胚胎干细胞因子Nanog、Oct4、Sox2与多囊卵巢综合征的相关性

目的探讨颗粒细胞中胚胎干细胞因子Nanog、Oct4、Sox2与多囊卵巢综合征(PCOS)发生的关系。方法选择接受体外受精/卵胞质内单精子注射-胚胎移植术(IVF/ICSI-ET)治疗的PCOS患者40例,同期输卵管因素或男方因素不孕患者40例作为对照组。利用Real time-PCR技术检测颗粒细胞中Nanog、Oct4、Sox2以及抗苗勒管激素(AMH)mRNA的表达水平,并用Western blotting分析蛋白表达水平与PCOS发生的关系。结果 PCOS组体质量指数(BMI)[(26.44±3.46)kg/m^2]、黄体生成素(LH)[(7.56±0.98)IU/L]、LH/卵泡刺激素(FSH)(1.06±0.15)及获卵数(28.7±3.9)较对照组[(22.43±3.17)kg/m^2、(4.26±0.34)IU/L、0.57±0.04、15.1±2.0]有显著升高(P均<0.05)。Real time-PCR结果显示,PCOS组胚胎干细胞因子Nanog(0.60±0.09)和Oct4(0.85±0.14)mRNA表达水平显著低于对照组(1.65±0.17,1.59±0.17)(P<0.05),AMH转录水平(1.28±0.11)显著高于对照组(0.89±0.07)(P<0.05)。Logistic回归分析结果显示,Nanog是PCOS发生的危险因素(OR=26.577,P=0.002),且Nanog与BMI、获卵数及AMH呈显著负相关(r1=-0.415,P=0.023;r2=-0.415,P=0.022;r3=-0.373,P=0.043),与Oct4呈显著正相关(r=0.684,P<0.01)。Western blotting结果显示,与对照组比较,PCOS组Nanog蛋白表达降低,AMH蛋白表达水平增高。结论 PCOS卵巢颗粒细胞中胚胎干细胞因子Nanog以及AMH的异常表达可能参与了PCOS的发生并影响卵母细胞的发育与成熟,为PCOS的临床治疗提供理论依据。

干性相关分子Nanog在食管鳞状细胞癌组织中高表达并促进食管鳞癌细胞的侵袭转移:基于激活TGF-β信号通路

目的 探讨食管鳞状细胞癌(鳞癌)中干性相关分子胚胎干细胞关键因子(Nanog)和侵袭迁移相关分子基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)蛋白的表达及其之间的调控关系。方法 运用免疫组织化学技术检测127例食管鳞癌组织和82例癌旁正常组织中Nanog和MMP-2/MMP-9蛋白的表达情况,分析Nanog和MMP-2/MMP-9蛋白的表达水平、相关性及与食管鳞癌患者的临床病理参数和预后之间的关系;采用GEO数据库分析Nanog等干性相关分子主要富集通路;应用TIMER在线网站分析食管癌中TβR1和MMP-2及MMP-9的相关性。在体外运用siRNA转染的方式将食管鳞癌细胞株分为正常对照组、Nanog低表达组,采用划痕实验分析其对食管鳞癌细胞迁移的影响,qRT-PCR及Western blotting检测两组之间TβR1、p-Smad2/3、MMP-2/MMP-9的表达情况。结果 Nanog和MMP-2/MMP-9蛋白在食管鳞癌组织中表达均上调(χ^(2)=70.475,P<0.01;χ^(2)=34.415,P<0.01;χ^(2)=46.605,P<0.01),且呈正相关(r=0.205,P=0.045;r=0.307,P<0.001)。Nanog和MMP-2/MMP-9蛋白表达水平与食管鳞癌的浸润深度相关(χ^(2)=23.9,P<0.01;χ^(2)=6.029,P<0.05;χ^(2)=11.89,P<0.05),有淋巴结转移的样本中,MMP-2/MMP-9蛋白表达水平高于无淋巴结转移的样本(χ^(2)=10.08,P<0.01;χ^(2)=5.731,P<0.05)。MMP-2/MMP-9蛋白表达与食管鳞癌患者的年龄、性别和肿瘤分化程度无相关性(P>0.05)。Kaplan-Meier生存分析显示,Nanog和MMP-2/MMP-9蛋白高表达组的食管鳞癌患者生存时间短于低表达组(P<0.001;P=0.004;P=0.017);生物信息学分析显示,Nanog等干性相关分子主要富集在转化生长因子-β(TGF-β)信号通路,MMP-2/MMP-9与TβR1表达在食管癌中呈正相关关系。体外划痕实验结果显示,Nanog促进食管鳞癌细胞系的迁移;qRT-PCR及Western blotting结果显示,敲低Nanog后,TβR1、p-Smad2/3、MMP-2/MMP-9的表达水平明显降低。结论 Nanog和MMP-2/MMP-9蛋白在食管鳞癌患者组织中高表达且呈正相关,其与食管鳞癌患者的肿瘤浸润深度、淋巴结转移及预后密切相关,且Nanog通过TGF-β信号通路影响MMP-2/MMP-9蛋白的表达。

