水牛PGC和前精原细胞的体外培养

分别对取自50~95日龄水牛胎儿的原生殖细胞和前精原细胞进行体外培养,观察其生物学行为,并检测其碱性磷酸酶(AP)活性和Oct-4蛋白特性,探讨利用这些生殖细胞建立干细胞系的可行性和检测方法。结果表明水牛原生殖细胞及前精原细胞分别在体外培养时,均能形成细胞克隆;克隆与周围细胞分界明显,但克隆中细胞相互间界限不清;部分克隆有分隔现象,形如多个克隆共同组成一个大克隆;细胞克隆均至少能培养4代以上;原生殖细胞和前精原细胞及其来源的细胞克隆均呈AP阴性和Oct-4蛋白阴性,其中部分克隆表现为AP假阳性。研究结果显示水牛原生殖细胞和前精原细胞均可用于建立干细胞系;体外培养时,AP活性和Oct-4蛋白不适宜用来检测这些细胞及其来源的细胞克隆。     

无血清培养关中奶山羊胚胎生殖细胞的研究

山羊胚胎生殖细胞是一种来源于胎儿原始性腺的多能干细胞,建立该细胞体外稳定分离培养体系对研究山羊繁殖育种具有重要价值。本试验通过酶消化法和组织培养法分离培养关中奶山羊胚胎生殖细胞,检测无血清培养基对细胞体外增殖的影响。结果发现,该培养基可以分离得到山羊胚胎生殖细胞,细胞集落形态典型,表达AKP、Oct4、TERT及SSEA-1。经体外分化试验表明,细胞可以分化为类胚体、成纤维样细胞、成脂细胞和卵母细胞样形态。无血清培养基可以用于山羊胚胎生殖细胞的分离与培养,本试验对进一步建立山羊胚胎生殖细胞长期培养体系提供了新的参考。     

兔胎儿成纤维细胞的分离培养和鉴定

本研究旨在建立稳定传代的兔胎儿成纤维细胞系及其鉴定方法。取配种后14~15d的兔胎儿,0.25%胰酶消化胎儿皮肤获得兔胎儿成纤维细胞,体外传代培养;通过观察细胞形态、测定生长曲线、免疫荧光染色、核型分析等鉴定兔胎儿成纤维细胞,PCR扩增SRY基因鉴定2株细胞系性别。研究结果表明,兔胎儿成纤维细胞呈梭形,可在体外快速生长,符合"潜伏期-对数期-平台期"的生长规律;免疫荧光染色结果显示,波形蛋白与纤维连接蛋白染色为阳性,细胞角蛋白染色为阴性,表明兔胎儿成纤维细胞纯度高,活性好;染色体检测结果显示,在传至第5代时,80%的成纤维细胞核型正常,为2n=44,属正常范畴;PCR法检测的2株细胞系均来源于兔雄性胎儿。综上表明,本研究建立了兔胎儿成纤维细胞系体外培养、传代、鉴定及其性别鉴定方法,可为转基因兔、克隆兔、兔诱导性多能性干细胞等研究提供充足、可靠的成纤维细胞来源。     

绵羊羊水、胎儿肾脏组织中表达Oct-4细胞的分离培养及其分子学特性研究简

试验旨在建立从绵羊羊水、胎儿肾脏组织中分离培养表达Oct-4细胞的新方法,同时,通过实时荧光定量PCR（Real-time PCR）初步探索二者在分子生物学特性方面的联系及差异。采用0.01%胰酶培养液,从同一只受孕中期绵羊的羊水和胎儿肾脏组织中各分离1株细胞。Real-time PCR分析结果表明,从绵羊羊水、胎儿肾脏组织中分离的细胞均表达胚胎干细胞相关标志基因Oct-4、胚胎生殖细胞标志基因SSEA-1、主要组织相容性抗原MHC-Ⅱ、细胞凋亡基因Bax及抑制细胞凋亡基因Bcl-2,不表达MHC-Ⅰ、Nanog及TERT,因此,将羊水中表达Oct-4的细胞暂命名为羊水来源多潜能干细胞(amniotic fluid stem cells,AFSC),胎儿肾脏组织中表达Oct-4的细胞暂命名为肾脏组织干细胞(renal tissue stem cells,RTSC)。相对定量分析结果显示,二者的Oct-4、Bax及Bcl-2基因的相对表达量存在明显差异。绵羊羊水中表达Oct-4的细胞（AFSC）和胎儿肾脏组织中表达Oct-4的细胞（RTSC）体外悬浮培养7 d均能形成类胚体。试验成功建立了从绵羊羊水、胎儿肾脏组织中分离培养表达Oct-4细胞的新方法,实时荧光定量PCR分析初步显示绵羊羊水中表达Oct-4的细胞和胎儿肾脏组织中表达Oct-4的细胞具有相同的起源,并在体内处于一种动态的变化之中。     

