人胚胎干细胞系chHES-20表达生殖细胞分化相关基因

目的 探讨人胚胎干细胞系chHES-20能否表达生殖细胞分化相关基因. 方法 反转录-聚合酶链式反应(RT-PCR)检测chHES-20细胞系生殖细胞特异基因的表达情况,以人正常睾丸组织作为阳性对照,以人成纤维细胞为阴性对照. 结果 胚胎干细胞系chHES-20表达全能性相关基因OCT4、NANOG和SOX2,同时表达减数分裂前生殖细胞标志基因DAZL和FRAGILIS;不表达减数分裂特异标志基因联会复合体基因3(SCP3)和生殖细胞分化后期标志基因VASA. 结论 chHES-20胚胎干细胞系具有向生殖细胞诱导分化的潜能.     

人胚胎干细胞表达生殖细胞分化相关基因的研究

目的 探讨人胚胎干细胞(hES)系H1生殖细胞分化相关基因的表达.方法 免疫荧光、碱性磷酸酶检测和核型分析等方法联合鉴定H1的基本生物学特性,RT-PCR方法检测未分化H1的生殖细胞分化相关基因的基本表达模式,分别以人正常睾丸组织和人胚胎成纤维细胞株HFF-1作为阳性和阴性对照组织.结果 H1保持正常核型并维持未分化状态.未分化H1表达减数分裂前生殖细胞标志基因STELLAR、DAZL、FRAGILIS、PUM1、PUM2和GDF3;不表达原始生殖细胞(PGCs)标志基因BLIMP-1、减数分裂特异标志基因SCP1、减数分裂启动标志基因STRA8以及生殖细胞分化后期标志基因VASA.结论 未分化Hl细胞表达生殖细胞减数分裂前标志基因,但不表达分化后期基因,BLIMP-1可能成为区分未分化hES细胞与PGCs的特异性标志基因.     

生殖细胞核因子在精原干细胞体外培养分化中的表达

目的 将分离纯化的小鼠精原干细胞(SSCs)体外培养并诱导分化,检测生殖细胞核因子(GCNF)在小鼠SSCs诱导分化前后的表达.方法 用干细胞因子(SCF)诱导小鼠SSCs向精母细胞分化,通过间接免疫荧光染色和RT-PCR,检测GCNF在小鼠SSCs诱导分化前后的表达.结果 小鼠SSCs向精母细胞诱导分化前后,在倒置相差显微镜下进行形态学观察,细胞形态未发生明显变化,仍然呈圆形或椭圆形,核较大;间接免疫荧光及RT-PCR结果均显示,原代培养的小鼠SSCs呈现GCNF阴性,但是向精母细胞诱导分化2d后,GCNF开始表达.结论 GCNF在体外培养小鼠SSCs向精母细胞分化的早期有表达,提示GCNF可能参与了SSCs的早期分化.     

维甲酸定向诱导小鼠核移植胚胎干细胞向雄性生殖细胞分化

目的 初步探讨体外条件下定向诱导小鼠核移植胚胎干细胞分化为雄性生殖细胞的条件,建立有效诱导分化技术平台.方法 分离BALB/c小鼠NT-ESCs样集落并鉴定.NT-ESCs消化后分4组:1)采用维甲酸诱导NT-EBs向雄性生殖细胞方向分化;2)未分化的NT-ESCs;3)分化培养前的NT-EBs;4)自主分化的NT-EBs;5)小鼠胚胎成纤维细胞.RT-PCR法检测各组Oct-4、VASA、Scp3、Sry、Tex14和β-actin的mRNA表达;免疫荧光染色法检测VASA、Scp3蛋白表达.结果 NT-ESCs集落性生长,符合小鼠胚胎干细胞的一系列特性.NT-EBs维甲酸诱导组Oct-4、VASA、Scp3、Sry、Tex14和β-actin均有表达,对照组为阴性;NT-EBs细胞诱导分化后Scp3荧光强度为(+);实验组VASA荧光强度(+)也优于对照组的(±).结论 采用维甲酸诱导可促进核移植胚胎来源NT-ESCs分化为雄性生殖细胞.     

