subcutaneous injection into the immunosuppressed chicks. The amount of differentiated tissues varied among individual teratomas. Each teratoma contained a broad variety of tissues, including neural cells, cartilage, forming bone, adipocytes, blood vessels, smooth muscle, striated muscle and secretory epithelia. There was no teratoma formed in chicks without immunosuppression or no EG cells injection. The above results indicated that the immunodeficiency chick model could be made by fed with immunosuppressive agents cyclosporine, and the present study using this chick model showed that the chicken EG cells could form teratomas containing a variety of differentiated tissues and cell types, as well as some patches of undifferentiated cells in vivo. 3. Formation of EBs from chicken EG cells in vitroEG colonies in culture were picked up and dissociated into single cells, transferred onto gelatin-coated plates for a 30 min period to remove contaminating somatic cells and then transferred onto Ultra Low Attachment plates to allow their aggregation and prevent adherence to the plate. Usually about 106 EG cells were incubated in each 35 mm plate accompanied by the withdrawal of LIF and 2-mercaptoethanol from the medium. During this period, the cells grew into compact aggregates and became simple EBs within 2-5 days, especially more easily accompanied by 15% FCS and 2 mmol/L Gln, then turned to cystic EBs with the formation of a central cavity. Inner structure of EBs were observed by acridine orange staining or HE staining of the EBs sections on Day 7 and Day 17 of suspension culture respectively. HE staining analysis of EB sections showed heterogeneous structure with the ability to differentiate into multiple cell types, including neural tube like structure, vascular structures, connective tissues, and glandular-like cells. Furthermore, expression of the lineage-specific markers of three germ layers, including endoderm specific gene characterized by AFP, mesoblast specific gene characterized by Gata6, ectoderm specific gene characterized by Sox3 and the trophectoderm specific gene characterized by Cdx2 was detected in EBs, suggesting their differentiation potential in vitro. In addition, we demonstrated the expression profiles of pluripotency-associated markers, including chicken Oct4 homologue PouV (cPouV), chicken Nanog (cNanog) and Sox2 genes during the formation of EBs, which were required for the maintenance of pluripotency in ES cells. All of these genes were expressed in undifferentiated EG cells, and down regulated during the formation of EBs, suggesting their presumable effects in the inhibition of chicken EG cells differentiation. The above results demonstrate the formation potential of EG cells into EBs, which will facilitate the establishment of the differentiation model of EG cells in vitro.3. Multipledifferentiation regulation of chicken EG cells in vitroFor the generation of the neural cells, EBs were plated onto gelatin coated tissue culture plates at Day 4. Neural progenitors were induced in serum-free medium containing 10-6 M RA,2 mmol/L glutamine,5 mmol/L HEPES,25μg/ml insulin,100μg/ml transferrin, and 30 nmol/L sodium selenite for 2 days. Results showed that, RA treatment together with serum free culture condition significantly increased levels of neural specific gene expression. After additional 7 days in culture, cells possessing typical neuronal morphology were fixed and proved to be NSE, TH, ChAT, GFAP and CNP positive by immunocytochemical staining. RT-PCR analysis also showed that after induction, nestin was down-regulated, whereas NSE,β-tubulinⅢ, ChAT, TH, GFAP and CNP became positive, which displayed mature neuron, astrocyte and oligodendrocyte differentiation. To induce mature neural cells, these progenitors were continually cultured for 7 days. Adipogenic differentiation was induced by culture 7-day-old EBs in medium supplemented with 10% FCS,10μg/ml insulin,1μmol/L dexamethasone,0.2 mmol/L indomethacin for 10 days. The accumulation of lipid vacuoles in the cells was first detected 5 days after the addition of adipogenic induction medium and determined by staining for Oil Red O 10 days after induction. As differentiation progressed, expression of PPARγ, GLUT1 were significantly increased. Mature adipocytes marker gene lipoprotein lipase (LPL) was detected 10 days after induction, thereby confirming the mature adiogenic differentiation. Fetal chicken liver-derived cells were prepared as adherent cells from 7-day-old chicken embryos, after which 14-day-old EBs were plated onto the gelatin-coated culture dishes and incubated in the liver cells-conditioned medium. The AFP mRNA expression was detected in 14-day-old EBs. However, the expression of cytochrome P450 7a1 (CYP7A1), hepatocyte nuclear factor la (HNFla) and hepatocyte nuclear factor 4a (HNF4a), possible markers for embryonic endoderm-derived mature hepatocytes, were only observed after a co-culture with fetal liver cell derived conditioned medium. Immunocytochemical staining together with histochemical results demonstrated a high ratio of ALB and PAS positive cells 10 days after induction of hepatocyte differentiation, thus showing that co-culture of EG cells with liver cells-derived conditioned medium guided the differentiation toward mature and functional hepatocytes. We also investigated the effect of 5-azacytidine (5-aza) on EG cells to differentiate into cardiomyocytes. EBs were formed after 10 days with 5-aza in suspension culture, beating cell clusters were observed. Results showed that 5-aza (0～10μmol/L) promoted the production of beating cell clusters of EG cells in a dose-dependent manner. In response to 5μmol/L of 5-aza, EG cells expressed cardiac markers such as GATA-6, Nkx2.5, cardiac troponin T, a-actinin, MHC6, Desmin, ANF, CASQ, SMHC and SMA were up-regulated in a time-dependent manner after induction. Immunocytochemistry revealed the expression of smooth muscle SMA, sarcomeric a-actinin, cardiac troponin T and connexin 43 respectively.Through these studies we successfully isolated positive cells for chicken PGC-specific markers from genital ridges. The positive cells proliferated to form colonies on somatic cells during primary culture. Firmly packed colonies with ES cell-like morphology could be observed after subculture. These cells showed important features of pluripotent stem cells especially the expression of the pluripotency-associated genes cPouV, cNanog and Sox2 and form teratoma in vivo. After suspension culture, simple and cystic EBs were formed from single EG cells and 15% FCS and 2 mmol/L Gln could promote their formation. The expression of the lineage-specific markers of three germ layers and the expression profiles of cPouV, cNanog and Sox2 genes during the EBs development in vitro were also examined. Results showed that cPouV, cNanog and Sox2 were down-Tregulated during the formation of EBs, and the the lineage-specific markers were expressed in 14-day-old EBs, suggesting their presumable effects in their regulatory effects in maintaining the pluripotency during differentiation of chicken EG cells. The differentiation potency into mature neuron, glial cells, adipocytes, hepatocytes and cardiomyocytes of EBs fully provide evidence that, chicken PGC-derived EG cells possess the pluripotency to differentiate into precursor cell lineages of all three germ layers and then into mature cells after the directed induction methods in vitro, thus making them a potential experimented model for further study the cellular mechanisms of tissue-specific differentiation and regeneration, that will help to devise strategies for restoring function to damaged or diseased tissues in humans.更多还原