人类体细胞核移植胚胎纺锤体形态及其相关蛋白的检测
发布时间：2019-06-07 07:22
【摘要】：研究背景：核有丝分裂器蛋白(Nuclear Mitotic Apparatus Protein, NuMA)、纺锤体驱动蛋白(kinesin spindle protein, Eg5)及γ-tubulin在人类成纤维细胞增殖,人类卵母细胞成熟和人类体细胞核移植胚胎早期发育过程中起重要作用。NuMA是一种分子量为236-240kDa的大分子核蛋白,具有微管聚合作用,能使微管锚定于纺锤体极。Eg5是驱动蛋白家族成员之一,动力蛋白介导运输并定位NuMA于纺锤体极,通过NuMA远端指导聚集微管蛋白。γ-tubulin是中心粒周围物质(Pericentriolar material, PCM)中一种具代表性的蛋白,具有起始微管晶核形成,调节纺锤体组装的作用。相关研究证明上述任一种蛋白的缺失或过表达均可导致纺锤体形态异常,最终导致细胞分裂异常。Simerly等在非人灵长类体细胞核移植实验中发现,所检测的体细胞核移植胚胎中NuMA缺失,驱动蛋白Eg5分布紊乱纺锤体形态异常,因此作者认为,非人灵长类体细胞核移植实验失败的主要原因是由于卵母细胞去核过程中造成了NuMA及驱动蛋白Eg5损失。后来的研究显示,大部分猴子体细胞核移植胚胎NuMA分布基本正常。人类不同成熟阶段卵母细胞及体细胞核移植胚胎纺锤体及其相关蛋白分布如何?日前还未见相关文献报道。 目的：为了寻求人类胚胎成纤维细胞、不同成熟阶段人类卵母细胞及人类体细胞核移植胚胎纺锤体形态及其相关蛋白分布规律,探讨人类卵母细胞去核过程中纺锤体及其相关蛋白的损失是否是导致人类体细胞核移植效率低下的原因,以期解决人类体细胞核移植研究面临的技术难题,加速人类治疗性克隆应用的步伐。 方法：使用间接免疫荧光染色方法在普通荧光显微镜或激光共聚焦荧光显微镜下对人类胚胎成纤维细胞、不同成熟阶段人类卵母细胞及人类体细胞核移植胚胎纺锤体及其相关蛋白分布进行观察。 生发泡(Gv)期、MⅠ期的人类卵母细胞来源于中信湘雅生殖与遗传专科医院ICSI患者治疗过程中废弃的卵母细胞；MⅡ期人类卵母细胞大部分来源于ICSI患者治疗废弃的卵母细胞体外成熟培养,另一部分来源于多囊卵巢(PCOS)患者穿刺治疗捐献的卵母细胞。人类胚胎成纤维细胞来源于人类干细胞国家工程研究中心。使用人类胚胎成纤维细胞为供核细胞,在偏振光显微镜下去除MⅡ期人类卵母细胞核,使用内径为8-10μm的显微注射针将供核细胞显微注射于去核的MⅡ期人类卵母细胞胞质内,体外化学激活(A23187联合6-DMAP)后对人类体细胞核移植胚胎进行G1.5/G2.5序贯培养。 结果：1、在人类胚胎成纤维细胞分裂间期及人类卵母细胞Gv期,NuMA分布于细胞核内除核仁外的位置,α-tubulin呈网状分布于细胞质内；人类胚胎成纤维细胞有丝分裂中期及人类卵母细胞MⅠ及MⅡ期,NuMA凝集分布于纺锤体两极。 2、在人类胚胎成纤维细胞有丝分裂间划,Eg5分布于核质内,γ-tubulin呈一凝集点状分布于核膜附近；在人类胚胎成纤维细胞有丝分裂中期,Eg5及γ-tubulin均集中分布于纺锤体两极,Eg5在纺锤体中央区及细胞质中也有分布。 3、去核后人类卵母细胞胞质未检测到NuMA及α-tubulin的表达,去除的核胞体中染色后发现正常规则的纺锤体,人类卵母细胞染色体整齐排列在赤道板上,NuMA定位在纺锤体两极。注入供核细胞后2小时(核移植胚胎激活前),供核细胞核出现成熟前染色体凝集,短暂纺锤体结构形成,NuMA聚集于纺锤体两极。原核形成时期,NuMA分布于除核仁外的核质内,α-tubulin分布于细胞质内；人类体细胞核移植胚胎第一次有丝分裂中期,NuMA主要分布于纺锤体两极。人类体细胞核移植发育阻滞胚胎可检测到NuMA分布在间期细胞核内。 结论：在人类胚胎成纤维细胞分裂间期及人类卵母细胞Gv期NuMA分布于核质内,人类胚胎成纤维细胞分裂中期及人类卵母细胞MⅠ、MⅡ期NuMA分布于纺锤体两极。在不同发育阶段的人类体细胞核移植胚胎中可检测到NuMA表达,细胞分裂间期,NuMA分布于核质内,α-tubulin分布于细胞质内,细胞分裂中期,NuMA聚集于纺锤体两极。人类体细胞核移植去核过程中,人类卵母细胞中NuMA连同纺锤体一并去除,但供核细胞提供了人类体细胞核移植胚胎NuMA的来源。因此认为人类体细胞核移植去核过程中NuMA蛋白丢失是导致人类体细胞核移植胚胎发育欠佳原因的可能性不大。
[Abstract]:Background: The nuclear mitosis protein (NuMA), the spindle-driven protein (Eg5) and the antigen-tubuin play an important role in the proliferation of human fibroblasts, the maturation of human oocytes and the early development of human somatic cell nuclear transfer embryos. NuMA is a macromolecular nucleoprotein with a molecular weight of 236-240 kDa, with microtubule polymerization, which can anchor the microtube to the spindle pole. Eg5 is one of the members of the drive protein family, which mediates transport and location of the NuMA in the spindle and directs the aggregation of tubulin through the distal tip of the NuMA. 1-tubelin is a representative protein in Pericentriolar material (PCM), which has the function of starting microtubule nucleation and regulating spindle assembly. The related study demonstrated that either deletion or overexpression of any of the above proteins could result in abnormal spindle morphology, and ultimately resulted in an abnormal cell division. in a non-human primate somatic cell nuclear transfer experiment, Simmerly et al., The main cause of the failure of the non-human primate cell nuclear transfer experiment was the loss of the NuMA and the drive protein Eg5 due to the enucleation of the oocyte. Later studies have shown that most of the monkey somatic cell nuclear transfer embryos, the NuMA distribution, is substantially normal. How to distribute that spindle of the embryo and its associated protein in the oocyte and somatic cell nuclear transfer in different stage of human maturation? No relevant literature has been reported before. Objective: To study the morphology and related protein distribution of human oocytes and human somatic cell nuclear transfer embryos in human embryonic fibroblasts and different mature stages. The paper discusses whether the loss of spindle and its associated protein in the process of enucleation of human oocytes is the cause of the low efficiency of human somatic cell nuclear transfer, in order to solve the technical problems of human somatic cell nuclear transfer and to accelerate the application of human therapeutic cloning. Methods: