本文關鍵詞： 細胞 周期 蛋白 依賴 激酶 抑制 因子 誘導型 多能 干細胞 形成 作用　出處：《南京大學》2010年博士論文　論文類型：學位論文

【摘要】：干細胞研究作為一項新興的學科,憑借其全新的生物學概念和良好的臨床應用前景,正在受到越來越大的關注。干細胞從來源上可分為胚胎干細胞和成體干細胞兩種。胚胎干細胞是從處于囊胚期的胚胎的內(nèi)細胞層分離出來后,在體外培養(yǎng)傳代形成的干細胞。它具有快速增殖的特性,并能夠分化成三個胚層的細胞和形成個體。這種能力被認為是胚胎干細胞特有的多向分化潛能。成體干細胞則來自成熟個體的各個器官,為所在的器官提供特定的細胞類型以維持該器官的功能。由于獲取胚胎干細胞存在倫理學問題(需要摧毀胚胎)和成體干細胞在單個器官中的數(shù)量極其稀少、收集上存在難度,這兩種干細胞在臨床的治療前景并不十分樂觀。目前對這兩種干細胞的研究更多的是對其干細胞生物學特性的研究。由于上述的問題和生物學技術的進步,一種“人工”干細胞的概念正逐步興起。和天然的干細胞不同,“人工”干細胞可以從機體的任何細胞中“制作”出來,而“加工”出的細胞能獲得和胚胎干細胞相似的多向分化潛能和自我更新的能力。目前有三種方法可以制作“人工”干細胞。最早提出的方法是通過核移植將未受精的卵細胞的細胞核替換成成熟的體細胞的細胞核,利用卵細胞內(nèi)特有的細胞微環(huán)境來改變植入的體細胞胞核的基因表達,從而將該細胞核轉變?yōu)榕咛ジ杉毎募毎恕Ｍㄟ^該方法獲得的干細胞在基因表達、DNA甲基化程度方面、多向分化潛能和自我更新能力和同樣來源的胚胎干細胞極為接近。因此,從這一點看,它最能替代胚胎干細胞。然而,由于它需要熟練的核移植技術、昂貴的設備和捐獻卵子,這種方法在人類細胞上始終未能實現(xiàn)。另一種方法和核移植的方法類似,它是通過核融合的方法,將一個胚胎干細胞和一個體細胞相融合,利用胚胎干細胞內(nèi)的特定微環(huán)境改變體細胞核的基因表達狀態(tài)。這個方法存在的缺點是最終得到的細胞為4倍體的細胞,因此這種細胞上也無法在人體上應用。最后一種方法,是用病毒作為載體向已經(jīng)分化成熟的體細胞內(nèi)導入特定的4個轉錄因子(Oct4, Sox2, Klf4和c-Myc.),以此來改變體細胞的基因表達狀態(tài)。被導入的細胞在4個轉錄因子的作用下,其基因的表達將返回胚胎干細胞的狀態(tài),進而再次獲得胚胎干細胞特有的多向分化潛能。通過這種方法制作的干細胞被稱為誘導型多能干細胞(induced pluripotent stem cells, iPSCs)。這種方法由于簡單、經(jīng)濟,并能使用病人的皮膚細胞直接制作,具有極好的臨床應用前景。目前利用這種方法來誘導多能干細胞存在誘導效率低、具體分子機制不明等問題。其中,阻礙誘導型多能干細胞形成的已知的分子機制之一是抗癌基因p53激活后的細胞周期抑制和細胞凋亡�？紤]到為臨床服務的需要,克服誘導效率低的缺點是當前研究的主要方向之一。本研究首先考察了使用病毒做載體制作誘導型多能干細胞的可行性,并驗證了所誘導出的細胞具有胚胎干細胞特性。然后,從細胞周期的調(diào)節(jié)通路入手,探索其他細胞周期調(diào)節(jié)因子在誘導性多能干細胞形成過程中的作用。第一部分誘導型多能干細胞的制作研究背景：通過向分化成熟的體細胞導入4個轉錄因子(Oct4、Sox2、Klf4和c-Myc)可誘導出具有多向分化潛能的干細胞。這種誘導型多能干細胞與其他“人工”干細胞的技術相比,有簡單、高效、能直接使用病人皮膚細胞制作的特點,具有良好的科研和臨床應用前景。實驗目的：實踐誘導多能干細胞的技術,即在分化成熟的細胞中誘導出多向分化潛能的干細胞,并考察其在誘導后獲得的多向分化潛能。實驗方法：使用感受態(tài)細菌制備含有四個轉錄因子編碼區(qū)的質(zhì)粒(Oct4、Sox2、Klf4和c-Myc)。原代分離、擴增小鼠胚胎成纖維細胞,并把它作為誘導的靶細胞。通過逆轉錄病毒導入基因的方法轉導小鼠胚胎成纖維細胞,將其誘導成誘導型多能干細胞。在多能干細胞成功誘導后,挑取單個細胞集落并擴增、培育形成獨立細胞系。通過多種方法考察所形成的誘導型多能干細胞系的多向分化潛能,包括胚胎干細胞的標識物的表達、畸胎瘤的形成能力和嵌合體形成的能力。