猪雄性生殖干细胞的分离培养及鉴定

本试验旨在探索猪雄性生殖干细胞(mGSCs)体外分离、培养的适宜条件,建立猪雄性生殖干细胞体外培养体系。采用两步酶消化法对新生小猪睾丸生殖干细胞进行了体外分离和初步的培养鉴定,并利用层黏连蛋白和明胶的不同贴壁特性,比较2种差易贴壁分选方法的富集效果,并对传代后的干细胞培养1周后进行碱性磷酸酶染色鉴定,通过免疫荧光技术检测培养细胞是否表达干细胞标志蛋白OCT-4。试验结果表明,层黏连蛋白更适用于猪生殖干细胞的富集、培养,细胞分选效率及增殖生长明显优于采用明胶分选的方法。培养的mGSCs拥有与小鼠mGSCs相同的形态、增殖及表达特征。鉴定结果显示,生长细胞克隆碱性磷酸酶染色呈阳性,支持细胞碱性磷酸酶染色呈阴性;培养的生殖干细胞克隆表达转录蛋白OCT-4,而饲养层支持细胞OCT-4抗体染色则呈阴性。结果表明培养的干细胞克隆仍保持较好的干细胞活性,保持正常的自我复制和分化潜能,初步建立了生殖干细胞培养体系。     

视黄酸诱导鸡胚胎干细胞向雄性生殖细胞分化的研究

体外胚胎干细胞（embryonic stem cells,ESCs）向生殖细胞分化可用于治疗不育症,同时也为揭示种系世代的分子机制提供最佳模型。试验旨在探讨视黄酸（retinoic acid,RA）诱导鸡胚胎干细胞（chicken embryonic stem cells,cESCs）向雄性生殖细胞（male germ cells,MGCs）分化的作用效果。利用胰蛋白酶消化法从新鲜种蛋X期鸡胚中分离胚胎干细胞,以鸡胚成纤维（chicken embryo fibroblast,CEF）细胞为滋养层,进行体外培养,利用形态法、碱性磷酸酶（alkaline phosphatase,AKP）染色和胚胎阶段特异性表面抗原（embryo specific surface antigen 1,SSEA-1）检测对获得的胚胎干细胞进行鉴定。结果表明,获得典型的呈巢状或岛状的cESCs克隆,细胞AKP染色呈蓝紫色,表明其具有较高的内源性AKP活性;SSEA-1鉴定结果呈阳性,显示cESCs克隆具有多能性。采用10^-5 mol/L RA诱导鸡胚胎干细胞向雄性生殖细胞分化,镜下观察细胞形态变化,分别于诱导第0、2、4、6、8、10天提取细胞总RNA,反转录成cDNA,用于实时荧光定量PCR检测生殖细胞标志基因的表达。结果表明,在此诱导过程中,作为胚胎干细胞标志基因Nanog、Sox2表达量持续显著下降,而生殖细胞特异性基因Dazl、Stra8、c-kit、integrin α6表达量呈持续上升趋势;免疫细胞化学检测可观察到特异基因相关蛋白的阳性克隆。本研究成功分离出cESCs,可体外培养并保持未分化状态及多能性。10^-5 mol/L RA能够促进cESCs向雄性生殖细胞方向分化,可以引起生殖细胞相应基因的表达,为进一步研究雄性生殖细胞的形成和调控机制提供参考。     