细胞生长因子联合诱导ES细胞向肾脏前体细胞分化的实验研究

目的:探讨细胞生长因子联合诱导胚胎干细胞(embryonic stem cell,ES细胞)向肾脏前体细胞定向分化的实验技术,为应用胚胎干细胞进行肾脏再生提供实验基础。方法:小鼠ES细胞悬浮培养2d制备成拟胚体,RA、activin-A、Bmp7三种生长因子诱导培养5d设为A组,不加生长因子培养设为对照(B组),生长因子培养5天后继续以肾脏上皮细胞生长培养液培养7d设为C组,免疫荧光检测Pax2、Bry、WT1蛋白表达情况,流式细胞技术检测Pax2、Bry、CD24阳性细胞比例,realtimePCR检测Pax2、Bry、Osr1、Lim1、WT1、Six2、Sall1、AQP1、CD24、PDGFR基因表达情况。结果:免疫荧光染色结果显示A组细胞Pax2、Bry、WT1的表达明显高于B组;流式细胞技术检测显示A组Pax2、Bry、CD24阳性细胞比例较B组增加,C组表达Pax2、Bry的阳性细胞比例较A组明显升高;realtimePCR检测显示A组Pax2、Bry、Osr1、Lim1、WT1表达较B组明显增加(P<0.05或P<0.01),C组表达CD24基因较A组明显升高(P<0.05)。结论:胚胎干细胞在体外生长因子联合诱导条件下能够分化为早期肾脏前体细胞,并表达部分成熟肾脏细胞的标记物。

雄激素受体和胚胎干细胞相关转录因子4在人表皮生长因子受体2过表达型乳腺癌中的表达及其临床意义

目的 探讨人表皮生长因子受体2（HER-2）过表达型乳腺癌中雄激素受体（AR）、胚胎干细胞相关转录因子4（NANOG）的表达情况,分析其相关性及其与患者临床病理特征的关系.方法 HER-2过表达型乳腺癌143例,采用免疫组织化学方法检测该类患者中AR、NANOG的表达.采用χ^2检验分析AR表达与患者临床病理特征的关系,采用Spearman等级相关分析AR表达和NANOG表达的相关性.结果143例HER-2过表达型乳腺癌患者中,AR阳性率为35.7%（51/143）,AR阳性率与年龄、月经状态无关（均P〉0.05）,与肿瘤最大径、TNM分期及淋巴结转移有关（均P〈0.05）.143例患者中NANOG阳性率为53.1%（76/143）,癌旁正常乳腺组织及良性乳腺病变组织不表达NANOG蛋白.NANOG阳性组的AR阳性率为27.6%（21/76）,NANOG阴性组为44.8%（30/67）,差异有统计学意义（χ^2=4.562,P=0.033）,Spearman等级相关分析提示,AR表达与NANOG表达呈负相关（r=-0.255,P=0.002）.结论 AR、NANOG有可能成为乳腺癌内分泌治疗和分子生物治疗的新靶标.

