【作者】 雷蕾；

【导师】 王华岩；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2013， 博士

【摘要】 山羊多能性干细胞包括：山羊胚胎干细胞(Embryonic Stem Cell, ESCs),山羊胚胎生殖细胞(Embryonic Germ Cell, EGCs)和山羊诱导性多能干细胞(Induced Pluripotent Stem cells, iPSc)。由于对山羊体外的培养体系和表观调控因素仍然不清楚,导致山羊ESCs和EGCs的研究仍没有成功建系的报道。而iPS细胞技术的出现,为家畜多能性细胞研究提供了新的途径和可替代的细胞来源。本实验以关中奶山羊为实验模型,通过从体内受精和发育的囊胚中分离出多能性细胞,尝试筛选适合山羊多能性细胞在体外培养的条件；另外,本实验采用山羊胎儿成纤维细胞(Goat Embryonic Fibroblasts, GEF)为供体细胞,通过逆转录病毒携带的Oct4/Sox2/Klf4/c-Myc四个转录因子,对山羊体细胞进行诱导重编程,并且将诱导重编程细胞定向转分化为神经样细胞和卵母细胞样细胞。主要获得以下结论：1.山羊多能性细胞培养体系的优化本实验共超排处理了25只关中奶山羊,总共获得胚胎253枚,其中发育到2-16细胞的胚胎39枚、桑椹胚113枚、囊胚94枚和孵化胚7枚。通过超排处理的每只山羊平均获得10.1枚胚胎。实验分别以HDMEM、DMEM/F12和Knockout DMEM为基础培养基,在添加血清(FBS)或血清替代品(KSR)条件下,探索山羊内细胞团(Inner Cell Mass, ICM)在体外培养的贴壁和增殖情况。另外,本实验还尝试在培养基中添加小分子化合物,如维生素C (Vitamin C, Vc),丙戊酸(Valproic Acid, VPA)和5-氮杂-2-脱氧胞苷(5-Aza-2’-Deoxycytidine,5-AzadC)等,探索小分子化合物对山羊多能性细胞在体外培养的影响。结果表明,含有15%血清的Knockout DMEM培养基可以促进ICM的贴壁和增殖效率,并且有利于山羊多能性细胞的体外传代。但是,血清中的部分成分同时又促进了多能性细胞的自发分化。在添加小分子化合物的实验中,添加小分子化合物实验组在原代分离培养的过程中与对照组无明显差异,而在传代过程中实验组的细胞细胞生长缓慢,但自发分化现象明显减少。在对山羊多能性细胞进行传代时,采用机械法与酶消化法相结合的传代方法,将山羊多能性细胞在体外培养最高传至18代。2.山羊成纤维细胞诱导重编程本实验使用携带转录因子Oct4、Sox2、Klf4和c-Myc的逆转录病毒载体系统,对山羊成纤维细胞(GEF)进行诱导,在体外诱导培养17天左右,获得了山羊诱导重编程细胞(giPSc)。实验中获得的重编程细胞具有两种状态,一种克隆形态与人ESCs相似,细胞集落扁平,边缘整齐,细胞的核质比较大,细胞核内含一个或多个核仁；另一种克隆形态与小鼠ESCs相似,细胞团间紧密,呈鸟巢状,集落突起,边缘清晰,核质比大,折光性强。获得的山羊iPS细胞的能在体外进行长期培养,稳定传代,并且碱性磷酸酶(AP)的检测呈阳性。免疫荧光检测实验结果表明,获得的山羊iPS细胞表达多能性因子和干细胞表面多能性标记蛋白Oct4、Sox2、SSEA-1和Tra-1-60,而SSEA-4和Tra-1-81表达呈阴性。山羊iPS细胞在体外可形成类胚体,具有发育分化为三胚层的潜能,但鉴于这些iPS细胞在裸鼠体内不能形成畸胎瘤,我们认为其尚处在不完全重编程阶段。3.山羊重编程细胞的定向转分化本实验在山羊成纤维细胞去分化后,分别向培养基中添加特定的诱导因子视黄酸(Retinoic Acid, RA)或牛卵泡液,作为定向转分化的诱导培养基。对重编程的细胞分别经过7天和18天的诱导,重编程过程中处于不稳定状态的去分化细胞转分化为神经样细胞和卵母细胞样细胞。实验结果表明,在逆转录病毒感染第6天后,成纤维细胞的特异性标记基因表达量逐渐下降,细胞进入去分化状态。分别对获得的神经样细胞和卵母细胞样细胞进行RNA和蛋白水平的检测。结果显示,对神经样细胞进行免疫荧光检测,细胞表达Ⅲ型β-微管蛋白(β-tublin Ⅲ, TUJ1),部分卵母细胞样细胞特异性基因表达上调。通过转分化获得的卵母细胞样细胞可通过显微操作仪进行固定操作,表明细胞具有卵母细胞的形态和结构。更多还原