视黄酸诱导鸡胚胎干细胞向雄性生殖细胞分化的研究

体外胚胎干细胞(embryonic stem cells,ESCs)向生殖细胞分化可用于治疗不育症,同时也为揭示种系世代的分子机制提供最佳模型。试验旨在探讨视黄酸(retinoic acid,RA)诱导鸡胚胎干细胞(chicken embryonic stem cells,cESCs)向雄性生殖细胞(male germ cells,MGCs)分化的作用效果。利用胰蛋白酶消化法从新鲜种蛋X期鸡胚中分离胚胎干细胞,以鸡胚成纤维(chicken embryo fibroblast,CEF)细胞为滋养层,进行体外培养,利用形态法、碱性磷酸酶(alkaline phosphatase,AKP)染色和胚胎阶段特异性表面抗原(embryo specific surface antigen 1,SSEA-1)检测对获得的胚胎干细胞进行鉴定。结果表明,获得典型的呈巢状或岛状的cESCs克隆,细胞AKP染色呈蓝紫色,表明其具有较高的内源性AKP活性;SSEA-1鉴定结果呈阳性,显示cESCs克隆具有多能性。采用10~(-5) mol/L RA诱导鸡胚胎干细胞向雄性生殖细胞分化,镜下观察细胞形态变化,分别于诱导第0、2、4、6、8、10天提取细胞总RNA,反转录成cDNA,用于实时荧光定量PCR检测生殖细胞标志基因的表达。结果表明,在此诱导过程中,作为胚胎干细胞标志基因Nanog、Sox2表达量持续显著下降,而生殖细胞特异性基因Dazl、Stra8、c-kit、integrinα6表达量呈持续上升趋势;免疫细胞化学检测可观察到特异基因相关蛋白的阳性克隆。本研究成功分离出cESCs,可体外培养并保持未分化状态及多能性。10~(-5) mol/L RA能够促进cESCs向雄性生殖细胞方向分化,可以引起生殖细胞相应基因的表达,为进一步研究雄性生殖细胞的形成和调控机制提供参考。     

奶山羊胎儿成纤维细胞的分离培养及SRY基因性别鉴定

为了探索和建立奶山羊胎儿成纤维细胞体外分离培养及性别鉴定的技术方法,获得转基因克隆羊的供体细胞,本试验用组织块培养法分离纯化得到两株奶山羊胎儿成纤维细胞系,进行细胞形态观察、生长曲线及细胞周期和倍性分析。同时根据GenBank上发布的山羊SRY基因设计合成一对PCR引物作为性别鉴定引物,另外根据山羊BLG基因序列设计一对引物作为内参引物,建立PCR反应体系对两株胎儿成纤维细胞系进行性别鉴定。结果表明,分离的胎儿成纤维细胞活力良好,可在体外快速生长、增殖、稳定培养;阳性对照和山羊胎儿成纤维细胞系2经PCR扩增得到337 bp片段和498 bp的BLG基因片段,而阴性对照和山羊胎儿成纤维细胞系1经PCR扩增得到498 bp的β-乳球蛋白基因片段。将337 bp片段和pMD19-T载体连接,构建重组载体pSRY,通过测序证明337 bp片段为SRY基因片段。这说明有337 bp扩增带的细胞系为雄性,无337 bp扩增带的细胞系为雌性。本试验为转基因奶山羊新品种的培育奠定了基础。     

不同因素对山羊原始生殖嵴细胞体外分离培养与克隆的影响

从本地槐山羊胎儿中,将原始生殖细胞与其生殖嵴周围组织细胞共同分离,经传代培养后获得了具有干细胞特征的山羊胚胎生殖(EG)细胞。结果表明:高糖DMEM培养基和低糖DMEM培养基相比较,低糖DMEM更适宜于山羊EG细胞的分离与克隆;EG细胞在山羊胎儿成纤维细胞饲养层上生长效果较好,可传4代或5代,而在小鼠成纤维细胞饲养层上EG细胞仅传3代;联合添加白血病抑制因子(LIF)、干细胞因子(SCF)和碱性成纤维细胞生长因子(bFGF)能显著提高山羊EG细胞分离与克隆的效率;胎龄为30～45d的胎儿原代培养时可获得大量的细胞集落,克隆培养可传至5代,适合做山羊EG细胞的分离培养。     

