NuMA和γ-tubulin在核移植克隆胚胎中的动态变化和作用

核有丝分裂器蛋白(NuMA)和γ-微管蛋白(γ-tubulin)在卵母细胞成熟、受精和核移植胚胎早期发育过程中起重要作用。供体细胞γ-tubulin和NuMA参与了核移植克隆胚有丝分裂纺锤体的组装,NuMA主要参与了纺锤体极的装配和维持,而供体中心体调节核移植胚最初纺锤体的形成。核移植重构胚中心体结构异常经常伴随染色体异常排列、纺锤体结构异常,而中心体分布异常经常伴随异常卵裂。     

有丝分裂纺锤体及中间小体提取方法的研究

目的 有丝分裂纺锤体和中间小体均是以在细胞有丝分裂进程中出现的微管为结构基础的暂时性结构,对细胞有丝分裂的顺利进行起着至关重要的作用.本研究旨在建立成熟高效的提取细胞内有丝分裂纺锤体和中间小体的方法.方法 通过细胞周期同步化方法使细胞内出现有丝分裂纺锤体或中间小体,运用低渗肿胀和甘油梯度离心的原理提取纺锤体和中间小体.结果 经Western Blot和细胞免疫荧光染色法鉴定,证实提取物中含有有丝分裂纺锤体和中间小体.结论 建立从人宫颈癌细胞Hela中提取有丝分裂纺锤体及中间小体的方法,可为鉴定有丝分裂纺锤体及中间小体上的蛋白分子垫定实验基础,同时也对研究肿瘤细胞有丝分裂的分子调控机制具有重要意义.     

KIF11在恶性肿瘤发生、发展中的研究进展

驱动蛋白超家族(kinesin family, KIF)是存在于真核生物中一类沿着微管网络运输货物的分子马达，主要参与细胞分裂、胞内囊泡及细胞器的转运、微管细胞骨架重组等一系列细胞内活动。根据结构域和序列同源性将其分为14个亚家族(Kinesin1～14),其中Kinesin5亚家族成员KIF11(又称Eg5、KSP)为纺锤体驱动蛋白，是双极纺锤体的重要组成部分，在中心体分离和双极有丝分裂纺锤体形成中发挥重要作用。研究表明KIF11表达上调可通过多种信号通路导致中心体碎裂、染色体不稳定，从而使细胞分裂增殖旺盛，促进肺癌、胃癌、胰腺癌、结直肠癌、卵巢癌等多种肿瘤的发生、发展。该文现就KIF11在恶性肿瘤发生、发展中的作用作一综述，旨在为进一步分析KIF11提供线索和方向。     

Aurora-A在细胞中的功能以及与肿瘤的关系

Aurora蛋白激酶家族，在细胞的有丝分裂期发挥着重要作用。目前分为3种即Aurora-A、Aurom-B、Aurora-C。其中Aurora-A定位于中心体和纺锤体，主要参与中心体的成熟、分离和纺锤体形成。此外Aurora-A可以参与p53通路的调节，细胞凋亡和分裂之间平衡的调节等。Aurom-A高表达可引起中心体异常扩增，异倍体产生。在肿瘤组织中的研究显示它在多种肿瘤乳腺癌、卵巢癌、结肠癌、胃癌等组织中存在着高表达，并且与某些肿瘤的预后相关。Aurom-A可能通过多种机制参与了肿瘤的发生和发展。     

Aurora-A在细胞中的功能以及与肿瘤的关系

Aurora蛋白激酶家族,在细胞的有丝分裂期发挥着重要作用。目前分为3种即Aurora-A、Au-rora-B、Aurora-C。其中Aurora-A定位于中心体和纺锤体,主要参与中心体的成熟、分离和纺锤体形成。此外Aurora-A可以参与p53通路的调节,细胞凋亡和分裂之间平衡的调节等。Aurora-A高表达可引起中心体异常扩增,异倍体产生。在肿瘤组织中的研究显示它在多种肿瘤乳腺癌、卵巢癌、结肠癌、胃癌等组织中存在着高表达,并且与某些肿瘤的预后相关。Aurora-A可能通过多种机制参与了肿瘤的发生和发展。     

NuSAP的生物学特点及其与肿瘤的关系

核仁纺锤体相关蛋白(nucleolar spindle-associated protein,NuSAP)是一种微管结合蛋白,在微管聚集、纺锤体组装和染色体分离过程中发挥重要作用,为细胞分裂所必须.近年来,NuSAP备受关注,多组独立研究表明其在胚胎形成和肿瘤发生过程中起了重要作用.文章将对目前涵盖NuSAP的基础研究和肿瘤相关研究作一综述,旨在整合NuSAP生物学特点的信息,探讨其在临床应用前景,并就其是否具备作为肿瘤标志物的潜力进行讨论.     

