精原干细胞介导的基因编辑动物模型研究进展

合适的动物模型对于人类疾病的研究和药物开发至关重要。精原干细胞是位于睾丸组织曲细精管基底膜上的一类具有自我更新和分化潜能的成体干细胞,可以定向分化产生精子。利用精原干细胞作为基因编辑的对象,生产基因编辑的精子进行受精有望成为建立基因编辑动物疾病模型的一条有效途径。该文就精原干细胞的生物学特征、体外培养以及精原干细胞介导的基因编辑动物模型的进展和优缺点进行了阐述。     

精原干细胞niche的研究进展

精原干细胞(spermatogonial stem cells,SSCs)是指睾丸内位于曲精细管基膜上既能自我更新维持自身适量恒定,又能定向分化产生精母细胞的一类原始精原细胞。随着干细胞深入的研究,人们发现了一种控制着干细胞可塑性与命运的微环境,此微环境被称为干细胞niche,干细胞niche由niche细胞、细胞外基质、细胞因子等构成。精原干细胞niche是由黏附因子、生长因子、支持细胞、间质细胞以及小管周肌肉细胞组成。大量的研究表明支持细胞在睾丸中是主要的成体细胞,通过分泌可溶性的因子来影响精原干细胞niche的结构与功能,同时支持细胞还能够间接的影响其他的成体细胞。随着年龄的增长使得精原干细胞niche的功能下降。精原干细胞数量以及精原干细胞niche为我们研究组织特异性干细胞生物学以及保持再生组织平衡提供了很宝贵的线索,精原干细胞对于保持组织的自我更新具有很重要的作用,并且受到人们大量的关注,然而精原干细胞niche也起到很重要的作用,它为治疗一些疾病提供新途径.本文将综述精原干细胞niche及其变化对精原干细胞功能调节的相关研究进展。     

BRCA-1 基因与神经干细胞

乳腺癌易感基因(Breast cancer susceptibility gene,Brca-1)是肿瘤抑制基因家族中的一员,它是乳腺癌特异性抑癌基因,1994年Miki等[1]采用定位克隆方法首次将Brca-1分离出来。Brca-1能防止细胞过快地或失去控制地生长和分化,在调节细胞进程、DNA损伤修复、细胞生长与凋亡及转录活化和抑制等多种生物学途径都发挥重要作用,Korhonen等2003年报道Brca-1基因可促进体外培养的大鼠来源的神经干细胞的增殖。     

非人灵长类动物精原干细胞研究进展

精原干细胞（spermatogonial stem cells,SSCs）是维持精子发生的一类干细胞,由于与人亲缘关系的相近性,使得开展非人灵长类动物SSCs研究具有重要的理论意义和比较医学价值。本文综述了非人灵长类动物SSCs的生物学特性、鉴定方法、冷冻保存、移植及生精细胞缺失模型建立等方面的研究进展。     

利用精原干细胞法生产转基因动物的研究进展

综述了利用精原干细胞生产转基因动物的发展历程、方法以及最新研究进展.重点从精原干细胞移植法和曲细精管微注射法两个方面,系统分析了利用精原干细胞生产转基因动物的优缺点,以及当前存在的问题.     

精原干细胞移植研究进展

精子发生 (ｓｐｅｒｍａｔｏｇｅｎｅｓｉｓ)是一个特殊的细胞分化过程。在这一过程中 ,精原干细胞 (ｓｐｅｒ ｍａｔｏｇｏｎｉａｌｓｔｅｍｃｅｌｌ)经过增殖分化形成具有特殊形态的精子。由于精子发生是在一个特定的环境中进行 ,缺乏体内外的研究模型 ,阻碍了这一领域的进展。 1994年开始的精原干细胞移植技术近几年取得了较大进展 ,已成为研究精子发生机理的重要模型。而且这一技术为治疗一些类型的雄性不育、保存某些需要化疗的癌症患者的生育能力以及转基因牲畜生产开辟了新的途径。本文全面综述精原干细胞移植的发展过程、最新进展和…     