影响山羊类胚胎干细胞分离克隆的因素分析

将本地槐山羊胎儿的原始生殖细胞(PGCs)与其生殖嵴周围组织细胞共同分离,经传代培养后获得了具有干细胞特征的山羊类ES细胞。结果表明高糖DMEM培养基和低糖DMEM培养基相比较,低糖DMEM更适宜于山羊类ES细胞的分离与克隆;类ES细胞在山羊胎儿成纤维细胞饲养层上生长效果较好,可传4代或5代,而在小鼠成纤维细胞饲养层上,类ES细胞仅传3代;联合添加白血病抑制因子(LIF)、干细胞因子(SCF)和碱性成纤维细胞生长因子(bFGF)能显著提高山羊类ES细胞分离与克隆的效率;胎龄为30~45d的胎儿原代培养时可获得大量的细胞集落,克隆培养可传至5代,适合作山羊类ES细胞的分离培养。     

Enhancing effects of serum-rich and cytokine-supplemented culture conditions on developing blastocysts and deriving porcine parthenogenetic embryonic stem cells

The present study was conducted to develop an effective method for establishment of porcine parthenogenetic embryonic stem cells (ppESCs) from parthenogenetically activated oocyte-derived blastocysts. The addition of 10% fetal bovine serum (FBS) to the medium on the 3rd day of oocyte culturing improved the development of blastocysts, attachment of inner cell masses (ICMs) onto feeder cells, and formation of primitive ppESC colonies. ICM attachment was further enhanced by basic fibroblast growth factor, stem cell factor, and leukemia inhibitory factor. From these attached ICMs, seven ppESC lines were established. ppESC pluripotency was verified by strong enzymatic alkaline phosphatase activity and the expression of pluripotent markers OCT3/4, Nanog, and SSEA4. Moreover, the ppESCs were induced to form an embryoid body and teratoma. Differentiation into three germ layers (ectoderm, mesoderm, and endoderm) was confirmed by the expression of specific markers for the layers and histological analysis. In conclusion, data from the present study suggested that our modified culture conditions using FBS and cytokines are highly useful for improving the generation of pluripotent ppESCs.     

Isolation and culture of rabbit primordial germ cells

Primordial germ cells (PGCs) are embryonic precursors of the gametes of adult animals and are considered stem cells of the germline. Since their proliferation in vitro correlates well with the schedule of developmental changes in vivo, they might be interesting research tools for genomic imprinting, germ-cell tumors and fertility. Furthermore, once primordial germ cells are separated and placed on a feeder layer with cytokines, they become cultured pluripotent cell lines called embryonic germ (EG) cells. EG cells share several important characteristics with embryonic stem (ES) cells as they can also contribute to the germ line of chimeras. To investigate the characteristics of PGCs and establish rabbit EG (rEG) cells, we cultured rabbit PGCs (rPGCs) in vitro with various combinations of leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) and forskolin on inactivated mouse embryonic fibroblast (MEF) feeder layers. The present study found PGC proliferation in early cultures and induction of rEG-like colonies. These cells expressed pluripotent markers, such as alkaline phosphatase activity, OCT-4, Sox-2 and SSEA-1, in the undifferentiated state; however, the cells did not develop into a teratoma when injected into the kidney capsules of SCID mice, although the restricted differentiation potentials to neural cells were determined via embryoid body formation. From these characteristics and further characterization of the germ stem cell markers Vasa, SCP-1 and SCP-3, we suggested that these were hybrid cells with characteristics somewhere between PGC and EG cells.     