小鼠卵母细胞减数分裂过程中新型微管蛋白ε-tubulin稳定表达并与纺锤体的组装和维持相关

目的研究新型微管蛋白ε-tubulin在小鼠卵母细胞减数分裂进程中的蛋白表达、亚细胞定位及其与纺锤体的相关性。方法用Western blot法检测ε-tubulin在小鼠卵母细胞减数分裂各个时期的蛋白表达;免疫荧光染色分析ε-tubulin在减数分裂进程中的亚细胞定位模式及其与纺锤体的相关性。结果ε-tubulin在小鼠卵母细胞减数分裂各个时期均有稳定的蛋白表达,在减数分裂前期均匀分布在细胞核中,在生发泡破裂后直到第二次减数分裂中期始终保持与纺锤体微管的共定位特性,并持续存在于染色体上。ε-tubulin对纺锤体毒性药物Taxol和Nocodazole敏感并呈现出与微管一致的反应性。结论小鼠卵母细胞内新型微管蛋白ε-tubulin稳定表达并与纺锤体的组装和维持相关。     

核仁纺锤体相关蛋白在恶性肿瘤中作用的研究进展

核仁纺锤体相关蛋白(nucleolar spindle-associated protein, NuSAP)/NuSAP1是一种微管相关蛋白,具有维持着丝粒蛋白稳定性、调节纺锤体正确组装及染色体分离的作用。目前许多研究报道显示NuSAP过表达与结肠癌、肝癌、乳腺癌等多个恶性肿瘤的预后不良相关,并有望成为新的标志物及治疗靶点。该文将其结构、功能及在恶性肿瘤中作用的研究情况作一综述。     

Stathmin调控及与疾病的关系

Stathmin是细胞内重要的细胞周期相关的微管作用蛋白。在有丝分裂间期,Stathmin以有活性的去磷酸化形式存在,抑制微管聚合。当细胞进入分裂期,Stathmin被磷酸化失活,微管聚合形成纺锤体,进而染色体分离、细胞分裂。Stathmin的表达和活性受多种转录因子和激酶/磷酸化酶的调控,其表达和活性异常与细胞增殖、神经系统发育及肿瘤等病理生理过程密切相关。     

细胞凋亡与真核细胞骨架蛋白相关性的研究进展

真核细胞骨架是真核细胞中的蛋白质纤维网状结构,主要由微丝、微管以及中间纤维三种蛋白纤维组成。其中,微丝在细胞分裂时,参与纺锤体的组装和定位,微管的动态不稳定性则影响着细胞的复制和凋亡,中间纤维形态多样,具有丰富的结构和功能。此外,神经细胞应激与细胞骨架蛋白的研究也逐渐成为解决创伤后应激障碍(PTSD),抑郁症等相关精神疾病的基础。     

Derivation of cloned human blastocysts by histone deacetylase inhibitor treatment after somatic cell nuclear transfer with β-thalassemia fibroblasts

Derivation of embryonic stem cells from patient-specific cloned blastocysts by somatic cell nuclear transfer (SCNT) holds promise for both regenerative medicine and cell-based drug discovery. However, the efficiency of blastocyst formation after human SCNT is very low. The developmental competence of SCNT embryos has been previously demonstrated in several species to be enhanced by treatment with histone deacetylase inhibitors, such as trichostatin A (TSA), to increase histone acetylation. In this study, we report that treatment of SCNT embryos with 5 nM TSA for 10 h following activation incubation increased the developmental competence of human SCNT embryos constructed from β-thalassemia fibroblast cells. The efficiency of blastocyst formation from SCNT human embryos treated with TSA was approximately 2 times greater than that from untreated embryos. Cloned blastocysts were confirmed to be generated through SCNT by DNA and mitochondrial DNA fingerprinting analyses. Further, treatment of SCNT embryos with TSA improved the acetylation of histone H3 at lysine 9 in a manner similar to that observed in in vitro fertilized embryos.     