【Abstract】 Goat pluripotent stem cells include goat embryonic stem cells (ESCs), embryonic germ cells (EGCs) and induced pluripotent stem cells (iPS). Goat ESCs or EGCs lines have not yet been established because of unknown appropriate culture conditions in vitro and effect of epigenetic factors with goat pluripotent stem cells. Alternatively, iPS cell technology could provid a new method to generate pluripotent cells. The aim of this study was to select appropriate culture conitions for goat pluripotent cells in vitro utilizing deriving goat pluripotent cells from blastocysts which fertilized and developed in vivo. Also, we tried to induce goat iPS cells from goat embryonic fibroblasts (GEF) with retrovirus encoded by human four factors (Oct4/Sox2/Klf4/c-Myc). At the same time, the reprogrammed somatic cells were transdifferentiated specificly into neural-like cells and oocyte-like cells. The results were obtained as follow:1. Optimized in vitro cultrue condition for goat pluripotent cells25Guanzhong dairy goats were superovulated in total and253embryos were obtained by flushing uterus which included2-cell to16-cell embryos, morulae, blastocysts and hatched blastocysts. The numbers were39,113,94and7respectively. In this study, we obtained10.1embryos from every goat on average. Three different basic media were used which were Knockout DMEM, DMEM and DMEM/F12to detect the effect in sustain growth and proliferation of goat ICM with serum or not. Also, we tried to add small molecular compounds into media to find the effect of proliferation with goat pluripotent cells in vitro. Such as, vitamin C (Vc), Valproic Acid (VPA) and5-Aza-2’-Deoxycytidine (5-AzadC). The result indicated that ICM cells were accelerated the process of attachment and increased the proliferative efficiency cultured in Knockout DMEM with15%fetal bovine serum (FBS). It is also benefit with subculture for goat pluripotent cells in vitro. But goat pluripotent cells differentiated spontaneously with some unknown elements in FBS. We also found that adding small molecular conmpounds coud decrease differentiated spontaneously, but reduce proliferation of pluripotent cells. The goat pluripotent cell colonies were subcultured with mechanical methods up to18passages.2. Induced and reprogrammed from goat fibroblastsIn our study, goat induced pluripotent stem cells (iPS) were reprogrammed from goat embryonic fibroblasts (GEF) with retrovirus encoding human cDNAs of Oct4, Sox2, Klf4and c-Myc. After induced17days in vitro, we obtained goat iPS cells with two kinds of different morphology. One kind of colony was similar with human ESCs in morphology, which was flattened with a neat edge, high nuclear cytoplasmic ratio with one or more nucleolus. The other kind of colony had the characterization of mouse ESCs, which was smooth domed, compact with neat edge and characterised by a high nuclear to cytoplasmic. The goat iPS cells were positive with AP staining and could be cultured and passaged stably in vitro. To detect these goat iPS cells with immunofluorescence, the result indicated goat iPS cells expressed pluripotent transcription factors and stem cell surface antigen, such as Oct4, Sox2, SSEA-1and TRA-1-60, but negative with SSEA-4and TRA-1-81. Because these goat iPS cells could express critical exogenous genes and form embryonic bodies in vitro but without developed teratomas in nude mice, these goat iPS cells still stayed in partially reprogrammed phase.3. Reprogrammed goat fibroblasts transdifferentiated into neural-like and oocyte-like cellsIn this study, goat fibroblasts cells which were infected with retrovirus were transdifferentiated into neural-like cells and oocyte-like cells with RA or5%bovine follicle fluids condition cultue media. After induction for7days and18days, the reprogrammed cells that were stayed in unstable and dedifferentiated stage, and then transdifferentiated into neural-like cells and oocyte-like cells. The result indicated that gene marker which were specific expressed in fibroblasts were down-regulation after infection6days with retrovirus when fibroblasts were in dedifferentiated stage. To detect the neural-like cells and oocyte-like cells with RNA and protein, the result showed oocyte-like cells markers were up-regualtion and β-tublin III was positive in neural-like cells with immunofluorescence. In addition, the oocyte-like cells could be manipulated with micromanipulation that could prove the oocyte-like cells had the similar cell morphology and structer with oocyte.更多还原