八聚体结合转录因子4和胚胎干细胞转录因子对胰腺癌干细胞体内干性特征的影响

目的 观察八聚体结合转录因子4（Oct4）和胚胎干细胞转录因子（Nanog）对胰腺癌干细胞（PCSCs）在体内干性特征的影响.方法 利用流式细胞分选技术分选人胰腺癌细胞-1（PANC-1）中CD44^＋ CD24^＋上皮特异性抗原（ESA）＋胰腺癌干细胞,通过慢病毒载体介导特异性短发卡RNA （shRNA）沉默胰腺癌干细胞中的Oct4、Nanog基因,并通过Western blot检测干扰效率;将Oct4、Nanog沉默的胰腺癌干细胞、未沉默的胰腺癌干细胞及胰腺癌PANC-1细胞株分别接种于BALB/c裸鼠皮下及腹腔,建立裸鼠异位移植瘤模型,观察沉默Oct4、Nanog对胰腺癌干细胞体内致瘤性、侵袭性及耐药性的影响.结果 PANC-1细胞株中CD44^＋ CD24^＋ ESA^＋胰腺癌干细胞占细胞总数的0.1％ ～0.8％;shRNA沉默Oct4和Nanog的效率分别是（46.00 ±0.08）％和（78.00±0.12）％;体内实验结果显示,沉默Oct4、Nanog基因后,裸鼠的致瘤性和肿瘤转移性显著下降,对吉西他滨耐药性减弱.结论 沉默Oct4、Nanog表达可抑制裸鼠体内胰腺癌干细胞的干性特征.

上皮性卵巢癌组织中Nanog、FOXD3 mRNA及蛋白的表达变化

目的观察上皮性卵巢癌组织中胚胎干细胞关键因子(Nanog)、叉头框转录因子D3(FOXD3)mRNA及蛋白的表达变化,并探讨其临床意义。方法将手术获取的上皮性卵巢癌组织40例份纳入恶性组,卵巢良性上皮性肿瘤组织26例份纳入良性组,正常卵巢组织25例份纳入对照组。采用qRT-PCR法检测三组中的Nanog、FOXD3mRNA,采用免疫组化SP法检测三组中的Nanog、FOXD3蛋白。分析Nanog、FOXD3蛋白表达与上皮性卵巢癌临床病理参数的关系,及上皮性卵巢癌组织中Nanog、FOXD3蛋白表达的相关性。结果恶性组Nanog、FOXD3 mRNA相对表达量高于良性组及对照组,且良性组FOXD3 mRNA相对表达量高于对照组(P均<0.05)。恶性组Nanog、FOXD3蛋白阳性表达率高于良性组及对照组,且良性组FOXD3蛋白阳性表达率高于对照组(P均<0.05)。Nanog蛋白表达与上皮性卵巢癌的临床分期、分化程度有关,其中Ⅲ、Ⅳ期组织中Nanog蛋白表达高于Ⅰ、Ⅱ期组织,低分化组织中Nanog蛋白表达高于中、高分化组织(P均<0.05)。上皮性卵巢癌组织中Nanog、FOXD3蛋白表达呈正相关(r=0.652,P<0.01)。结论上皮性卵巢癌组织中Nanog、FOXD3 mRNA及蛋白表达增高,Nanog、FOXD3可能参与了上皮性卵巢癌的发生、发展,且二者之间可能存在协同作用。

WWP2 promotes degradation of transcription factor OCT4 in human embryonic stem cells