A comparative approach to somatic cell nuclear transfer in the rhesus monkey

BACKGROUND: Despite the potential utility of primate somatic cell nuclear transfer (SCNT) to biomedical research and to the production of autologous embryonic stem (ES) cells for cell- or tissue-based therapy, a reliable method for SCNT is not yet available. Employing the rhesus monkey as a clinically relevant animal model, we have compared a conventional electrofusion method for SCNT with a one-step micromanipulation (OSM) method. METHODS: A prospective, randomized trial was conducted using only oocytes that were mature [metaphase II (MII)] at collection and a fibroblast-like cell line as nuclear donor cells (fetal fibroblasts). The embryos produced were characterized for in vitro developmental potential, cell number, karyotype and expression of nuclear mitotic apparatus (NuMA) and OCT-4. RESULTS: An in vitro blastocyst development rate of 24.4% was achieved with the OSM method, significantly higher than the 12.2% obtained following electrofusion. SCNT-produced embryos expressed normal karyotypes, cell numbers and NuMA and OCT-4 proteins in most cases. SCNT with male nuclear donor cells resulted in the production of male, SCNT blastocysts, eliminating the possibility of a parthenogenetic origin. Of the four fibroblast cell lines tested as nuclear donor cells, two supported the routine production of blastocysts following SCNT. CONCLUSIONS: The application of a modified SCNT technique (OSM) followed by embryo culture in hamster embryo culture medium-10 (HECM-10) allows, for the first time, the routine production of SCNT blastocysts, most of which appear normal by immunochemical, cytochemical and in vitro developmental criteria. These embryos will provide a resource for isolating ES cells and for studies of nuclear reprogramming by monkey cytoplasts.     

Reprogramming following somatic cell nuclear transfer in primates is dependent upon nuclear remodeling

BACKGROUND: Somatic cell nuclear transfer (SCNT) requires cytoplast-mediated reprogramming of the donor nucleus. Cytoplast factors such as maturation promoting factor are implicated based on their involvement in nuclear envelope breakdown (NEBD) and premature chromosome condensation (PCC). Given prior difficulties in SCNT in primates using conventional protocols, we hypothesized that the ability of cytoplasts to induce nuclear remodeling was instrumental in efficient reprogramming. METHODS: NEBD and PCC in monkey (Macaca mulatta) SCNT embryos were monitored by lamin A/C immunolabeling. RESULTS: Initially, a persistent lamin A/C signal from donor cell nuclei after fusion with cytoplasts was observed indicative of incomplete NEBD following SCNT and predictive of developmental arrest. We then identified fluorochrome-assisted enucleation and donor cell electrofusion as likely candidates for inducing premature cytoplast activation and a consequent lack of nuclear remodeling. Modified protocols designed to prevent premature cytoplast activation during SCNT showed robust NEBD and PCC. Coincidently, over 20% of SCNT embryos reconstructed with fetal fibroblasts progressed to blastocysts. Similar results were obtained with other somatic cells. Reconstructed blastocysts displayed patterns of Oct-4 expression similar to fertilized embryos reflecting successful reprogramming. CONCLUSIONS: Our results represent a significant breakthrough in elucidating the role of nuclear remodeling events in reprogramming following SCNT.     

Telomere Elongation Facilitated by Trichostatin A in Cloned Embryos and Pigs by Somatic Cell Nuclear Transfer