Nrf2通过调控EZH2的表达促进DMSO诱导的MEL细胞红系分化

目的 探讨Nrf2/EZH2通路参与DMSO诱导MEL细胞红系分化的作用及相关分子机制.方法 以MEL细胞为靶细胞,DMSO为刺激源,联苯胺染色法检测细胞红系分化情况;免疫印迹检测EZH2和Nrf2蛋白表达水平.结果 DMSO可显著诱导MEL细胞红系分化,同时EZH2和Nrf2表达水平也明显升高.通过使用Nrf2诱导剂TBHQ及Nrf2 shRNA,证明Nrf2调控MEL细胞中EZH2的表达.敲低Nrf2和EZH2的表达能够抑制DMSO诱导的MEL细胞红系分化.结论 Nrf2可通过调控EZH2的蛋白水平从而发挥促进MEL细胞红系分化的作用.     

生殖细胞核因子及Oct-4在P 19细胞系诱导分化中的表达

目的 体外培养未成熟畸胎瘤细胞（P19细胞）并诱导分化,检测生殖细胞核因子（GCNF）及Oct-4在P 19细胞诱导分化前后的表达。
方法 用全反式维甲酸（ATRA）诱导P 19细胞分化,通过间接免疫荧光染色和RT-PCR方法检测GCNF及Oct-4在P 19细胞诱导分化前后的表达。
结果 Oct-4在P 19细胞诱导前呈强阳性表达,诱导2 d后表达下降;而GCNF在P 19细胞诱导前呈阴性表达,诱导2 d后呈阳性表达。
结论 GCNF参与了恶性生殖细胞肿瘤的体外分化。GCNF可能是通过下调Oct-4基因的表达参与了恶性生殖细胞肿瘤的分化。     

全反式维甲酸诱导P19细胞系长非编码RNA Neat1基因表达

目的研究全反式维甲酸(atRA)诱导小鼠畸胎瘤P19细胞分化模型中LncRNA Neat1表达的变化,探究组蛋白修饰对其表达的影响。方法用含终浓度为0.5μmol/L atRA的培养基诱导P19细胞分化,检测神经分化标志物Mash1的mRNA表达;利用RT-q PCR检测在P19细胞神经分化过程中LncRNA Neat1的表达变化;利用组蛋白修饰调控因子抑制剂曲古柳菌素(TSA)、烟酰胺(NAM)、腺苷二醛(Ad Ox)处理P19细胞,观察对Neat1表达的影响。结果成功建立atRA诱导的P19神经分化模型,神经分化标志物Mash1在P19分化过程中表达上调;诱导过程中Neat1表达显著上调(P0.01),Ⅰ类和Ⅱ类去乙酰化酶抑制剂TSA可诱导Neat1表达,但Ⅲ类去乙酰化酶抑制剂NAM和甲基转移酶抑制剂Ad Ox对Neat1的表达无显著影响。结论全反式维甲酸诱导P19分化过程中,LncRNA Neat1表达显著上调,维持组蛋白高乙酰化状态的去乙酰化酶抑制剂TSA可参与促进Neat1的表达。     

