细胞周期蛋白依赖激酶抑制因子对诱导型多能干细胞形成的抑制作用

发布时间：2018-01-24 17:13

本文关键词： 细胞周期蛋白依赖激酶抑制因子诱导型多能干细胞形成作用　出处：《南京大学》2010年博士论文　论文类型：学位论文

【摘要】：干细胞研究作为一项新兴的学科,凭借其全新的生物学概念和良好的临床应用前景,正在受到越来越大的关注。干细胞从来源上可分为胚胎干细胞和成体干细胞两种。胚胎干细胞是从处于囊胚期的胚胎的内细胞层分离出来后,在体外培养传代形成的干细胞。它具有快速增殖的特性,并能够分化成三个胚层的细胞和形成个体。这种能力被认为是胚胎干细胞特有的多向分化潜能。成体干细胞则来自成熟个体的各个器官,为所在的器官提供特定的细胞类型以维持该器官的功能。由于获取胚胎干细胞存在伦理学问题(需要摧毁胚胎)和成体干细胞在单个器官中的数量极其稀少、收集上存在难度,这两种干细胞在临床的治疗前景并不十分乐观。目前对这两种干细胞的研究更多的是对其干细胞生物学特性的研究。由于上述的问题和生物学技术的进步,一种“人工”干细胞的概念正逐步兴起。和天然的干细胞不同,“人工”干细胞可以从机体的任何细胞中“制作”出来,而“加工”出的细胞能获得和胚胎干细胞相似的多向分化潜能和自我更新的能力。目前有三种方法可以制作“人工”干细胞。最早提出的方法是通过核移植将未受精的卵细胞的细胞核替换成成熟的体细胞的细胞核,利用卵细胞内特有的细胞微环境来改变植入的体细胞胞核的基因表达,从而将该细胞核转变为胚胎干细胞的细胞核。通过该方法获得的干细胞在基因表达、DNA甲基化程度方面、多向分化潜能和自我更新能力和同样来源的胚胎干细胞极为接近。因此,从这一点看,它最能替代胚胎干细胞。然而,由于它需要熟练的核移植技术、昂贵的设备和捐献卵子,这种方法在人类细胞上始终未能实现。另一种方法和核移植的方法类似,它是通过核融合的方法,将一个胚胎干细胞和一个体细胞相融合,利用胚胎干细胞内的特定微环境改变体细胞核的基因表达状态。这个方法存在的缺点是最终得到的细胞为4倍体的细胞,因此这种细胞上也无法在人体上应用。最后一种方法,是用病毒作为载体向已经分化成熟的体细胞内导入特定的4个转录因子(Oct4, Sox2, Klf4和c-Myc.),以此来改变体细胞的基因表达状态。被导入的细胞在4个转录因子的作用下,其基因的表达将返回胚胎干细胞的状态,进而再次获得胚胎干细胞特有的多向分化潜能。通过这种方法制作的干细胞被称为诱导型多能干细胞(induced pluripotent stem cells, iPSCs)。这种方法由于简单、经济,并能使用病人的皮肤细胞直接制作,具有极好的临床应用前景。目前利用这种方法来诱导多能干细胞存在诱导效率低、具体分子机制不明等问题。其中,阻碍诱导型多能干细胞形成的已知的分子机制之一是抗癌基因p53激活后的细胞周期抑制和细胞凋亡。考虑到为临床服务的需要,克服诱导效率低的缺点是当前研究的主要方向之一。本研究首先考察了使用病毒做载体制作诱导型多能干细胞的可行性,并验证了所诱导出的细胞具有胚胎干细胞特性。然后,从细胞周期的调节通路入手,探索其他细胞周期调节因子在诱导性多能干细胞形成过程中的作用。第一部分诱导型多能干细胞的制作研究背景：通过向分化成熟的体细胞导入4个转录因子(Oct4、Sox2、Klf4和c-Myc)可诱导出具有多向分化潜能的干细胞。这种诱导型多能干细胞与其他“人工”干细胞的技术相比,有简单、高效、能直接使用病人皮肤细胞制作的特点,具有良好的科研和临床应用前景。实验目的：实践诱导多能干细胞的技术,即在分化成熟的细胞中诱导出多向分化潜能的干细胞,并考察其在诱导后获得的多向分化潜能。实验方法：使用感受态细菌制备含有四个转录因子编码区的质粒(Oct4、Sox2、Klf4和c-Myc)。原代分离、扩增小鼠胚胎成纤维细胞,并把它作为诱导的靶细胞。通过逆转录病毒导入基因的方法转导小鼠胚胎成纤维细胞,将其诱导成诱导型多能干细胞。在多能干细胞成功诱导后,挑取单个细胞集落并扩增、培育形成独立细胞系。通过多种方法考察所形成的诱导型多能干细胞系的多向分化潜能,包括胚胎干细胞的标识物的表达、畸胎瘤的形成能力和嵌合体形成的能力。实验结果：小鼠胚胎成纤维细胞经过14天的诱导后逐步获得了与胚胎干细胞相似的细胞集落形态。这些集落在形成独立细胞系以后,表达有胚胎干细胞特有的标记物Oct4、Sox2、Nanog和SSEA-1。这些诱导型多能干细胞在皮下注射入NOD/SCID免疫缺陷小鼠后,能形成畸胎瘤。将畸胎瘤进行常规病理染色后发现,畸胎瘤组织含有包括有外胚层、中胚层和内胚层在内的多种细胞类型。将这些诱导型多能干细胞植入胚胎后,该胚胎能形成健康个体。在这些个体身上,发现有由植入的诱导型多能干细胞来源的毛发。实验小结：使用分化成熟的小鼠胚胎成纤维细胞可以诱导出具有多向分化潜能的干细胞。通过这个方法诱导出的多能干细胞具有和胚胎干细胞类似的形态、标识物表达、向多胚层细胞分化的能力以及参与正常机体发育的能力。第二部分细胞周期蛋白依赖激酶抑制因子对诱导型多能干细胞的形成抑制作用研究背景：虽然在成熟的体细胞诱导多能干细胞有很好的临床应用前景,但是其诱导效率低的特点阻碍了它在临床上的应用。在诱导型多能干细胞形成的过程中,起阻碍作用的途经之一是由抗癌基因p53控制下的细胞周期抑制和细胞凋亡。而前者是通过上调细胞周期蛋白依赖激酶抑制因子p21来完成。细胞周期蛋白依赖激酶抑制因子为一多成员的抑制分子家族,包括有p16、p18、p19和p21、p27等因子,其中p18和p27在造血干细胞的作用是抑制其细胞周期和自我更新。我们设想p18和p27在iPSCs诱导过程中可能也会起一定作用。实验目的：研究和p21同属一类的细胞周期抑制因子：p18和p27在诱导型多能干细胞形成过程中所起的作用。实验方法：测定小鼠胚胎成纤维细胞和小鼠胚胎干细胞细胞周期的差别。使用SSEA-1这一胚胎干细胞的标识物标识在多能干细胞诱导过程中的被成功诱导的细胞,观察其细胞周期的变化。测定p18和p27在小鼠胚胎成纤维细胞、小鼠胚胎干细胞和诱导型多能干细胞中mRNA的表达和在诱导型多能干细胞形成过程中的表达水平。使用取材白p18或p27基因敲除小鼠的胚胎成纤维细胞制作诱导型多能干细胞,观察野生型、杂合体和纯合体的小鼠胚胎成纤维细胞在诱导成多能干细胞时诱导效率的不同。制作含p18或p27编码区的质粒。使用由该质粒制作的逆转录病毒分别感染p18缺陷型或p27缺陷型的小鼠胚胎成纤维细胞,观察在p18或p27在相应的基因缺陷型的细胞重新表达后它们对多能干细胞的诱导效率的影响。使用药物Roscovitine和PD-0332991分别直接抑制p27的靶分子CDK2或p18的靶分子CDK4/6,来观察诱导型多能干细胞形成时的诱导效率。实验结果：分化成熟的小鼠胚胎成纤维细胞的细胞周期周转缓慢,而胚胎干细胞的细胞周期活跃。在诱导型多能干细胞形成的过程中,被成功诱导的细胞逐步获得活跃的细胞周期特性。与之相应的是,细胞周期抑制因子p18和p27在小鼠胚胎成纤维细胞的mRNA表达量大于其在诱导型多能干细胞和胚胎干细胞的表达量。p18和p27的mRNA在多能干细胞诱导的过程中其表达量低于对照组。在把p18或p27的基因敲除后,小鼠胚胎成纤维细胞形成多能干细胞的诱导成功效率上升10倍以上,且该效率的提升有基因剂量依赖性。将p18或p27基因重新导入相应的基因缺陷型细胞后,其对诱导型多能干细胞形成的抑制作用可恢复。通过药物直接抑制p18或p27的靶分子,可模拟p18或p27对诱导型多能干细胞形成的抑制作用,提示p18或p27对诱导型多能干细胞形成的抑制作用是分别通过抑制其靶分子CDK4/6或CDK2、进而抑制所诱导细胞的细胞周期来实现的。实验结论：在体细胞内表达的p18和p27参与了对诱导型多能干细胞形成的抑制。这种抑制作用具有基因剂量依赖性。这种抑制作用分别是通过抑制靶分子CDK4/6或CDK2、进而抑制细胞周期来实现的。