Telomere attrition and genomic instability are associated with organism aging. Concerns still exist regarding telomere length resetting in cloned embryos and ntES cells, and possibilities of premature aging of cloned animals achieved by somatic cell nuclear transfer (SCNT). Trichostatin A (TSA), a histone deacetylase inhibitor, effectively improves the developmental competence of cloned embryos and animals, and recently contributes to successful generation of human ntES cells by SCNT. To test the function of TSA on resetting telomere length, we analyzed telomeres in cloned blastocysts and pigs following treatment of SCNT embryos with TSA. Here, we show that telomeres of cloned pigs generated by standard SCNT methods are not effectively restored, compared with those of donor cells, however TSA significantly increases telomere lengths in cloned pigs. Telomeres elongate in cloned porcine embryos during early cleavage from one-cell to four-cell stages. Notably, TSA facilitates telomere lengthening of cloned embryos mainly at morula-blastocyst stages. Knockdown of pTert by shRNA in donor cells reduces telomerase activity in cloned blastocysts but does not abrogate telomere elongation in the TSA-treated embryos (p?>?0.05). However, genes associated with recombination or telomerase-independent mechanism of alternative lengthening of telomeres (ALT) Rad50 and BLM show increased expression in TSA-treated embryos. These data suggest that TSA may promote telomere elongation of cloned porcine embryos by ALT. Together, TSA can elongate telomeres in cloned embryos and piglets, and this could be one of the mechanisms underlying improved development of cloned embryos and animals treated with TSA.     

Development of human cloned blastocysts following somatic cell nuclear transfer with adult fibroblasts

Nuclear transfer stem cells hold considerable promise in the field of regenerative medicine and cell-based drug discovery. In this study, a total of 29 oocytes were obtained from three young (20-24 years old) reproductive egg donors who had been successful in previous cycles. These oocytes, deemed by intended parents to be in excess of their reproductive needs, were donated for research without financial compensation by both the egg donor and intended parents after receiving informed consent. All intended parents successfully achieved ongoing pregnancies with the oocytes retained for reproductive purposes. Mature oocytes, obtained within 2 hours following transvaginal aspiration, were enucleated using one of two methods, extrusion or aspiration, after 45 minutes of incubation in cytochalasin B. Rates of oocyte lysis or degeneration did not differ between the two methods. Somatic cell nuclear transfer (SCNT) embryos were constructed using two established adult male fibroblast lines of normal karyotype. High rates of pronuclear formation (66%), early cleavage (47%), and blastocyst (23%) development were observed following incubation in standard in vitro fertilization culture media. One cloned blastocyst was confirmed by DNA and mitochondrial DNA fingerprinting analyses, and DNA fingerprinting of two other cloned blastocysts indicated that they were also generated by SCNT. Blastocysts were also obtained from a limited number of parthenogenetically activated oocytes. This study demonstrates, for the first time, that SCNT can produce human blastocyst-stage embryos using nuclei obtained from differentiated adult cells and provides new information on methods that may be needed for a higher level of efficiency for human nuclear transfer.     

不同类型的转基因细胞核供体对生产小鼠转基因克隆胚胎的影响

目的 利用体细胞核移植(SCNT)技术生产小鼠转基因克隆胚胎. 方法 利用所构建的含新霉素抗性(Neor)基因和增强型绿色荧光蛋白(EGFP)基因的双标记选择载体, 通过电穿孔的方法, 分别转染小鼠胎儿成纤维细胞和小鼠胚胎干细胞,经G418筛选14d后用于核移植.同时以未转基因小鼠卵丘细胞和成纤维细胞为核供体,进行体细胞核移植. 结果 转基因与未转基因胎鼠成纤维细胞的重构胚的囊胚(154/160)发育率为19.23%和22.91%,差异不显著(P＞0.05);转基因胚胎干细胞与胎鼠成纤维细胞的重构胚的囊胚(152/154)发育率为41.54%和19.23%,差异显著(P＜0.05);未转基因卵丘细胞与成纤维细胞的重构胚的囊胚(171/160)发育率为41.17%和22.91%,差异显著(P＜0.05). 结论 利用体细胞核移植技术,小鼠胎儿成纤维细胞和胚胎干细胞可以有效地生产小鼠转基因囊胚.     

