影响鸡原始生殖细胞分离克隆因素的研究(简报)

具有多向分化潜能的胚胎干细胞有两种来源:一是来自于早期胚胎内细胞团的胚胎干细胞(Em-bryonic Stem Cells,ESCs),另一种是来自于胚胎生殖腺原始生殖细胞(Primordial Germ Cells,PGCs)的胚胎生殖细胞(Embryonic Germ Cells,EGCs)。     

从原始生殖细胞分离克隆鸡胚胎生殖细胞的研究

从孵化 5 5天的鸡胚生殖腺中分离得到大量原始生殖细胞 (PGCs)集落 ,这些集落的细胞经多次克隆传代具有胚胎生殖细胞 (EG)的诸多特征 ,如有连续传代的能力 (传至第 9代 ) ,细胞集落有典型鸟巢状结构 ,PAS染色阳性 ,AKP染色阳性 ,在无饲养层无分化抑制因子LIF时可以自发分化成几种细胞类型 ,包括成纤维细胞、神经细胞、自律细胞等 ,悬浮培养时具有形成类胚体的能力。上述发现表明该细胞具EG细胞的诸多特性 ,为类EG细胞     

影响牛胚胎干细胞分离克隆因素的研究

采用与同源胎儿成纤维细胞共同培养及传统饲养层培养方式,以高糖DMEM,添加0.1mM2-巯基乙醇,犊牛血清,细胞因子为培养基,以4-13周龄屠宰牛胎儿为实验材料,探讨影响牛原始生殖细胞分离克隆胚胎干细胞的相关因素,结果发现：当犊牛血清为15%时效果最好;细胞因子添加与否对胚胎干细胞的分离及同源牛胎儿成纤维细胞的贴壁与生长影响并不显著,而在传代过程中中有一定影响;以0.2%胰酶 0.04?TA为细胞消化液效果最佳;以同源胎儿成纤维细胞共培养的方式分离克隆牛胚胎干细胞,本研究观察到效果最好。     

不同时期鸡胚原始生殖细胞分离的研究

采用Ficoll密度梯度离心,酶解离两种方法在鸡胚孵化的第14期、19期、28期,分离、培养鸡胚中的原始生殖细胞(PGCs)。探索PGCs分离、培养的适宜时期及方法,以期获得较多数量,较高活力的PGCs作介导生产转基因鸡。结果表明：1．提取、分离PGCs的最佳时期依次为19期、28期。2．两种分离方法均能分离到一定数量的PGCs细胞。但在19期和28期,酶解离法分离到的PGCs的相对数量较多,存活时间较长,是一种较适宜的分离方法。     

猪原始生殖细胞的分离、培养与鉴定

Embryonic germ cells (EG cells) are pluripotential undifferentiated stem cells isolated from cultured primordial germ cells (PGCs). Like ES cells, EG cells are of importance for gene targeting, therapeutical cloning and organ trans-plantation. The aim of this study was to isolate and characterize EG cells from porcine PGCs. The genital ridges from 24- 26 days old porcine embryos were treated in 0.02% EDTA for 20 min and pricked with a needle to release PGCs. The isolated PGCs were cultured on a SNL feeder layer in an EG cell medium. The EG cell medium consisted of Dulbecco‘‘s modified Eagle‘‘ s medium (DMEM) supplemented with 20 % Buffalo rat lever (BRL) cell-conditioned medium, 15 % fe-tal bovine serum, 1 mmol L-glutamine, 0.1 mol nonessential amino acids, 10 μmol β-mercaptoethanol and antibiotics.The freshly isolated PGCs were positive for alkaline phosphatase activity and Periodic acid-Schiff‘‘ s staining. Under this culture regime, PGCs could be maintained in an undifferentiated state and used for further cultures. One strain of the cul-tured PGCs was cultured 8 times, and alkaline phosphatase activity was detected in the colony formed from this strain.These cultured PGCs could spontaneously differentiate into fibroblast-like cells. These data suggested that we had success-fully isolated EG-like cells from oorcine PGCs.     