POU transcription factor OCT4 not only plays an essential role in maintaining the pluripotent and self-renewing state of embryonic stem （ES） cells but also acts as a cell fate determinant through a gene dosage effect. However, the molecular mechanisms that control the intracellular OCT4 protein level remain elusive. Here, we report that human WWP2, an E3 ubiquitin （Ub）-protein ligase, interacts with OCT4 specifically through its WW domain and enhances Ub modification of OCT4 both in vitro and in vivo. We first demonstrated that endogenous OCT4 in hu- man ES cells can be post-translationally modified by Ub. Furthermore, we found that WWP2 promoted degradation of OCT4 through the 26S proteasome in a dosage-dependent manner, and the active site cysteine residue of WWP2 was required for both its enzymatic activity and proteolytic effect on OCT4. Remarkably, our data show that the en- dogenous OCT4 protein level was significantly elevated when WWP2 expression was downregulated by specific RNA interference （RNAi）, suggesting that WWP2 is an important regulator for maintaining a proper OCT4 protein level in human ES cells. Moreover, northern blot analysis showed that the WWP2 transcript was widely present in diverse human tissues/organs and highly expressed in undifferentiated human ES cells. However, its expression level was quickly decreased after human ES cells differentiated, indicating that WWP2 expression might be developmentally regulated. Our findings demonstrate that WWP2 is an important regulator of the OCT4 protein level in human ES cells.

Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media： distinct requirements for TGF-β and IGF-Ⅱ

A paracrine regulation was recently proposed in human embryonic stem cells （hESCs） grown in mouse embryonic fibroblast （MEF）-conditioned media （MEF-CM）, where hESCs spontaneously differentiate into autologous fibroblastlike cells to maintain culture homeostasis by producing TGF-β and insulin-like growth factor-lI （IGF-Ⅱ） in response to basic fibroblast growth factor （bFGF）. Although the importance of TGF-β family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-Ⅱ. In order to ease hESC cul- ture conditions and to reduce xenogenic components, we sought （i） to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts （HFFs） and human mesenchymal stem cells （hM- SCs） rather than MEF-CM, and （ii） to analyze whether the cooperation of bFGF with TGF-β and IGF-Ⅱ to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell （MSC）-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors （FGFR1-4） and specifically produce TGF-β in response to bFGF. However, HFFs but not MSCs secrete IGF-Ⅱ. Despite the absence of IGF-Ⅱ in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-Ⅱ may be dispensable for hESC pluripotency. In fact, IGF-Ⅱ blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-Ⅱ-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-β and IGF-Ⅱ in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.

Effect of electroacupuncture on oocyte quality and pregnancy of patients with PCOS undergoing in vitro fertilization and embryo transfer

Objective To observe the impacts of electroacupuncture （EA） on oocyte quality and pregnant outcome for the patients with polycystic ovary syndrome （PCOS） undergoing in vitro fertilization and embryo transfer （IVF-ET） and explore its potential mechanism. Methods Sixty-six patients with PCOS and undergoing IVF-ET were divided into two groups randomly, including an observation group （34 cases） and a control group （32 cases）. Ethinylestradiol and cyproterone acetate tablets and gonadotropin-releasing hormone agonist were administered for long-program superovulation in either group. In the observation group, the intervention of EA was applied to Guānyuán （关元 CV 4）, Zhōngjí （中极 CV 3）, Sānyīnjiāo （三阴交 SP 6）, Zǐgōng （子宫 EX-CA 1） and Tàixī （太溪 KI 3） additionally for 30 min, once daily, 1 menstrual cycle before controlled ovarian hyperstimulation （COH） and during COH. The pregnant outcome, evaluation of kidney deficiency syndrome, blood hormone level on the day of human chorionic gonadotropin injection and the concentrations of stem cell factor （SCF） in the serum and follicular fluid on the day of oocyte collection were compared between the two groups. Results The score of kidney deficiency symptoms was reduced remarkably after treatment in either group and the improvement in the observation group was superior to that in the control group （P0.01）. The fertilization rate [（76.25？±？20.33）% vs （66.34？±？15.44）%], cleavage rate [（98.66？±？3.70）% vs （94.47？±？9.45）%] and the rate of high-quality embryos [（60.20？±？22.20）% vs （50.55？±？16.15）%] in the observation group were all superior to those in the control group separately （all P0.05）. Clinical pregnancy rate （46.67%, 14/30） in the observation group was higher than that （37.93%, 11/29） in the control group, but without statistical difference （P0.05）. SCF concentrations in the serum and follicular fluid on the day of oocyte collection in the observation group were higher obviously than those in the control group （both P0.05）. Conclusion Electroacupuncture plays an active role in the pregnant outcomes of PCOS patients undergoing IVF-ET and it can relieve the symptoms of kidney deficiency in terms of TCM and improve clinical pregnant rate. The mechanism is relevant to the overall adjustment of organic endocrinal system and the local micro-environment of ovary and the improvement of oocyte quality through the up-regulation of SCF concentration.