[Abstract]:Stem cell research as a new subject, with its new concept of biology and good clinical application prospect, is getting more and more attention. Stem cells from the source can be divided into embryonic stem cells and adult stem cells. Two kinds of embryonic stem cells from the blastocyst stage embryos in the inner cell layer after separated, in the passage formed by stem cells cultured in vitro. It has the characteristics of rapid proliferation, and can differentiate into three germ cells and the formation of the individual. This ability is thought to be multipotent embryonic stem cell specific. Adult stem cells from various organs of mature individuals, specific cell types in order to maintain the function of organs for the organs. Due to the existence of embryonic stem cells (ethical issues need to destroy the embryo) and adult stem cells in a single organ in the number of extremely rare, are difficult to collect Of these two types of stem cells in clinical treatment in the future is not very optimistic. The current of the two kinds of stem cell research is more on the biological characteristics of stem cell research. Because of the above problems and the development of biological technology, an "artificial" stem cell concept is gradually rising and different natural. The stem cells, "artificial" stem cells from any cell in the body in the "production", and "processing" of the cell can be obtained and the ability of embryonic stem cell differentiation and self-renewal similar. At present, there are three ways to make "people" stem cells. The earliest methods proposed is not fertilized by nuclear transfer of the nucleus of an egg cell replacement into mature somatic cell nuclei, micro environment to change into somatic cell nucleus gene expression using the characteristic of eggs within the cells, and the nuclear transformation As an embryonic stem cell obtained by this method. The expression of stem cell gene, DNA methylation, multilineage differentiation potential and self-renewal ability and also the source of embryonic stem cells is very close. Therefore, from this point of view, it can replace embryonic stem cells. However, since it requires nuclear transplantation skilled, expensive equipment and the method of egg donation, not always in human cells. Another method and nuclear transplantation is similar, it is through the method of nuclear fusion, an embryonic stem cell and somatic cell fusion, using embryonic stem cells in specific microenvironment changes in somatic cell nuclear gene expression. This method has the disadvantages of the resulting cells is 4 times of body cells, so the cells can not in human applications. Finally a method is used as the carrier to have virus The 4 transcription factors differentiated somatic cells into specific (Oct4, Sox2, Klf4 and c-Myc.), in order to change the expression of somatic cell gene was introduced into the cell. In 4 transcription factors under the action of the gene will return the embryonic stem cell state, and again won the multi the differentiation potential of embryonic stem cell specific production. The stem cell known as induced pluripotent stem cells by this method (induced pluripotent stem cells, iPSCs). Since this method is simple, economic, and can use the patient's skin cells directly, has excellent clinical application prospect. At present the method of induced pluripotent stem cells induced by low efficiency, specific molecular mechanism is unknown. Among them, one of the molecular mechanisms of block induced pluripotent stem cell formation is known to the cell cycle of tumor suppressor gene p53 after activation and inhibition Cell apoptosis. Taking into account the need to provide clinical