鸡胚不同发育时期原始生殖细胞的分离方法

采用Ficoll密度梯度离心法和EDTA-胰酶酶解法两种方法,分别提取第14期(孵化53h)血液、第19期(孵化72h)和第28期(孵化132h)生殖腺中的PGCs,以比较两种分离方法对3个发育时期的鸡胚原始生殖细胞在相同体外培养条件下存活时间的差异。结果发现,两种分离方法均能分离到一定数量的原始生殖细胞,但是酶解法分离到的原始生殖细胞的相对数量较Ficoll密度梯度离心法的多,存活时间较长,是一种适宜的分离方法;对鸡胚发育第14、19、28期3个时期提取的原始生殖细胞进行体外培养,存活时间分别为：72h、88h和80h,三者之间差异显著。结果表明：在鸡胚孵化的第19期,因原始生殖细胞大量聚集在肢体后端的生殖嵴原基处,因而较容易收集,体外培养较为适宜,具有较强的可操作性[动物学报49(6)：835～842,2003]。     

鸡胚胎原始生殖细胞体外培养

以14-15期鸡胚血液为材料,采用Ficoll密度梯度离心方法,提取鸡胚胎原始生殖细胞(primordial germ cells,PGCs),在无基质细胞和基质细胞上分别进行体外培养。从实验结果可以看出：在含有胎牛血清(fetal bovine serum,FBS)、鸡血清(chicken serum,CS)、碱性成纤维细胞生长因子(bFGF)、人胰岛素样生长因子(hIGF-1)、小鼠白血病抑制因子(mLIF)和青,链霉素双抗的M199培养液中培养时,鸡PGCs最多能够存活4天：当采用细胞因子和5天鸡胚胎性腺基质细胞共培养时能存活23代且每代细胞增殖可达近10倍。提纯后的PGCs细胞冻存复苏后,经台盼蓝染色鉴定存活率可达80％左右。     

细胞因子对鸡胚胎原始生殖细胞（EPGCs）增殖的影响

采用MTT法分别检测mLIF、bFGF、hSCF、hIL-11四种细胞因子协同作用对体外培养的第19、28期鸡EPGCs生长的影响。结果表明：与对照组相比较,19期的EPGCs体外培养72h后, mLIF、hSCF、bFGF、hIL-11对鸡EPGCs的增殖影响显著（P<0.05）。mLIF的最佳作用剂量是10~20ng/ml,hSCF的最佳作用剂量是15~20ng/ml,bFGF的最佳作用剂量是10～20ng/ml。hSCF、bFGF的联合使用优于单因子作用的结果（P<0.05）。单独使用hIL-11时,细胞的增殖情况比其他三因子单独使用的效果较差,但OD均值有随剂量增高而上升的趋势。在与其他因子联合使用的情况下, hIL-11的最佳作用剂量为0.10~0.20ng/ml。     

鸡胚原始生殖细胞的分离和鸡鸭嵌和体的制备

探索了鸡胚12～17期血液中的原始生殖细胞(PGCs)迁移数量变化规律,将其在液氮中冷冻保存.并以Ficoll密度梯度离心、MiniMACS磁气分离、滤膜三种方法对PGCs进行分离,结果发现12～17期血液中均有PGCs存在,13期达到高峰约47.1±10.5个/μl,在血液中比例为0.0126%.冷冻保存3个月后解冻成活率达80%以上.三种分离方法所得的分离效果分别为95.7%、39.2%、63.0%,纯度为27.5%、8.4%、3.1%.将分离的原始生殖细胞以微注射法转移至14～15期麻鸭胚胎中制备了鸡鸭种间嵌和体,获得8只雏鸭(8/110).以鸡W染色体探针原位杂交法在早期鸭胚性腺中检测到鸡原始生殖细胞,嵌和率达84.2%(16/19).表明鸡原始生殖细胞能够迁移定居到鸭胚性腺中,并有可能增殖分化成有功能的配子.     