Transposable elements at the center of the crossroads between embryogenesis, embryonic stem cells, reprogramming,and long non-coding RNAs

Transposable elements(TEs) are mobile genomic sequences of DNA capable of autonomous and nonautonomous duplication. TEs have been highly successful,and nearly half of the human genome now consists of various families of TEs. Originally thought to be non-functional,these elements have been co-opted by animal genomes to perform a variety of physiological functions ranging from TE-derived proteins acting directly in normal biological functions, to innovations in transcription factor logic and influence on epigenetic control of gene expression. During embryonic development, when the genome is epigenetically reprogrammed and DNA-demethylated, TEs are released from repression and show embryonic stage-specific expression, and in human and mouse embryos, intact TEderived endogenous viral particles can even be detected. Asimilar process occurs during the reprogramming of somatic cells to pluripotent cells: When the somatic DNA is demethylated, TEs are released from repression. In embryonic stem cells(ESCs), where DNA is hypomethylated, an elaborate system of epigenetic control is employed to suppress TEs, a system that often overlaps with normal epigenetic control of ESC gene expression. Finally, many long non-coding RNAs(lnc RNAs) involved in normal ESC function and those assisting or impairing reprogramming contain multiple TEs in their RNA. These TEs may act as regulatory units to recruit RNA-binding proteins and epigenetic modifiers. This review covers how TEs are interlinked with the epigenetic machinery and lnc RNAs, and how these links influence each other to modulate aspects of ESCs,embryogenesis, and somatic cell reprogramming.

Four recombinant pluripotency transcriptional factors containing a protein transduction domain maintained the in vitro pluripotency of chicken embryonic stem cells

Long-term in vitro maintenance of embryonic stem cell (ESC) pluripotency enables the pluripotency and differentiation of ESCs in animals to be investigated. The ability to successfully maintain and differentiate chicken embryonic stem cells (cESCs) would provide a useful tool for avian biology research and would be a resource directly applicable to agricultural production. In this study, endogenous chicken pluripotency transcription factors, POUV, Sox-2, Nanog and Lin28 were cloned and expressed as recombinant proteins containing a nine consecutive arginine protein transduction domain (PTD). cESCs were cultured with these recombinant proteins to maintain cESC pluripotency in vitro. Cultured cESCs exhibited typical characteristics of pluripotency, even after six generations of rapid doubling, including positive staining for stage-specific embryonic antigen I, and strong staining for alkaline phosphatase. Expression levels of the pluripotency markers, POUV, Nanog, C-Myc, Sox-2 and Lin28 were the same as in uncultured stage X blastoderm cells, and most significantly, the formation of embryoid bodies (EBs) by 6th generation cESCs confirmed the ability of these cultured cESCs to differentiate into cells of all three embryonic germ layers. Thus, transcription factors could be translocated through the cell membrane into the intracellular space of cESCs by using a PTD of nine consecutive arginines and the pluripotency of cESCs could be maintained in vitro for at least six generations.

Interplay of transcription factors and microRNAs during embryonic hematopoiesis

Hematopoietic stem cells (HSCs), which are localized in the bone marrow of adult mammals, come from hematopoietic endothelium during embryonic stages. Although the basic processes of HSC generation and differentiation have been described in the past, the epigenetic regulation of embryonic hematopoiesis remains to be fully described. Here, by utilizing an in vitro differentiation system of mouse embryonic stem cells (ESCs), we identified more than 20 microRNAs that were highly enriched in embryonic hematopoietic cells, including some (e.g. miR-10b, miR-15b, and miR-27a) with previously unknown functions in blood formation. Luciferase and gene expression assays further revealed combinational binding and regulation of these microRNAs by key transcription factors in blood cells. Finally, bioinformatics and functional analyses supported an interactive regulatory control between transcription factors and microRNAs in hematopoiesis.