Determination of Feeder Cell‐Based Cellular Niches Supporting the Colonization and Maintenance of Spermatogonial Stem Cells from Prepubertal Domestic Cat Testes

Recently, isolation and in vitro culture of putative spermatogonial stem cells (SSCs) in the domestic cat have been conducted. However, the cellular niche conditions that facilitate the establishment and long‐term maintenance of feline SSCs (FSSCs) have not been described. Therefore, we investigated the type of feeder cells used to stimulate colony formation and growth of FSSCs among the various factors in the FSSC niche. Spermatogonial stem cells isolated from feline testes were cultured on mitotically inactivated testicular stromal cells (TSCs) derived from cats, dogs and mice, and mouse embryonic fibroblasts (MEFs). The formation and growth of colonies derived from SSCs cultured on each type of feeder cell were identified at passage 0, and the morphology, alkaline phosphatase (AP) activity and expression of SSC‐specific genes in surviving colonies were investigated at passage 4. Among these diverse feeder cells, TSCs from cat showed the greatest colony formation, growth and maintenance of FSSCs, and SSC colonies cultured by passage 4 showed a typical dome‐shaped morphology, AP activity and expression of SSC‐specific genes (NANOG, OCT4, SOX2 and CD9). Accordingly, these results demonstrate that feline TSCs could be used as feeder cells to support the establishment and maintenance of SSCs from domestic cats.     

Isolation, culturing and characterization of feeder-independent amniotic fluid stem cells in buffalo (Bubalus bubalis)

Heterogeneous amniotic fluid contains various cell types. The aim of this study was to characterize and differentiate some of the key stemness attributes of the amniotic fluid-derived cells in buffalo (Bubalus bubalis). The amniotic fluid (AF) cells were cultured without feeder cells, in DMEM containing 15% FBS, 1% non-essential amino acids, 1% penicillin/streptomycin/ampicillin, 1% vitamin solution, and 1% l-glutamine in 5% CO(2) in humidified air at 38.5±0.5 °C. After 6 days of culture different types of cells viz., star shaped (62.7%), spherical without nucleus (1.9%), spherical with nucleus (26.4%), pentagonal (0.4%), and free floating/rounded cells (8.3%) were observed. Most of the cells started anchorage-dependent growth after day 7 of the culture. Expression of alkaline phosphatase (AP) and Oct-4, Nestin and FGF-5 were observed from the AF cells at different passages. Using species-specific primers, a PCR amplicon of 200, 296 and 210 bp were observed for Oct-4, Nestin and FGF-5, respectively. The cells were found to have a normal karyotype at different passages. These results may contribute towards establishing non-embryonic pluripotent stem cells for various therapeutic and reproductive biotechnological applications in the species.     

绵羊胚胎成纤维细胞饲养层用于培养人诱导性多能干细胞的效果研究

试验在体外分离培养1月龄绵羊胚胎成纤维细胞（sheep embryonic fibroblast，SEF），经丝裂霉素C处理后探讨其作为诱导性多能干细胞（induced pluripotent stem cells，iPSC）体外培养饲养层的可行性。试验以SNL饲养层细胞为对照，人iPSC（hiPSC）为培养对象，通过形态学观察、碱性磷酸酶（AP）染色、以及实时荧光定量PCR和免疫细胞化学对hiPSC标志基因mRNA和蛋白表达的检测，比较了SEF细胞和SNL细胞作为干细胞饲养层的效果。结果表明，在试验期内，与SNL饲养层体外培养的hiPSC相似，SEF饲养层体外培养的hiPSC在形态上呈集落样生长，增殖速度快；AP染色呈蓝紫色，能够维持未分化状态；能正常表达多能性标志基因。两种饲养层细胞培养的hiPSC多能性标志基因c-Myc、Klf4、OCT4和SOX2 mRNA的表达以及OCT4、SOX2、SSEA4和TRA-1-60蛋白的表达并无显著差异（P>0.05）。本研究结果初步表明SEF可作为体外培养iPSC的饲养层细胞，为进一步建立可表达促生长因子的基因修饰SEF细胞系奠定了基础。     

绒山羊原始生殖细胞继代培养研究

本研究对原代培养至6d时的鸟巢状绒山羊类胚胎干细胞(ES细胞)集落进行了传代培养。其结果:这些集落的细胞经6代克隆传代具有胚胎干细胞的诸多特征。细胞集落有典型的鸟巢状结构,AKP染色阳性,核型分析结果染色体正常。在透射电镜下细胞体积小,核大,有多个核仁而且核仁明显,细胞形态不规则,无明显界限。细胞质中核糖体较多,内质网、线粒体发达,部分细胞的核膜上出现小泡、染色质边集而形成凋亡小体。上述结果表明该细胞具有多能性,类似ES细胞。