定向诱导小鼠原始生殖细胞向肝细胞分化的研究

目的探索诱导小鼠原始生殖细胞向肝细胞定向分化的最佳条件。方法分离提取小鼠的原始生殖细胞，体外培养扩增后，接种到6孔培养板中。向不同孔中分别加入不同浓度的bFGF、EGF、β-NGF、RA、肝细胞提取液和与胎肝组织分隔培养，进行诱导分化。用靛青绿摄入试验和α-1-抗胰蛋白酶（AAT）、白蛋白（ALB）免疫细胞化学染色鉴定原始生殖细胞向肝细胞分化的状况。结果与胎肝组织分隔培养2d，可见原始生殖细胞向肝样细胞分化，分化细胞呈圆形和星形，圆形细胞多呈簇状，可以摄入靛青绿呈绿色，并呈AAT和ALB免疫阳性着色，培养12d时分化率达80％。肝细胞提取液也可诱导原始生殖细胞向肝样细胞分化，12d时分化率达70％以上；0．5g／L和1g／L的肝细胞提取液的诱导分化率无显著差异；但是0．5g／L提取液诱导组圆形细胞较多；而1g／L肝提取液诱导组，星状分化细胞多。β-神经生长因子（β-NGF）诱导4d，可见肝样细胞出现，随诱导时间延长，分化细胞增多，圆形分化细胞较多，12d时分化率达60％以上；20μg/L和50μg/L两种浓度的β-NGF诱导分化率无显著差异。bFGF、EGF、RA诱导组未见ALB免疫反应阳性细胞。RA可以增强β-NGF的诱导分化作用，使星状细胞增多明显。结论β-NGF和肝细胞因子可诱导原始生殖细胞向肝细胞定向分化。     

人诱导性多能干细胞定向分化为神经干细胞的实验研究

目的 观察人诱导性多能干细胞（iPS细胞）定向分化为神经干细胞（NSCs）的潜能。方法 用维甲酸（RA）诱导人iPS细胞向NSCs分化。倒置显微镜下观察iPS细胞的形态变化,RT-PCR检测NANOG, OCT4, SOX2的表达;免疫组织化学方法检测NESTIN、SOX2、β-TUBULIN Ш和GFAP的表达。结果RA诱导后第4天,贴壁的拟胚体出现了早期神经祖细胞特有的神经管样结构并不断增多,细胞表达神经巢蛋白NESTIN,而对照组未观察到神经管样结构。神经管样结构内的细胞能分化为β-TUBULIN Ш阳性的神经元,但GFAP阳性的星形胶质细胞少见。结论 人iPS细胞具有定向分化为NSCs的潜能,并能模拟神经发育过程。     

胚胎干细胞向生殖细胞分化的研究进展

近年来,人们探索如何在体外诱导胚胎干细胞获得配子,以了解性别决定的基因调控和环境影响.这些研究都是用胚胎干细胞（embryonic stem cell,ES）建立1个类原始生殖细胞（primordial germ cell-like cells）的细胞群体,然后移植到睾丸或继续培养从而获得雄性或雌性配子.原始生殖细胞的迁移和向生殖干细胞的分化依赖于自身基因表达以及与周围细胞的相互作用.本文将对原始生殖细胞分化为雄性生殖细胞（有少许为雌性生殖细胞）的关键环节、原始生殖细胞和生殖干细胞分化过程中的标志物做一综述.     

Oct-4和人端粒酶逆转录酶在不同胎龄人胚原始生殖细胞的表达变化

目的 检测不同胎龄人胚原始生殖细胞中Oct-4、人端粒酶逆转录酶(hTERT)的表达变化.方法 取人胚胎生殖嵴,通过RT-PCR检测5～13周龄人胚生殖嵴中Oct-4、hTERT的表达变化;同时,取人胚胎生殖嵴,经石蜡切片,HE染色、免疫组织化学染色等技术,观察不同胎龄人胚原始生殖细胞的形态,检测Oct-4、hTERT的表达情况.结果 胎龄6～7周时,生殖嵴中可见少量原始生殖细胞,且表达Oct-4及hTERT;胎龄8～12周时,生殖嵴中原始生殖细胞数量增多,Oct-4及hTERT表达增强(P<0.05);胎龄13周以后,生殖嵴中Oct-4阳性的原始生殖细胞数量逐渐减少,Oct-4表达减弱,但hTERT仍然高表达.结论 不同胎龄人胚原始生殖细胞中Oct-4的表达呈动态变化,胎龄8～12周时表达较强,但hTERT的表达量始终维持在较高水平,没有明显变化.     