Stra 8基因的激活与精原干细胞的特异性分化研究

视黄酸对维持正常的雄性睾丸结构和功能起着重要的作用。近来的研究发现,在雄性生殖腺发育过程中有一组基因,它们可以被视黄酸特异性的诱导活化,称为Stra(Stimulated by Retinoic Acid)基因。从鼠源分离得到的Stra8基因编码一种细胞质蛋白,该基因只特异性的在成熟雄性生殖细胞中表达,其功能被认为与精子形成有关。为研究Stra8基因的表达特性,我们从小鼠的基因组中克隆了Stra8基因的启动子序列(1.4kb)。将Stra8基因的1.4kb启动子序列克隆到pEGFP-1载体的EGFP基因之前,构建成由Stra8基因1.4kb启动子序列调控表达绿色荧光蛋白的pStra8-EGFP载体。将其分别转化到不同类型的细胞中,如小鼠ES-129细胞、人胎儿胰腺干细胞、小鼠骨髓间充质干细胞和小鼠精原干细胞等,通过荧光显微镜观察发现,绿色荧光蛋白只在小鼠精原干细胞中表达,表明Stra8基因是组织特异性表达的基因。将pStra8-EGFP转化小鼠骨髓间充质干细胞,经G418筛选2周后,用视黄酸诱导,12h培养后,有一部分转化pStra8-EGFP载体的细胞表达绿色荧光蛋白。RT-PCR证明这些细胞中有精原干细胞特异表达基因Stra8的转录,还有生殖细胞特异表达基因CyclinA8和Oct4的转录,这些结果说明小鼠骨髓间充质细胞经视黄酸的诱导可以向生殖细胞方向分化。     

逆转录病毒载体介导的LacZ基因在NIH-3T3细胞中的稳定表达

精原干细胞(SSCs)介导的转基因技术很可能成为制作转基因动物及治疗雄性不育的一条新途径。为了研究逆转录病毒载体介导法转染体外培养SSCs的可行性,用脂质体介导法将携带LacZ基因的重组逆转录病毒载体pLNCL导入包装细胞PA317,用含G418的培养液筛选得到5株稳定转染的产毒细胞。收集这些克隆的产毒上清,过滤后进行倍比稀释,用NIH-3T3细胞通过X-gal染色测定其浓缩前病毒滴度。结果显示,PA3173培养上清中病毒的浓缩前滴度最高,达1.1×103CFU/mL。再将筛选到的稳定转染的NIH-3T3细胞培养至单层,进行X-gal染色检测β-半乳糖苷酶的表达。结果显示,大多数稳定转染的NIH-3T3细胞均为X-gal ,表明这些细胞成功表达了目的基因LacZ。本研究结果为后期工作中用该载体感染体外培养SSCs奠定了基础。     

精原干细胞与睾丸生殖衰老的研究进展

雄性睾丸内精子的生成及其质量随年龄增长逐渐降低。精原干细胞是精子生成的起点,其数量和质量决定了精子的生成,而精原干细胞niche是调节精原干细胞自我更新与分化的重要因素。在衰老过程中,干细胞微环境退化,精原干细胞自我更新和分化失衡,被认为是衰老导致睾丸生殖功能衰退的的主要因素。本文将综述衰老引起的精原干细胞与niche变化及其对生殖的影响相关研究进展。     

Stem cell protein Piwil2 modulates expression of murine spermatogonial stem cell expressed genes

