【摘要】：研究背景：核有絲分裂器蛋白(Nuclear Mitotic Apparatus Protein, NuMA)、紡錘體驅(qū)動(dòng)蛋白(kinesin spindle protein, Eg5)及γ-tubulin在人類成纖維細(xì)胞增殖,人類卵母細(xì)胞成熟和人類體細(xì)胞核移植胚胎早期發(fā)育過(guò)程中起重要作用。NuMA是一種分子量為236-240kDa的大分子核蛋白,具有微管聚合作用,能使微管錨定于紡錘體極。Eg5是驅(qū)動(dòng)蛋白家族成員之一,動(dòng)力蛋白介導(dǎo)運(yùn)輸并定位NuMA于紡錘體極,通過(guò)NuMA遠(yuǎn)端指導(dǎo)聚集微管蛋白。γ-tubulin是中心粒周圍物質(zhì)(Pericentriolar material, PCM)中一種具代表性的蛋白,具有起始微管晶核形成,調(diào)節(jié)紡錘體組裝的作用。相關(guān)研究證明上述任一種蛋白的缺失或過(guò)表達(dá)均可導(dǎo)致紡錘體形態(tài)異常,最終導(dǎo)致細(xì)胞分裂異常。Simerly等在非人靈長(zhǎng)類體細(xì)胞核移植實(shí)驗(yàn)中發(fā)現(xiàn),所檢測(cè)的體細(xì)胞核移植胚胎中NuMA缺失,驅(qū)動(dòng)蛋白Eg5分布紊亂紡錘體形態(tài)異常,因此作者認(rèn)為,非人靈長(zhǎng)類體細(xì)胞核移植實(shí)驗(yàn)失敗的主要原因是由于卵母細(xì)胞去核過(guò)程中造成了NuMA及驅(qū)動(dòng)蛋白Eg5損失。后來(lái)的研究顯示,大部分猴子體細(xì)胞核移植胚胎NuMA分布基本正常。人類不同成熟階段卵母細(xì)胞及體細(xì)胞核移植胚胎紡錘體及其相關(guān)蛋白分布如何?日前還未見(jiàn)相關(guān)文獻(xiàn)報(bào)道。 目的：為了尋求人類胚胎成纖維細(xì)胞、不同成熟階段人類卵母細(xì)胞及人類體細(xì)胞核移植胚胎紡錘體形態(tài)及其相關(guān)蛋白分布規(guī)律,探討人類卵母細(xì)胞去核過(guò)程中紡錘體及其相關(guān)蛋白的損失是否是導(dǎo)致人類體細(xì)胞核移植效率低下的原因,以期解決人類體細(xì)胞核移植研究面臨的技術(shù)難題,加速人類治療性克隆應(yīng)用的步伐。 方法：使用間接免疫熒光染色方法在普通熒光顯微鏡或激光共聚焦熒光顯微鏡下對(duì)人類胚胎成纖維細(xì)胞、不同成熟階段人類卵母細(xì)胞及人類體細(xì)胞核移植胚胎紡錘體及其相關(guān)蛋白分布進(jìn)行觀察。 生發(fā)泡(Gv)期、MⅠ期的人類卵母細(xì)胞來(lái)源于中信湘雅生殖與遺傳�？漆t(yī)院ICSI患者治療過(guò)程中廢棄的卵母細(xì)胞；MⅡ期人類卵母細(xì)胞大部分來(lái)源于ICSI患者治療廢棄的卵母細(xì)胞體外成熟培養(yǎng),另一部分來(lái)源于多囊卵巢(PCOS)患者穿刺治療捐獻(xiàn)的卵母細(xì)胞。人類胚胎成纖維細(xì)胞來(lái)源于人類干細(xì)胞國(guó)家工程研究中心。使用人類胚胎成纖維細(xì)胞為供核細(xì)胞,在偏振光顯微鏡下去除MⅡ期人類卵母細(xì)胞核,使用內(nèi)徑為8-10μm的顯微注射針將供核細(xì)胞顯微注射于去核的MⅡ期人類卵母細(xì)胞胞質(zhì)內(nèi),體外化學(xué)激活(A23187聯(lián)合6-DMAP)后對(duì)人類體細(xì)胞核移植胚胎進(jìn)行G1.5/G2.5序貫培養(yǎng)。 結(jié)果：1、在人類胚胎成纖維細(xì)胞分裂間期及人類卵母細(xì)胞Gv期,NuMA分布于細(xì)胞核內(nèi)除核仁外的位置,α-tubulin呈網(wǎng)狀分布于細(xì)胞質(zhì)內(nèi)；人類胚胎成纖維細(xì)胞有絲分裂中期及人類卵母細(xì)胞MⅠ及MⅡ期,NuMA凝集分布于紡錘體兩極。 2、在人類胚胎成纖維細(xì)胞有絲分裂間劃,Eg5分布于核質(zhì)內(nèi),γ-tubulin呈一凝集點(diǎn)狀分布于核膜附近；在人類胚胎成纖維細(xì)胞有絲分裂中期,Eg5及γ-tubulin均集中分布于紡錘體兩極,Eg5在紡錘體中央?yún)^(qū)及細(xì)胞質(zhì)中也有分布。 3、去核后人類卵母細(xì)胞胞質(zhì)未檢測(cè)到NuMA及α-tubulin的表達(dá),去除的核胞體中染色后發(fā)現(xiàn)正常規(guī)則的紡錘體,人類卵母細(xì)胞染色體整齊排列在赤道板上,NuMA定位在紡錘體兩極。注入供核細(xì)胞后2小時(shí)(核移植胚胎激活前),供核細(xì)胞核出現(xiàn)成熟前染色體凝集,短暫紡錘體結(jié)構(gòu)形成,NuMA聚集于紡錘體兩極。原核形成時(shí)期,NuMA分布于除核仁外的核質(zhì)內(nèi),α-tubulin分布于細(xì)胞質(zhì)內(nèi)；人類體細(xì)胞核移植胚胎第一次有絲分裂中期,NuMA主要分布于紡錘體兩極。人類體細(xì)胞核移植發(fā)育阻滯胚胎可檢測(cè)到NuMA分布在間期細(xì)胞核內(nèi)。 結(jié)論：在人類胚胎成纖維細(xì)胞分裂間期及人類卵母細(xì)胞Gv期NuMA分布于核質(zhì)內(nèi),人類胚胎成纖維細(xì)胞分裂中期及人類卵母細(xì)胞MⅠ、MⅡ期NuMA分布于紡錘體兩極。在不同發(fā)育階段的人類體細(xì)胞核移植胚胎中可檢測(cè)到NuMA表達(dá),細(xì)胞分裂間期,NuMA分布于核質(zhì)內(nèi),α-tubulin分布于細(xì)胞質(zhì)內(nèi),細(xì)胞分裂中期,NuMA聚集于紡錘體兩極。人類體細(xì)胞核移植去核過(guò)程中,人類卵母細(xì)胞中NuMA連同紡錘體一并去除,但供核細(xì)胞提供了人類體細(xì)胞核移植胚胎NuMA的來(lái)源。因此認(rèn)為人類體細(xì)胞核移植去核過(guò)程中NuMA蛋白丟失是導(dǎo)致人類體細(xì)胞核移植胚胎發(fā)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，

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