小鼠胚胎成纤维细胞重编程为多能干细胞的实验研究

发布时间：2018-05-09 05:05

  本文选题：胚胎成纤维细胞 + 逆转录病毒　； 参考：《南方医科大学》2012年硕士论文

【摘要】：多潜能干细胞(pluripotent stem cells)是指能够自我更新并具有分化成多种细胞潜能的细胞,它在细胞替代治疗、基因治疗、发育生物学等研究中具有独特的作用及优越性。目前,最为人类所熟知的多潜能性干细胞是胚胎干细胞(embryonic stem cells, ES细胞),它一般是从囊胚的内细胞团经过适当的培养而产生,也可通过体细胞核转移或细胞融合等途径得到多潜能性干细胞。然而胚胎干细胞的研究,尤其是人胚胎干细胞,都需要破坏胚胎或卵细胞,引发诸多伦理争议,应用于临床时还将面临免疫排斥等问题。诱导性多能干细胞(induced pluripotent stem cells, iPS细胞)的出现为解决上述难题提供了希望。与ESCs相比,iPS细胞有其独到的优势：①iPS由体细胞诱导产生,不存在ESCs面临的伦理问题；②iPS细胞来源广泛,iPS可来自患者体细胞(包括病变体细胞),经过克隆增殖分化可满足临床移植治疗数量上的要求；③来自患者的iPS避免了一直困扰再生医学界的免疫排斥难题。因此自iPS技术诞生以来,其在诱导效率、安全性及临床应用等相关研究中取得了巨大的进展。本研究利用逆转录病毒介导的方法将三个干细胞因子Oct4、Sox2、Klf4导入小鼠胚胎成纤维细胞(mouse embryonic fibroblasts, MEFs),成功获得了小鼠iPS细胞,并对其干细胞标记的表达及全能性进行了分析鉴定。 目的：1.利用Yamanaka三因子(Oct4、Sox2、Klf4)构建小鼠诱导性多能干细胞系。2.对所获得的iPS细胞进行鉴定及全能性分析,以期获得与胚胎干细胞类似的多能干细胞,为本课题后续将iPS细胞进一步定向分化为胰岛样细胞提供实验基础和技术平台。 方法： 1.逆转录病毒的制备：包含Oct4,Sox2和K1f4三个基因的逆转录病毒质粒pMXs-Oct3、pMXs-Sox2、pMXs-Klf4及参照质粒pMXs-EGFP由Addgene公司购买,使用DH5α细菌进行扩增后将4个质粒分别转染至逆转录病毒包装细胞Plat-E,48h后收集病毒上清,过滤、浓缩后用于感染MEFs。 2.鼠胚成纤维细胞的分离与培养：无菌分离孕13.5d的BALB/C鼠胚,消化后接种至平皿培养。1-3代内的细胞用于iPS细胞的诱导。 3.逆转录病毒感染MEF及iPS细胞的产生：将收集的病毒上清感染生长良好的MEFs,约1周时可首次看到iPS克隆,20d左右iPS克隆可长至做够大被挑取扩大培养。 4.iPS细胞的生物学特性鉴定：通过镜下观察、碱性磷酸酶染色、反转录PCR(RT-PCR)、免疫荧光实验及畸胎瘤形成实验等对iPS细胞形态、多能性基因表达情况、干细胞表面标记及全能性等进行鉴定分析。 结果： 1.逆转录病毒的制备：逆转录病毒质粒转染Plat-E细胞48h后,荧光显微镜下可见绿色荧光表达(pMXs-EGFP),转染效率约在50-70%之间,细胞状态良好。 2.鼠胚成纤维细胞的分离与培养：MEFs原代培养在接种后3-4h贴壁,细胞呈长梭形、多边形或不规则形,中间1-2个圆形或卵圆形的核,原代MEF杂细胞较多,随着传代次数的增多,其纯度增高,一般第二代后即可得到较纯的细胞。 3.逆转录病毒感染MEFs及iPS细胞的产生：包含三个干细胞基因的逆转录病毒感染MEFs后大约一周左右可以在镜下首次看到iPS克隆,而后克隆继续增殖,12d左右形成肉眼可见的iPS集落,16-20d时克隆长至足够大,此时进行挑取、消化、接种至饲细胞上传代培养。 4.iPS细胞的生物学特性鉴定：从MEFs获得的iPSCs镜下观察呈典型的克隆状生长,圆形或椭圆形,与饲细胞分界清楚；RT-PCR、碱性磷酸酶染色及免疫荧光检测iPS细胞高表达胚胎干细胞相关基因及蛋白；种植在免疫缺陷鼠体内能够形成向内中外三个胚层分化的畸胎瘤,表明iPSCs具有多潜能性。未感染逆转录病毒的MEFs不具有上述ES相关的细胞特性。 结论： 1.通过转导三个重编程因子(Oct4、Sox2、Klf4)可以将小鼠胚胎成纤维细胞诱导为多能干细胞,即iPS细胞。 2.该iPS细胞具有ES样的生物学特性,并能长期多次传代而维持,表明初步建立了小鼠iPS细胞系。
[Abstract]:Pluripotent stem cells is a cell that is self renewing and has the potential to differentiate into multiple cells. It has a unique role and superiority in cell replacement therapy, gene therapy and developmental biology. At present, the most human known pluripotent stem cells are embryonic stem cells (embryonic stem CE). LLS, ES cells), it is usually produced by proper culture of the inner cell mass of the blastocyst, and can also obtain pluripotent stem cells through the transfer of somatic cell nucleus or cell fusion. However, embryonic stem cells, especially human embryonic stem cells, need to destroy embryo or egg cells and cause a lot of ethical disputes to be applied to the clinic. The emergence of inducible pluripotent stem cells (induced pluripotent stem cells, iPS cells) provides hope for solving the above problems. Compared with ESCs, iPS cells have their unique advantages: (1) iPS is induced by somatic cells, and there is no ethical problem in ESCs face; (2) iPS cells are widely derived and iPS can come. From patients' somatic cells (including somatic cells), cloned proliferation and differentiation can meet the requirements of clinical transplantation treatment. (3) iPS from patients has avoided the problem of immune rejection that has been puzzling the regenerative medicine community. Since the birth of iPS technology, it has made great achievements in the related research of induction efficiency, safety and clinical application. Great progress. In this study, three stem cell factors Oct4, Sox2, and Klf4 were introduced into mouse embryonic fibroblasts (mouse embryonic fibroblasts, MEFs) by retrovirus mediated method. The mouse iPS cells were successfully obtained and the expression and omnipotency of the stem cell markers were analyzed and identified.
Objective: 1. using Yamanaka three factor (Oct4, Sox2, Klf4) to construct mouse induced pluripotent stem cell line.2. to identify and analyze the acquired iPS cells, in order to obtain the pluripotent stem cells similar to embryonic stem cells, and provide experimental basis and technology for further directing the differentiation of iPS cells into islet like cells. Platform.
Method锛，

本文编号：1864694