The piwi family genes are highly conserved during evolution and play essential roles in stem cell self-renewal, gametogenesis, and RNA interference in diverse organisms ranging from Arabidopsis to human. Piwil2, known also as Mili gene, is one of three mouse homologues of piwi. Piwil2 was found in germ cells of adult testis, suggesting that this gene functions in spermatogonial stem cell self-renewal. In order to find molecular mechanisms underlying stem cell activity mediated by Piwil2 gene, an in vitro gain of function cell culture model was established. Messenger RNAs isolated from cells expressing Piwil2 and mRNAs isolated from cells without Piwil2 expression were compared using a stem cell array technique. It was shown that Piwil2 modulates expression of stem cell specific genes, including platelet-derived growth factor receptor, beta polypeptide (Pdgfrb), solute carrier family 2 member 1 (Slc2a1), gap junction membrane channel protein alpha 7 (Gja7), and spermatogonial cell surface markers Thy-1 (CD90), integrin alpha 6 (Itga6), CD9, and spermatogonia specific markers heat shock protein 90 alpha (Hsp90a), and stimulated by retinoic acid gene 8 (Stra8). These molecules play essential role in stem cells proliferation (Pdgfrb), energy metabolism (Slc2a1), cell adhesion, cell-cell interaction (Itga6, Gja7, Thy-1, and CD9), and germ cell differentiation (Stra8). The expression of these markers in spermatogonial stem cells and other nongerminal stem cells suggests that these cells share elements of common molecular machinery with stem cells in other tissues which are modulated by stem cell protein Piwil2.     

植物干细胞决定基因WUS的研究进展

WUS(WUSCHEL)基因编码一转录因子,它的存在使周围细胞具有干细胞的特征,与之相关的信号系统近年逐步被阐明.在茎尖分生组织内WUS和CLV(CLAVATA)之间形成一个反馈调节环,使得干细胞保持自我更新,维持茎尖的顶端优势.在胚胎分生组织内,CLV3的表达只依赖于WUS的存在,然而在胚以后的发育中,CLV3的表达受到WUS和STM(SHOOTMERISTEMLESS)的双重调节,启动器官发生.在花分生组织中,WUS和LFY(LEAFY)共同激活AG(AGAMOUS)基因的表达,WUS受AG的反馈抑制.由WUS建立的信号体系还参与胚珠的发育.当WUS蛋白和生长素共存时,可以高效启动体细胞胚的发生.细胞对WUS信号的感应性与细胞所处的微环境有关,WUS在不同环境条件下可以启动不同的下游基因表达.     

鸡胚ESC和SSCs特定基因表达差异的研究

目的:探讨鸡胚胎干细胞(ESC)和精原干细胞(SSCs)在基因表达水平上的差异.方法:传至二代的ESC和SSCs,采用免疫荧光和碱性磷酸酶检测法联合鉴定其干细胞特性,RT-PCR方法检测两者相关基因的表达差异.结果:两种处于未分化状态时的干细胞基因表达存在差异:未分化的ESC表达基因GDF3基因和Nanog基因;SSCs表达特定基因c-kit、Cvh和Stra8基因.结论:两种干细胞在基因表达水平上有差异,为ESC与SSCs在基因水平上的鉴定提供参考依据.     

Efficient generation of transgenic chickens using the spermatogonial stem cells in vivo and ex vivo transfection

The highly efficient novel methods to produce transgenic chickens were established by directly in-jecting the recombinant plasmid containing green fluorescent protein (GFP) gene into the cock's testis termed as testis-medianted gene transfer (TMGT), and transplanting transfected spermatogonial stem cells (TTSSCs). For the TMGT approach,four dosages of pEGFP-N1 DNA/cationic polymer complex were injected intratesticularly. The results showed: (1) 48 h after the injection,the percentages of testis cells expressing GFP were 4.0%, 8.7%, 10.2% and 13.6% in the 50, 100, 150 and 200 μg/mL group, re-spectively. The difference from the four dosage groups was significant (P<0.05). On day 25 after the injection, a dosage-dependent and time-dependent increase in the number of transgenic sperm was observed. The percentages of gene expression reached the summit and became stable from day 70 to 160, being 12.7%, 12.8%, 15.9% and 19.1%, respectively. The difference from the four dosage groups was also significant (P<0.05). (2) 70 d after the injection, strong green fluorescent could be observed in the seminiferous tubules by whole-mount in-situ hybridization. (3) 70 d after the injection, the semen was collected and used to artificially inseminate wild-type females. The blastoderms of F1 and F2 transgenic chicken expressed GFP were 56.2% (254/452) and 53.2% (275/517), respectively. The detec-tion of polymerase chain reaction (PCR) of F1 and F2 transgenic chicken blood genomic DNA showed that 56.5% (3/23) of F1 and 52.9% (9/17) of F2 were positive. Southern blot showed GFP DNA was in-serted in their genomic DNAs. (4) Frozen whole mount tissue sections of F1 and F2 transgenic chicken liver, heart, kidney and muscle showed that the rates of green fluorescent positive were between 50.0% and 66.7%. (5) With the TTSSCs method, SSCs ex vivo transfected with GFP were transplanted into recipient roosters whose endogenic SSCs had been resoluted. The donor SSCs settled and GFP ex-pression became readily detectable in the frozen whole mount tissue sections of recepient testes. Moreover, sperms carrying GFP could be produced normally. The results of artificially inseminating wild-type females with these sperms showed 12.5% (8/64) of offspring embryo expressed GFP and 11.1% (2/18) hatched chicks were tested transgenic. Our data therefore suggest TMGT and TTSSCs are the feasible methods for the generation of transgenic chickens.     