EZH1 and EZH2 cogovern histone H3K27 trimethylation and are essential for hair follicle homeostasis and wound repair

Polycomb protein group (PcG)-dependent trimethylation on H3K27 (H3K27me3) regulates identity of embryonic stem cells (ESCs). How H3K27me3 governs adult SCs and tissue development is unclear. Here, we conditionally target H3K27 methyltransferases Ezh2 and Ezh1 to address their roles in mouse skin homeostasis. Postnatal phenotypes appear only in doubly targeted skin, where H3K27me3 is abolished, revealing functional redundancy in EZH1/2 proteins. Surprisingly, while Ezh1/2-null hair follicles (HFs) arrest morphogenesis and degenerate due to defective proliferation and increased apoptosis, epidermis hyperproliferates and survives engraftment. mRNA microarray studies reveal that, despite these striking phenotypic differences, similar genes are up-regulated in HF and epidermal Ezh1/2-null progenitors. Featured prominently are (1) PcG-controlled nonskin lineage genes, whose expression is still significantly lower than in native tissues, and (2) the PcG-regulated Ink4a/Inkb/Arf locus. Interestingly, when EZH1/2 are absent, even though Ink4a/Arf/Ink4b genes are fully activated in HF cells, they are only partially so in epidermal progenitors. Importantly, transduction of Ink4b/Ink4a/Arf shRNAs restores proliferation/survival of Ezh1/2-null HF progenitors in vitro, pointing toward the relevance of this locus to the observed HF phenotypes. Our findings reveal new insights into Polycomb-dependent tissue control, and provide a new twist to how different progenitors within one tissue respond to loss of H3K27me3.     

Nanos2 suppresses meiosis and promotes male germ cell differentiation

In mouse fetal gonads, retinoic acid (RA) induces meiosis in the female germ cells, whereas the male germ cells never enter meiosis due to Cyp26b1-mediated RA metabolism. We show here that Nanos2 plays critical roles in the differentiation of male germ cells. We find that Nanos2 maintains the suppression of meiosis by preventing Stra8 expression, which is required for premeiotic DNA replication, after Cyp26b1 is decreased. We also demonstrate that Nanos2 activates a male-specific genetic program, which is supported by the inhibition of meiosis and the induction of male-type differentiation in female germ cells following the forced expression of Nanos2.     

Stem cell based therapeutical approach of male infertility by teratocarcinoma derived germ cells

Infertility affects 13-18% of couples and growing evidence from clinical and epidemiological studies suggests an increasing incidence of male reproductive problems. There is a male factor involved in up to half of all infertile couples. The pathogenesis of male infertility can be reflected by defective spermatogenesis due to failure in germ cell proliferation and differentiation. We report here in vitro generation of a germ cell line (SSC1) from the pluripotent teratocarcinoma cells by a novel promoter-based sequential selection strategy and show that the SSC1 cell line form mature seminiferous tubule structures, and support spermatogenesis after transplantation into recipient testes. To select differentiated germ cell population, we generated a fusion construct (Stra8-EGFP) harbouring the 1.4 kb promoter region of germ line specific gene Stra8 and coding region of enhanced green fluorescence protein. This region was sufficient to direct gene expression to the germinal stem cells in testis of transgenic mice. The purified cells expressed the known molecular markers of spermatogonia Rbm, cyclin A2, Tex18, Stra8 and Dazl and the beta1- and alpha6-integrins characteristic of the stem cell fraction. This cell line undergoes meiosis and can develop into sperm when transplanted into germ cell depleted testicular tubules. Sperm were viable and functional, as shown by fertilization after intra-cytoplasmic injection into mouse oocytes. This approach provides the basis that is essential for studying the development and differentiation of male germ line stem cell, as well as for developing new approaches to reproductive engineering and infertility treatment.     