實驗結果：小鼠胚胎成纖維細胞經(jīng)過14天的誘導后逐步獲得了與胚胎干細胞相似的細胞集落形態(tài)。這些集落在形成獨立細胞系以后,表達有胚胎干細胞特有的標記物Oct4、Sox2、Nanog和SSEA-1。這些誘導型多能干細胞在皮下注射入NOD/SCID免疫缺陷小鼠后,能形成畸胎瘤。將畸胎瘤進行常規(guī)病理染色后發(fā)現(xiàn),畸胎瘤組織含有包括有外胚層、中胚層和內(nèi)胚層在內(nèi)的多種細胞類型。將這些誘導型多能干細胞植入胚胎后,該胚胎能形成健康個體。在這些個體身上,發(fā)現(xiàn)有由植入的誘導型多能干細胞來源的毛發(fā)。實驗小結：使用分化成熟的小鼠胚胎成纖維細胞可以誘導出具有多向分化潛能的干細胞。通過這個方法誘導出的多能干細胞具有和胚胎干細胞類似的形態(tài)、標識物表達、向多胚層細胞分化的能力以及參與正常機體發(fā)育的能力。第二部分細胞周期蛋白依賴激酶抑制因子對誘導型多能干細胞的形成抑制作用研究背景：雖然在成熟的體細胞誘導多能干細胞有很好的臨床應用前景,但是其誘導效率低的特點阻礙了它在臨床上的應用。在誘導型多能干細胞形成的過程中,起阻礙作用的途經(jīng)之一是由抗癌基因p53控制下的細胞周期抑制和細胞凋亡。而前者是通過上調(diào)細胞周期蛋白依賴激酶抑制因子p21來完成。細胞周期蛋白依賴激酶抑制因子為一多成員的抑制分子家族,包括有p16、p18、p19和p21、p27等因子,其中p18和p27在造血干細胞的作用是抑制其細胞周期和自我更新。我們設想p18和p27在iPSCs誘導過程中可能也會起一定作用。實驗目的：研究和p21同屬一類的細胞周期抑制因子：p18和p27在誘導型多能干細胞形成過程中所起的作用。實驗方法：測定小鼠胚胎成纖維細胞和小鼠胚胎干細胞細胞周期的差別。使用SSEA-1這一胚胎干細胞的標識物標識在多能干細胞誘導過程中的被成功誘導的細胞,觀察其細胞周期的變化。測定p18和p27在小鼠胚胎成纖維細胞、小鼠胚胎干細胞和誘導型多能干細胞中mRNA的表達和在誘導型多能干細胞形成過程中的表達水平。使用取材白p18或p27基因敲除小鼠的胚胎成纖維細胞制作誘導型多能干細胞,觀察野生型、雜合體和純合體的小鼠胚胎成纖維細胞在誘導成多能干細胞時誘導效率的不同。制作含p18或p27編碼區(qū)的質(zhì)粒。使用由該質(zhì)粒制作的逆轉錄病毒分別感染p18缺陷型或p27缺陷型的小鼠胚胎成纖維細胞,觀察在p18或p27在相應的基因缺陷型的細胞重新表達后它們對多能干細胞的誘導效率的影響。使用藥物Roscovitine和PD-0332991分別直接抑制p27的靶分子CDK2或p18的靶分子CDK4/6,來觀察誘導型多能干細胞形成時的誘導效率。實驗結果：分化成熟的小鼠胚胎成纖維細胞的細胞周期周轉緩慢,而胚胎干細胞的細胞周期活躍。在誘導型多能干細胞形成的過程中,被成功誘導的細胞逐步獲得活躍的細胞周期特性。與之相應的是,細胞周期抑制因子p18和p27在小鼠胚胎成纖維細胞的mRNA表達量大于其在誘導型多能干細胞和胚胎干細胞的表達量。p18和p27的mRNA在多能干細胞誘導的過程中其表達量低于對照組。在把p18或p27的基因敲除后,小鼠胚胎成纖維細胞形成多能干細胞的誘導成功效率上升10倍以上,且該效率的提升有基因劑量依賴性。將p18或p27基因重新導入相應的基因缺陷型細胞后,其對誘導型多能干細胞形成的抑制作用可恢復。通過藥物直接抑制p18或p27的靶分子,可模擬p18或p27對誘導型多能干細胞形成的抑制作用,提示p18或p27對誘導型多能干細胞形成的抑制作用是分別通過抑制其靶分子CDK4/6或CDK2、進而抑制所誘導細胞的細胞周期來實現(xiàn)的。實驗結論：在體細胞內(nèi)表達的p18和p27參與了對誘導型多能干細胞形成的抑制。這種抑制作用具有基因劑量依賴性。這種抑制作用分別是通過抑制靶分子CDK4/6或CDK2、進而抑制細胞周期來實現(xiàn)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.

【學位授予單位】：南京大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R329.2

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