services, to overcome the disadvantage of low efficiency of induction is one of the main direction of current research. This study first investigated the carrier making induced pluripotent stem cells, the feasibility of using the virus, it is verified that the induced cells with embryonic stem cell like properties. Then, starting from the cell cycle. Pathways to explore other cell cycle regulatory factors in the pathogenesis of induced pluripotent stem cells. The first part of induced pluripotent stem cells, making the research background: through to the differentiation and maturation of somatic cells into 4 transcription factors (Oct4, Sox2, Klf4 and c-Myc) can induce pluripotent stem this kind of cells. Induced pluripotent stem cells and other "artificial" stem cell technology compared to a simple, efficient and can be used directly with characteristics of skin cell production, has the good scientific research and Clinical application. Objective: the practice of induced pluripotent stem cells, which induce multipotential stem cells in differentiation of mature cells, and investigate its in induced differentiation potential. Methods: using the competent bacteria preparation containing four transcription factor encoding plasmid (Oct4 Sox2, Klf4, and c-Myc). Primary isolation, amplification of mouse embryonic fibroblasts, and take it as the target cell induced by retroviral gene transfection method. The transduction of mouse embryonic fibroblast cells, the induction of inducible pluripotent stem cells. After induction of pluripotent stem cells, from single cells colony and amplification, foster the formation of independent cell lines. Multipotential differentiation induced pluripotent stem cell lines formed by a variety of methods to investigate, including the expression of embryonic stem cell marker, teratoma formation ability And the chimera formation ability. Results: mouse embryonic fibroblast cells after 14 days of induction after gradually obtained with embryonic stem cell similar colony morphology. The colony after the formation of independent cell lines, the expression of embryonic stem cell specific markers Oct4, Sox2, Nanog and SSEA-1. of the inducible pluripotent stem cells in immunodeficient mice after subcutaneous injection into NOD/SCID, can form teratomas. The teratoma were routine pathological staining found, teratoma tissue contains including ectoderm, mesoderm and endoderm, a variety of cell types. These induced pluripotent stem cells implantation embryo, the embryo can form healthy individuals in these individuals., found by implantation of induced pluripotent stem cells. Conclusion: the use of hair the mature differentiation of mouse embryonic fibroblasts can induce to produce more The differentiation potential of stem cells. By this method the induced pluripotent stem cells and embryonic stem cells have similar morphology and marker expression, ability to mesoderm cell differentiation and ability to participate in normal body development. The second part of the cyclin dependent kinase inhibitor due to the formation of induced pluripotent stem cells, inhibition of background: Although in the mature cell induced pluripotent stem cells have good prospects for clinical application, but its low inducing efficiency hinders its clinical application. The induced pluripotent stem cell formation process, one of the obstacles is passing by the cell cycle inhibitory and apoptosis gene under the control of p53. While the former is through upregulation of cyclin dependent kinase