DPPA2基因的研究进展

DPPA2（Developmental pluripotency．associatedgene2）是近年来发现的一种在多能性细胞和某些癌组织中特意表达的基因。它与早期胚胎发育密切相关,参与维持胚胎干细胞的多能性及自我更新,还在体细胞重编程为多能性诱导干细胞的过程中发挥了作用。此外,它还是一种新的肿瘤抗原,有望成为某些恶性肿瘤的特异性免疫治疗新靶点。本文就DPPA2的结构、功能,以及它与胚胎发育、恶性肿瘤、体细胞重编程的关系等方面的研究进展做一综述。     

microRNAs和体细胞重编程

体细胞重编程与microRNAs(miRNAs)均为近年来研究的热点问题。到目前为止,能成功诱导体细胞形成多能性干细胞的体细胞重编程方法有核移植(nuclear transfer,NT)和外源因子诱导形成多能干细胞(induced pluripotent stem cells,iPSc)两种,这两种方法让人们看到了体细胞重编程在细胞治疗方面具有诱人的应用前景。miRNAs是真核生物中存在的一类长度为22nt左右起调控作用的内源性非编码RNA,它在转录后水平调节靶基因的表达,是细胞内基因表达的基本调控机制之一。近年的研究结果表明,miRNAs在干细胞干性维持和分化过程中具有重要的调节作用,从miRNAs角度研究体细胞重编程机理将对体细胞重编程的应用具有重要意义。     

微核糖核酸簇miR-290-295促进体细胞重编程

目的：探究干细胞中表达丰度最高的微核糖核酸簇miR-290-295对体细胞重编程的影响。方法：使用逆转录病毒载体将miR-290-295簇在小鼠体细胞中过表达,研究其促进体细胞重编程为诱导性多能干细胞(induced pluripotent stem cells, iPSCs)以及此过程对细胞功能的影响。结果：miR-290-295簇的过表达在三因子(Sox2、Klf4、Oct4)诱导体系中能够显著提高小鼠体细胞重编程的效率;过表达miR-290-295簇能够促进重编程中多能性标记基因的上调与体细胞标记基因的下调,同时也会促进间质-上皮细胞转化(mesenchymal-epithelial transition, MET)标记基因的表达。结论：miR-290-295簇对小鼠体细胞重编程具有促进作用,这有助于深入理解干细胞多能性和重编程的RNA调节机制,为开发新型诱导体系提供了新视角。     

哺乳动物DNA甲基化及其在体细胞诱导重编程中的作用

诱导多能干细胞(Induced pluripotent stem cell, iPS)技术提供了将终末分化的细胞逆转为多潜能干细胞的可能, 在干细胞基础理论研究和再生医学中具有重要意义。然而, 目前体细胞诱导重编程方法效率极低, 常发生不完全的重编程。研究表明, 在不完全重编程的细胞中存在体细胞的表观遗传记忆, 而DNA甲基化作为相对长期和稳定的表观遗传修饰, 是影响重编程效率和iPS细胞分化能力的重要因素之一。哺乳动物DNA甲基化是指胞嘧啶第五位碳原子上的甲基化修饰, 常发生于CpG位点。DNA甲基化能够调节体细胞特异基因和多能性基因的表达, 因此其在哺乳动物基因调控、胚胎发育和细胞重编程过程中发挥着重要作用。此外, 异常DNA甲基化可能导致iPS细胞基因印记的异常和X染色体的失活。文章重点围绕DNA甲基化的机制、分布特点、及其在体细胞诱导重编程中的作用进行了综述。     

MiR-302/367在体细胞向多能性干细胞重编程中的研究

MicroRNA-302/367(miR-302/367)发现于2003年,是一类长度在21～22 nt的miRNA簇,与多能性干细胞自我更新及多向分化有重要关系.在体细胞向多能性干细胞重编程中具有重要作用. miR-302/367簇中各miRNA具有相对保守的种子区及靶基因,主要通过抑制靶基因蛋白质的翻译,从而促进间质-上皮转化（mesenchymal epithelial transition, MET）、抑制细胞周期、调控细胞分化相关基因及表观遗传水平等方式促进体细胞向多能性细胞重编程.本文对miR 302/367的发现、结构、miR 302/367在多能性细胞中的作用及在体细胞向多能性干细胞重编程中的作用及其机理等做一综述.     

细胞融合致体细胞重编程机制研究进展

已分化的体细胞能够通过重编程转化回多能干细胞,在细胞移植、疾病细胞模型的制备以及药物筛选等领域具有重要意义。通过干细胞和体细胞的细胞融合,可使体细胞重编程。细胞融合致体细胞重编程速度快、效率高,是一种研究重编程机制的重要手段。对细胞融合致体细胞重编程的机制作一综述。     

MBD3调节干细胞多能性和重编程机制的研究进展

MBD3(methyl CpG binding domain 3)是甲基CpG结合域蛋白家族的成员之一,也是NuRD(nucleosome remodeling and deacetylase complex)的核心亚单位之一。MBD3蛋白可以结合非甲基化DNA,通过MBD蛋白结构域或与NuRD结合发挥作用。MBD3通过参与调节染色质结构和激活转录过程,调节胚胎干细胞的多能性和谱系分化,对于胚胎发育和分化十分关键。MBD3在体细胞和神经干细胞重编程中也发挥着重要作用。此外,在缺氧环境下MBD3还能影响细胞代谢调控。该文围绕MBD3诱导DNA去甲基化、调节染色质结构、调控转录、调节胚胎干细胞的多能性和谱系分化、在重编程中的作用以及缺氧环境中的对细胞代谢的影响等展开论述,以期为多能干细胞的表观遗传研究及重编程技术的优化提供参考。     

