克服多能干细胞移植免疫排斥反应的实验研究

发布时间：2018-01-18 05:26

  本文关键词：克服多能干细胞移植免疫排斥反应的实验研究　出处：《第四军医大学》2009年博士论文　论文类型：学位论文

  更多相关文章： 多能干细胞 胚胎干细胞 人白细胞抗原G 免疫排斥 重编程

【摘要】： 多能干细胞(Pluripotent Stem Cells)主要包括胚胎干细胞和诱导多潜能干细胞(induced pluripotent stem cell, iPS),均具有自我复制及多潜能分化的能力。理论上,它们能够为细胞移植治疗提供无限的供体细胞来源,因此是理想的“种子细胞”。然而,免疫排斥反应是细胞移植治疗面临的共同问题,当然是多能干细胞移植治疗首先需要考虑的问题。本课题根据两种多能干细胞的不同特点,为解决免疫排斥进行了以下两个方面尝试: 一、通过在人胚胎干细胞上稳定表达人白细胞抗原G(Human Leucocyte Antigen G,HLA-G),获得具有免疫耐受能力的通用的种子细胞。HLA-G是母胎免疫及器官移植免疫耐受的重要分子。本研究建立了稳定表达HLA-G1的hES细胞系。该细胞系核型正常,仍具有自我复制和多潜能性(表达干细胞特异性的转录因子Oct4和Sox2,接种SCID小鼠后能够形成畸胎瘤,其中有三胚层的分化细胞)。由HLA-G1+hESCs分化为神经前体细胞(Neural Progenitor cells,NPCs)的过程中,HLA-G1持续表达,通过细胞毒实验及混合淋巴细胞反应证实HLA-G1对由hESCs分化获得的NPCs具有明显的免疫保护作用。 二、提高建立iPS细胞(induced pluripotent stem cells,iPS细胞)的效率,同时探讨iPS细胞形成的机制。iPS细胞是个体特异性多能干细胞,可完全避免个体的免疫排斥反应。本研究采用人胚胎神经干细胞作为重编程对象,与传统的四分子转染体系不同,我们用Oct4进行单分子转染。转染1周后出现iPS细胞克隆,2周统计iPS细胞的形成效率为0.015±0.001,比成纤维细胞效率高10倍。对获得的iPS细胞与hES细胞进行比较鉴定后发现,二者在表达谱、重要标志物以及DNA甲基化方式和多潜能分化能力等方面都非常相似。由此,本研究仅采用了一个分子就获得了人iPS细胞,同时神经干细胞重编程获得iPS细胞的效率远高于成纤维细胞。
[Abstract]:The pluripotent stem cells (Pluripotent Stem cells) mainly include embryonic stem cells and induced multipotential stem cells. Induced pluripotent stem cell. IPSs are capable of self-replication and multipotential differentiation. In theory, they provide an unlimited source of donor cells for cell transplantation therapy and are therefore ideal "seed cells". Immune rejection is a common problem in cell transplantation therapy, of course, it is the first problem to be considered in the treatment of pluripotent stem cell transplantation. According to the different characteristics of two kinds of pluripotent stem cells. In order to solve the immune rejection, the following two aspects have been attempted: Firstly, human Leucocyte Antigen HLA-Gs were stably expressed on human embryonic stem cells. HLA-G is an important molecule in maternal and fetal immunity and organ transplantation tolerance. A stable hES cell line expressing HLA-G1 was established in this study. The karyotype was normal. Still self-replicating and multipotent (expressing stem cell-specific transcription factors Oct4 and Sox2), teratoma can be formed after inoculation with SCID mice. In the process of differentiation from HLA-G1 hESCs to neural Progenitor cells (NPCs). The expression of HLA-G1 was sustained. The results of cytotoxicity assay and mixed lymphocyte reaction confirmed that HLA-G1 had a significant protective effect on NPCs derived from hESCs differentiation. Second, to improve the efficiency of establishing iPS cells induced pluripotent stem cells. The mechanism of iPS cell formation. IPS cells are individual specific pluripotent stem cells, which can completely avoid individual immune rejection. In this study, human embryonic neural stem cells were used as reprogramming objects. Different from the traditional four-molecule transfection system, we used Oct4 for monolayer transfection. After one week of transfection, iPS cell clones appeared. At 2 weeks, the efficiency of iPS cells was 0.015 卤0.001, which was 10 times higher than that of fibroblasts. The iPS cells were compared with hES cells. They are very similar in expression profile, important markers, DNA methylation mode and multipotential differentiation ability. Therefore, human iPS cells were obtained by using only one molecule in this study. At the same time, neural stem cells were reprogrammed to obtain iPS cells more efficiently than fibroblasts.
【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R329

【参考文献】

相关期刊论文 前1条

1 Martin Oudega;Bas Blits;;GENETIC ENGINEERING NEURAL STEM CELL MODIFIED BY LENTIVIRUS FOR REPAIR OF SPINAL CORD INJURY IN RATS[J];Chinese Medical Sciences Journal;2006年02期

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本文编号：1439650