A key role for EZH2 and associated genes in mouse and human adult T-cell acute leukemia

In this study, we show the high frequency of spontaneous γδ T-cell leukemia (T-ALL) occurrence in mice with biallelic deletion of enhancer of zeste homolog 2 (Ezh2). Tumor cells show little residual H3K27 trimethylation marks compared with controls. EZH2 is a component of the PRC2 Polycomb group protein complex, which is associated with DNA methyltransferases. Using next-generation sequencing, we identify alteration in gene expression levels of EZH2 and acquired mutations in PRC2-associated genes (DNMT3A and JARID2) in human adult T-ALL. Together, these studies document that deregulation of EZH2 and associated genes leads to the development of mouse, and likely human, T-ALL.     

以转染白血病抑制因子基因的人胚肺成纤维细胞为饲养层培养人胚胎生殖细胞

目的 以转染白血病抑制因子(LIF)基因的人胚肺成纤维细胞为饲养层培养人胚胎生殖细胞,为建立无动物成分污染的人胚胎生殖细胞培养体系奠定基础.方法 将LIF真核表达载体pcDNA3.1( )-LIF转染到人胚肺成纤维细胞中,通过筛选和鉴定获得表达LIF的阳性细胞.将原始生殖细胞(PGCs)种植到转染后细胞制备而成的饲养层上,在不添加外源性LIF的条件下培养,并对PGCs来源的细胞集落进行鉴定.结果 经RT-PCR及Western blotting 鉴定证实,转染pcDNA3.1( )-LIF的人胚肺成纤维细胞表达LIF基因.在转染后人胚肺成纤维细胞上生长的PGCs可形成典型的鸟巢状集落,经检测集落碱性磷酸酶活性呈强阳性,表达胚胎阶段特异性抗原SSEA-1、SSEA-4、TRA-1-60、TRA-1-81,及未分化标志Oct-4.结论 用转染LIF基因后的人胚肺成纤维细胞作为饲养层能支持PGCs来源人胚胎生殖细胞的生长,维持自我更新.     

Gene expression in the axolotl germ line: Axdazl, Axvh, Axoct-4, and Axkit.

Primordial germ cells (PGCs) in embryos of mammals and urodele amphibians are formed by induction in the absence of germ plasm. We describe expression of four germ cell-related genes through the germ cell cycle of the axolotl. The orthologs of vasa and daz-like are up-regulated in PGCs of tail bud embryos before the gonad forms and are expressed throughout the female germ cell cycle. Mammalian Oct-4 is a marker of pluripotency in embryonic cells. Axolotl Oct-4 has higher homology to Oct-4 than that found in other vertebrates. It is expressed in the equivalent of the mouse epiblast, in the posterior mesoderm of late gastrulae that gives rise to PGCs, and in diplotene growing oocytes, but not in presumptive PGCs after gastrulation. Finally, a c-kit homolog is expressed in gonadal oogonia and growing oocytes as in mice but is also not found in PGCs. The expression pattern in urodele gonadal germ cells is similar to that of other vertebrates, although the pattern in pregonadal PGCs is distinctly different from that of mice. We conclude that PGCs are restricted to the germ line later in urodeles than in mice or lack migration and proliferation programs.     

叙利亚地鼠原始生殖细胞体外培养方法的初步研究

为建立叙利亚地鼠原始生殖细胞的体外培养方法．在体外无菌分离孕龄10．5d的叙利亚地鼠胚胎的生殖嵴，采用酶机械分离方法获取原始生殖细胞，在条件培养基中培养于饲养层细胞上，然后用酶组织化学染色方法鉴定碱性磷酸酶（ALP）的活性。去除分化抑制因素并诱导原始生殖细胞体外分化后．观察拟胚体和分化细胞的产生。实验结果显示孕龄10．5d的地鼠胚胎生殖嵴．用酶机械分离方法能成功地分离出具有发育多能性的原始生殖细胞，ALP染色鉴定呈强阳性，体外诱导分化后可观察到3种胚层来源的细胞。结果提示该细胞体外培养体系的建立，为生物医学工程学提供了一种新的细胞来源。