inhibitor p21. Cyclin dependent kinase inhibitor is a member of the tumor For molecules, including p16, P18, P19 and p21, p27 and other factors, including P18 and p27 in hematopoietic stem cells inhibits the cell cycle and self-renewal. We assume that P18 and p27 during iPSCs induction may also play a role. Objective: To study p21 and belong to the same class cell cycle inhibitor: P18 and p27 in induced pluripotent stem cells, which will play a role in the process of experiment. Methods: Determination of fibroblast and mouse embryonic stem cell cycle differences. The application of SSEA-1 in the embryonic mouse embryonic stem cell marker identification in induced pluripotent stem cell process was successfully induced cells, observe the change of cell cycle. The determination of P18 in mouse embryonic fibroblasts and p27 mouse embryonic stem cells and induced pluripotent stem cells and the expression of mRNA in induced pluripotent stem cells formed in the process of expression The level of P18 or p27 were used. The white gene knockout mouse embryonic fibroblasts produced induced pluripotent stem cells was observed in wild-type mouse embryos, heterozygote and homozygote fibroblasts induced pluripotent stem cells induced into efficiency in the different production. Plasmid containing P18 or p27 encoding region of mouse. The use of fiber cells produced by the retrovirus plasmid were infected with P18 defective or defective p27, observed in the P18 or p27 gene defects in the corresponding cell re expression of pluripotent stem cells induced by efficiency. The influence of target molecules of CDK4/6 using the drug Roscovitine and PD-0332991 respectively the direct inhibition of p27 target molecules of CDK2 or P18, to observe the induction efficiency of induced pluripotent stem cells during the formation. Results: the differentiation of mouse embryonic mature into cell cycle and the slow turnover of embryonic stem cells The active cell cycle. In the process of induced pluripotent stem cell formation, was successfully induced cells gradually get cell cycle characteristics of active. Correspondingly, the cell cycle inhibitor of P18 and p27 in mouse embryonic fibroblasts mRNA expression induced pluripotent stem than in the expression of.P18 and p27 cells and embryonic stem cells in mRNA induced pluripotent stem cell in its expression level is lower than the control group. The P18 or p27 gene knockout mouse embryonic fibroblast cells, formation of pluripotent stem cells successfully induced efficiency increases more than 10 times, and the efficiency of gene dose dependent. The P18 or p27 gene deficient cells re import the corresponding after its inhibitory effect on induced pluripotent stem cell formation can be restored. The target molecule drugs through direct inhibition of P18 or p27, can simulate the P18 or p27 on the induction type Inhibition of stem cell formation, suggesting that inhibition of P18 or p27 on induced pluripotent stem cells are formed respectively by inhibiting the target molecules of CDK4/6 or CDK2, and the inhibition of cell cycle induced by the cell to achieve. Conclusion: expression in somatic cells of P18 and p27 were involved in induced pluripotent stem the inhibition of cell formation. This inhibition is dose dependent gene. This inhibition is through inhibition of target molecules of CDK4/6 or CDK2, and inhibit the cell cycle to achieve.

【学位授予单位】：南京大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R329.2

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