The embryonic spindle and related proteins of human embryonic fibroblasts, human oocytes and human somatic cell nuclear transfer embryos were distributed by indirect immunofluorescent staining method under a common fluorescent microscope or a laser confocal fluorescence microscope. It was observed that the human oocytes during the period of birth (Gv) and M I were derived from the oocytes discarded during the treatment of the ICSI patients in Citic Xiangya and the genetic special hospital. The majority of the human oocytes from the phase M were derived from the ICSI patients for the treatment of the discarded oocytes. The other part of the external mature culture is derived from the patients with polycystic ovary (PCOS). Oocyte. Human embryonic fibroblasts are derived from human stem cell national workers A human embryo fibroblast is used as the nuclear donor, and the nucleus of the M phase 鈪，
本文编号：2494615
[Abstract]:Background: The nuclear mitosis protein (NuMA), the spindle-driven protein (Eg5) and the antigen-tubuin play an important role in the proliferation of human fibroblasts, the maturation of human oocytes and the early development of human somatic cell nuclear transfer embryos. NuMA is a macromolecular nucleoprotein with a molecular weight of 236-240 kDa, with microtubule polymerization, which can anchor the microtube to the spindle pole. Eg5 is one of the members of the drive protein family, which mediates transport and location of the NuMA in the spindle and directs the aggregation of tubulin through the distal tip of the NuMA. 1-tubelin is a representative protein in Pericentriolar material (PCM), which has the function of starting microtubule nucleation and regulating spindle assembly. The related study demonstrated that either deletion or overexpression of any of the above proteins could result in abnormal spindle morphology, and ultimately resulted in an abnormal cell division. in a non-human primate somatic cell nuclear transfer experiment, Simmerly et al., The main cause of the failure of the non-human primate cell nuclear transfer experiment was the loss of the NuMA and the drive protein Eg5 due to the enucleation of the oocyte. Later studies have shown that most of the monkey somatic cell nuclear transfer embryos, the NuMA distribution, is substantially normal. How to distribute that spindle of the embryo and its associated protein in the oocyte and somatic cell nuclear transfer in different stage of human maturation? No relevant literature has been reported before. Objective: To study the morphology and related protein distribution of human oocytes and human somatic cell nuclear transfer embryos in human embryonic fibroblasts and different mature stages. The paper discusses whether the loss of spindle and its associated protein in the process of enucleation of human oocytes is the cause of the low efficiency of human somatic cell nuclear transfer, in order to solve the technical problems of human somatic cell nuclear transfer and to accelerate the application of human therapeutic cloning. Methods: The embryonic spindle and related proteins of human embryonic fibroblasts, human oocytes and human somatic cell nuclear transfer embryos were distributed by indirect immunofluorescent staining method under a common fluorescent microscope or a laser confocal fluorescence microscope. It was observed that the human oocytes during the period of birth (Gv) and M I were derived from the oocytes discarded during the treatment of the ICSI patients in Citic Xiangya and the genetic special hospital. The majority of the human oocytes from the phase M were derived from the ICSI patients for the treatment of the discarded oocytes. The other part of the external mature culture is derived from the patients with polycystic ovary (PCOS). Oocyte. Human embryonic fibroblasts are derived from human stem cell national workers A human embryo fibroblast is used as the nuclear donor, and the nucleus of the M phase 鈪，
本文编号：2494615
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