鸡胚胎原始生殖细胞的培养和传代

本文旨在探索鸡胚胎原始生殖细胞(Primordialgermcells,PGCs)培养、传代以及各种因素对PGCs培养的影响。实验结果表明,在添加10%的胎牛血清、2%的鸡血清、2mmol/LL谷氨酰胺、1mmol/L丙酮酸钠、5.5×10-5mol/Lβ巯基乙醇、10μl/ml非必需氨基酸、以及5ng/ml人干细胞生长因子(Humanstemcellfactor,hSCF)、10U/ml鼠白血病抑制因子(Mouseleukemiainhibitoryfactor,mLIF)、10ng/ml碱性成纤维生长因子(Fibroblastgrowthfactorbasic,bFGF)、0.04ng/ml人白细胞介素11(Humaninterleukin11,hIL11),10ng/ml胰岛素样生长因子(Humaninsulinlikegrowthfactor,hIGF)的高糖DMEM培养体系中,以多次离散法进行传代获得5-6代鸡胚胎PGCs,有效地维持了PGCs的未分化状态和正常二倍体核型,同时能够定向地诱导分化为神经细胞,具有作为多能性胚胎干细胞的特征     

第二十八期鸡胚原始生殖细胞的冷冻保存

采用Ficoll密度梯度离心 ,提取第 2 8期 (孵化 13 2h)性腺中的PGCs ,对其应用不同的冷冻保护剂和不同的平衡方法进行冷冻保存 ,并于复苏后进行体外培养。复苏后的PGCs用台盼蓝染色检测其存活率 ,结果发现 :从第 2 8期性腺中获取的PGCs在同一种冷冻保护液下 ,采用不同的平衡方法进行冷冻 ,对PGCs的存活率有显著影响 (P <0 .0 5 ) ;平衡方法相同 ,在不同冷冻保护液条件下 ,冷冻保护液III的冻存效果最好 ,与其他保护液之间存在显著 (P <0 .0 5 )或极显著 (P <0 .0 1)差异。冷冻保护液V和冷冻保护液VI二者对PGCs的冻存效果次之。冷冻保护液I、冷冻保护液II和冷冻保护液IV三者对PGCs的冻存效果最差。PGCs经体外培养 2 4小时后再进行冷冻保存 ,复苏后其存活率、体外培养存活时间均极 (P <0 .0 1)显著短于分离后直接冷冻的PGCs。     

Expression patterns and miRNA regulation of DNA methyltransferases in chicken primordial germ cells

DNA methylation is widespread in most species, from bacteria to mammals, and is crucial for genomic imprinting, gene expression, and embryogenesis. DNA methylation occurs via two major classes of enzymatic reactions: maintenance-type methylation catalyzed by DNA (cytosine-5-)-methyltransferase (DNMT) 1, and de novo methylation catalyzed by DNMT 3 alpha (DNMT3A) and -beta (DNMT3B). The expression pattern and regulation of DNMT genes in primordial germ cells (PGCs) and germ line cells has not been sufficiently established in birds. Therefore, we employed bioinformatics, RT-PCR, real-time PCR, and in situ hybridization analyses to examine the structural conservation and conserved expression patterns of chicken DNMT family genes. We further examined the regulation of a candidate de novo DNA methyltransferase gene, cDNMT3B by cotransfection of cDNMT3B 3'UTR- and cDNMT3B 3'UTR-specific miRNAs through a dual fluorescence reporter assay. All cDNMT family members were differentially detected during early embryonic development. Of interest, cDNMT3B expression was highly detected in early embryos and in PGCs. During germ line development and sexual maturation, cDNMT3B expression was reestablished in a female germ cell-specific manner. In the dual fluorescence reporter assay, cDNMT3B expression was significantly downregulated by four miRNAs: gga-miR-15c (25.82%), gga-miR-29b (30.01%), gga-miR-383 (30.0%), and gga-miR-222 (31.28%). Our data highlight the structural conservation and conserved expression patterns of chicken DNMTs. The miRNAs investigated in this study may induce downregulation of gene expression in chicken PGCs and germ cells.     