浅谈精原干细胞研究进展

精原干细胞是雄性动物体内精子发生过程中起重要作用的精原细胞类型,不但具有干细胞特性,还能定向分化为雄性配子将自身基因传递给后代。除此之外,体外培养和鉴定精原干细胞为移植和转基因提供了基础。我们对精原干细胞的生物学特性、分离培养、鉴定、移植及精原干细胞介导的转基因进行简要概述。     

ERas基因在胃癌中的表达及其作用机制的研究进展

胚胎干细胞（EScells）为多能干细胞,来自哺乳动物胚胎早期。ES细胞表达的Ras（ERas）基因促进其体外增殖和肿瘤形成。该基因产物Ras蛋白关联和激活多个下游效应,调控多种细胞反应来参与细胞增殖,存活与分化。ERas基因位于x染色体短臂（Xpll．23）,其cDNA编码的蛋白包含227个氨基酸,与传统ras基因Hras,Kras和Nras分别有43％,46％和47％的相似性,故属于新的ras家族成员,与传统ras基因不同的是ERas基因非常活跃但不带有任何突变。近几年发现ERas基因的表达与胃癌密切相关,本文就ERas基因在人胃癌细胞和组织中的表达及其机制的最新进展做一综述,主要包括三个方面：1,ERas基因在胃癌细胞和组织中的表达情况及其功能。2,ERas基因在胃癌细胞中的表观遗传调控。3,ERas基因与胃癌肝和淋巴结转移的关系。     

E-cadherin as a novel surface marker of spermatogonial stem cells

Spermatogenesis is a fundamental biological process that ensures the transition of a gene from one ganeration to another via male gametes. This process relies on a rare population of testicular cells called spermatogonial stem cells (SSCs), which self-renew throughout adult male life and differentiate into mature gametes. Despite the longstanding study of SSCs, their biological properties remain largely unknown, which is partly due to the very limited availability of these cells. Here, we show that cell adhesion protein E-cadherin is a highly specific surface marker of mouse SSCs that can be successfully used to enrich them.     

Gene delivery to embryonic stem cells

Since the establishment of embryonic stem (ES) cells and the identification of tissue-specific stem cells, researchers have made great strides in the analysis of the natural biology of such stem cells for the development of therapeutic applications. Specifically, ES cells are capable of differentiating into all of the cell types that constitute the whole body. Thus, ES cell research promises new type of treatments and possible cures for a variety of debilitating diseases and injuries. The potential medical benefits obtained from stem cell technology are compelling and stem cell research sees a bright future. Control of the growth and differentiation of stem cells is a critical tool in the fields of regenerative medicine, tissue engineering, drug discovery, and toxicity testing. Toward such a goal, we present here an overview of gene delivery in ES cells, covering the following topics: significance of gene delivery in ES cells, stable versus transient gene delivery, cytotoxicity, suspension versus adherent cells, expertise, time, cost, viral vectors for gene transduction (lentiviruses, adenoviruses, and adeno-associated viruses, chemical methods for gene delivery, and mechanical or physical gene delivery methods (electroporation, nucleofection, microinjection, and nuclear transfer).