卵母细胞裂解液逆转化体细胞为多能干细胞的研究进展

采用卵母细胞裂解液重编程体细胞为多能干细胞是体细胞重编程的一个新思路,该方法主要是通过体细胞与卵母细胞裂解液的共孵育,使得体细胞在形态、基因和功能等方面发生改变,趋向于胚胎干细胞的方向发展。就卵母细胞裂解液重编程体细胞的研究现状、影响因素及存在的问题进行综述。     

iPSCs遗传稳定性与重编程机制的研究进展

诱导性多能干细胞(Induced pluripotent stem cells, iPSCs)是采用特定转录因子,将体细胞重编程为具有多能性的干细胞。iPSCs已成功由多种体细胞诱导出来,不仅具有发育多能性还能避免胚胎干细胞(Embryonic stem cells, ESCs)的伦理道德问题,已成为生命科学领域不可或缺的研究工具,具有广阔的应用前景。但获得高质量、遗传稳定的iPSCs是当前亟须解决的问题。文章对iPSCs重编程机制和遗传稳定性的研究进展进行了综述,以期为提高iPSCs的诱导效率、降低诱导成本、掌握iPSCs质量控制的关键点提供参考,从而推进多能性干细胞临床应用的发展。     

细胞提取物介导的体细胞重编程

将完全分化的细胞重编程,不经胚胎阶段而直接逆转至多能干细胞状态,这从法律.道德,伦理等方面均被人们所接受,重新点燃了人们对体细胞重编程的热情,点燃了再生医学研究的新希望.现重点阐述细胞提取物介导的体细胞重编程的原理及其应用前景,并详细介绍体细胞重编程的最新方法:细胞核移植入卵母细胞:体细胞与胚胎干细胞或胚胎癌细胞融合;在体细胞中强制性过表达特定的转录因子;用卵细胞、胚胎干细胞或多能癌细胞的细胞提取物处理体细胞等.     

Alteration in genes expression patterns during in vitro differentiation of mouse spermatogonial cells into neuroepithelial-like cells

Pluripotent stem cells derived from testis is a new, natural, and unlimited source for cell therapy in regenerative medicine and represent a possible alternative to replacing of all cells in the body. Here, we designed a simple co-culture system of spermatogonia cells with Sertoli cells for the generation of embryonic stem-like cells from mouse testis. The importance of our simple method will be clear when we compared it with other complex and time-consuming methods. Embryonic stem-like colonies with sharp border confirmed by real-time PCR, immunocytochemistry and flow cytometry assessments. Embryonic stem-like colonies were immunopositive for pluripotency markers. Transition of spermatogonia cells to embryonic stem-like cells was accompanied by extensive changes in gene expression. These changes included significant increase in pluripotency genes expression and significant decrease in germ cell-specific genes expression. Also, we proved the differentiation capacity of embryonic stem-like cells to neuroepithelial-like cells which were immunoreactive to Nestin and Neurofilament 68. Evaluation of genes expression during in vitro differentiation into neuroepithelial-like cells showed high-level expression of Nestin whether this gene approximately has no expression in undifferentiated embryonic stem-like cells. Also, expression of pluripotency genes has significantly decreased in neuroepithelial-like cells compared with embryonic stem-like cells. This study shows that embryonic stem-like cells derived from testis are capable to differentiate into neuroepithelial-like cells that may provide a cellular reservoir usable for neurodegenerative disorders.     

Restricted expression of LUZP in neural lineage cells: A study in embryonic stem cells

A novel protein LUZP with 3 leucine zipper motifs at its amino terminus is predominantly expressed in the adult brain. A modified gene targeting approach was employed in an attempt to establish in vitro and in vivo models in which Luzp is knock-out (KO) for phenotype assessment and a reporter gene lacZ is knock-in (KI) for tracing its expression. We report in this study the molecular cloning of the Luzp gene, its targeting vector construction and Luzp-KO/lacZ-KI embryonic stem (ES) clone selection. Since LUZP is also expressed in ES cells, the possibility of embryonic lethality cannot be excluded when attempting to establish Luzp-null mutant mice. We have therefore examined the development of homozygous Luzp-KO/lacZ-KI clones in nude mice. Tissue types derived from all three embryonic germ layers were observed in teratomas developed in nude mice. In situ X-gal staining further revealed restricted expression of LUZP in neural lineage cells.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

小鼠胚胎干细胞的培养

目的:建立小鼠胚胎干细胞(embryonic stem cells,ES)的培养方法。方法:制备G418抗性的原代小鼠胚胎成纤维细胞,经丝裂霉素C处理后成滋养层细胞,将小鼠胚胎干细胞复苏后,应用含白血病抑制因子的ES细胞培养液,培养小鼠ES细胞,观察集落的生长情况,并在光镜下观察细胞形态。结果:小鼠胚胎成纤维细胞生长良好,ES细胞呈克隆状生长,且保持未分化状态。结论:建立了小鼠胚胎干细胞培养的有效方法,为下一步基因打靶奠定基础。     

小鼠胚胎干细胞建系技术研究进展

目前,对小鼠胚胎干细胞的研究较为深入,并已成为研究细胞分化及信号转导、新基因发现及功能鉴定、器官发生、人类疾病和药物开发等的有效手段。胚胎干细胞建系是一项基础性工作。虽然技术日趋成熟,有些品系小鼠的胚胎干细胞建系已是常规技术,但不同品系小鼠胚胎干细胞的建系效率仍有很大差异,建系途径和方法各有特点,一个品系胚胎干细胞的建系方法不一定都适用于其他品系。本文从小鼠胚胎干细胞建系的途径、分离操作技术、培养体系等方面进行综述,并就与之相关的有些问题提出思考和对策。