Cloned human embryonic stem cells for tissue repair and transplantation

One approach to overcome transplant rejection of human embryonic stem (ES) cells is to derive ES cells from nuclear transfer of the patient’s own cells. Because an efficient protocol for human somatic cell nuclear transfer (SCNT) has not been reported, several critical factors need to be determined and optimized. Our experience with domestic animals indicate that reprogramming time (the period of time between cell fusion and oocyte activation), activation method and in vitro culture conditions each play a critical role in chromatin remodeling and the developmental competence of SCNT embryos. In this review, we describe the optimization of human SCNT and derivation of human cloned ES cells. In our study, about approx 25% of human reconstructed embryos developed into blastocysts when we allowed 2 h for reprogramming to support proper embryonic development. Since sperm-mediated activation is absent in SCNT, an artificial stimulus is needed to initiate embryo development. Incubation with 10 μM calcium ionophore for 5 min followed by incubation with 2.0 μM 6-dimethyl amino purine was found to be the most efficient chemical activation protocol for SCNT using human oocytes. In order to overcome inefficiencies in embryo culture, we prepared human modified synthetic oviductal fluid with amino acids (hmSOFaa) by supplementing mSOFaa with human serum albumin and fructose instead of bovine serum albumin and glucose, respectively. Culturing human SCNT-derived embryos in G1.2 medium for the first 48 h followed by hmSOFaa medium produced more blastocysts than culturing in G1.2 medium for the first 48 h followed by culture in G2.2 medium or culturing continuously in hmSOFaa medium. The protocol described here produced cloned blastocysts at rates of 19–29%, which is comparable with the rates in cattle (approx 25%) and pigs (approx 26%) using established SCNT methods. A total of 30 SCNT-derived blastocysts were cultured, 20 inner cell masses (ICMs) were isolated by immunosurgical removal of the trophoblast, and one human cloned ES cell line (SCNT-hES1) with typical ES cell morphology and pluripotency was derived. Our approach opens the door for the use of autologous cells derived from nuclear transfer ES (ntES)-derived cells in transplantation medicine.     

Microtubular spindle dynamics and chromosome complements from somatic cell nuclei haploidization in mature mouse oocytes and developmental potential of the derived embryos

BACKGROUND: The aim of this study was to investigate haploidization of somatic cell nuclei in non-enucleated mature oocytes regarding spindle formation, chromosomes and developmental potential. METHODS: Mouse cumulus cells were injected into metaphase II oocytes. Some injected oocytes were examined for morphological changes of chromosomes and the spindle immediately, and at 30 min, 1 h or 2 h after the injections. The remaining oocytes were activated by Sr(2+) after various incubation periods and observed for formation of a second polar body and pseudo-polar body. Cytogenetic analysis was performed for some of the resulting zygotes. The progress to blastocysts in vitro and the possibility of conception in vivo were assessed. RESULTS: Immediately after injection, the cumulus cell nucleus was still in interphase without spindle formation. The occurrence of premature chromosome condensation (PCC) and spindle formation increased as the incubation time increased. The percentages of activated oocytes increased with the incubation time after nuclear transfer, but the difference was not significant between 1 (58%) and 2 h (62%). The incidence of chromosomal aberrations was high for the derived embryos. Development in vitro was poor, and no procreation of pups occurred after transfer of the 324 embryos. CONCLUSIONS: The PCC and spindle formation induced by cumulus cell nuclei in mature oocytes was time dependent, as was the chance for successful activation. The chromosomal abnormalities from segregation errors presented one obvious cause, apart from the potential epigenetic defects, of developmental failure of the semi-cloned embryos.     

The status of human nuclear transfer

Human therapeutic cloning is a recently emerged application of somatic cell nuclear transfer (SCNT), which is currently being performed to produce patient-specific stem cell lines for future stem cell therapies. The advantages in producing human nuclear transfer (NT) embryos to derive NT stem cell lines are that these can be tailor-made (i.e., are autologous in nature) for the patient and may overcome the need to administer life-long immunosuppression following stem cell transplantation. Although the rationale for using NT embryos is not for reproductive purposes, human NT remains clouded in ethical, moral, and religious controversies. The recent retraction of high-impact factor publications in the field of human NT from a research group in South Korea has placed stem cell research in a delicate situation. These heavily publicized issues may hinder the progress of this research and may threaten to bring current research to a complete halt. This review outlines the recent status of human NT, its continuing progress and the difficulties the field faces. Of most concern are the ethical issues, which surround obtaining human oocytes for research. Recent evidence suggests that failed-to-fertilize oocytes are poor sources for human SCNT, but obtaining fresh, viable oocytes may be even more problematic. The current status of human SCNT is outlined in this review with particular reference made to, lessons learnt from animal research, the oocyte dilemma and optimization of human NT.