Differentiation of female chicken primordial germ cells into spermatozoa in male gonads

In avian species, the developmental fate of different-sex germ cells in the gonads is unclear. The present study attempted to confirm whether genetically female germ cells can differentiate into spermatozoa in male gonads using male germline chimeric chickens produced by the transfer of primordial germ cells (PGC), and employing molecular biological methods. As a result of Southern hybridization, specific sequences of the W chromosome (the female specific sex chromosome in birds) were detected in the genomic DNA extracted from one out of four male germline chimeric chickens. When two-color in situ hybridization was conducted on the spermatozoa of this germline chimera, 0.33% (average) of the nuclei of each semen sample showed the fluorescent signal indicating the presence of the W chromosome. The present study shows that female PGC can differentiate into spermatozoa in male gonads in the chicken. However, the ratio of produced W chromosome-bearing (W-bearing) spermatozoa fell substantially below expectations. It is therefore concluded that most of the W-bearing PGC could not differentiate into spermatozoa because of restricted spermatogenesis.     

Expression and knockdown analysis of glucose phosphate isomerase in chicken primordial germ cells

Glucose is an important monosaccharide required to generate energy in all cells. After entry into cells, glucose is phosphorylated to glucose-6-phosphate and then transformed into glycogen or metabolized to produce energy. Glucose phosphate isomerase (GPI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. Without GPI activity or fructose-6-phosphate, many steps of glucose metabolism would not occur. The requirement for GPI activity for normal functioning of primordial germ cells (PGCs) needs to be identified. In this study, we first examined the expression of chicken GPI during early embryonic development and germ cell development. GPI expression was strongly and ubiquitously detected in chicken early embryos and embryonic tissues at Embryonic Day 6.5 (E6.5). Continuous GPI expression was detected in PGCs and germ cells of both sexes during gonadal development. Specifically, GPI expression was stronger in male germ cells than in female germ cells during embryonic development and the majority of post-hatching development. Then, we used siRNA-1499 to knock down GPI expression in PGCs. siRNA-1499 caused an 85% knockdown in GPI, and PGC proliferation was also affected 48 h after transfection. We further examined the knockdown effects on 28 genes related to the glycolysis/gluconeogenesis pathway and the endogenous glucose level in chicken PGCs. Among genes related to glycolysis/gluconeogenesis, 20 genes showed approximately 3-fold lower expression, 4 showed approximately 10-fold lower, and 2 showed approximately 100-fold lower expression in knockdown PGCs. The endogenous glucose level was significantly reduced in knockdown PGCs. We conclude that the GPI gene is crucial for maintaining glycolysis and supplying energy to developing PGCs.     

Ontogenesis of primordial germ cells

In sexually reproducing animals all gametes of either sex arise from primordial germ cells (PGC). PGC represent a small cell population, appearing early during embryo development. They represent a key cell population responsible for the survival and the evolution of a species. Indeed, the production of gametes will assure fertilisation and therefore the establishment of the next generation. Until recently only few laboratories were working on PGC biology. A new interest emerged since these cells have the ability to function as pluripotent stem cells when established as cell lines. Indeed, like embryonic stem cells (ESC), embryonic germ cells (EGC) are able to differentiate in a wide variety of tissues. In vivo, EGC are able, after injection into a host blastocyst cavity to colonise the inner cell mass and to participate in embryonic development. In vitro studies in human and mouse have also shown their capacity to differentiate into a large variety of cell types allowing the study of processes involved in cardiomyocyte, haematopoietic, neuronal and myogenic differentiation pathways. We present here the last updates of PGC ontogeny focusing mainly on the murine model.     

Immunomagnetic isolation of primordial germ cells and the establishment of embryonic germ cell lines in the mouse

The stage-specific embryonic antigen 1 (SSEA-1) is a cell marker of primordial germ cells (PGCs). In the present study, it is shown that isolation and purification of PGCs from 8.5-11.5 days post coitum (dpc) embryos can be achieved by a immunomagnetic cell sorting method using SSEA-1 antibody-conjugated magnetic beads, and then the sorted PGCs can be used for long-term culture under strict culture conditions to derive embryonic germ (EG) cell lines. Five independent EG cell lines with male karyotypes have been established. They show both a strong alkaline phosphatase activity and expression of the SSEA-1 antigen, and are karyotypically stable with a modal number of chromosomes in more than 80% of the cells. One of the EG cell lines from 8.5-dpc embryos produced chimeras after injections of the cells into 8-cell host embryos. These procedures could provide a useful and simple method for isolation of undifferentiated cells from a heterogeneous cell population and for establishment of embryo-derived stem cell lines.