利用TALEN技术建立低免疫原性的人类胚胎干细胞

发布时间：2017-12-28 00:24

本文关键词：利用TALEN技术建立低免疫原性的人类胚胎干细胞　出处：《湖北工业大学》2017年硕士论文　论文类型：学位论文

【摘要】：器官或细胞移植是治疗脏器器官终末病变的根本方法,然而供体细胞表面的人类白细胞抗原(Human leukocyte antigens,HLAs)与受体间的不兼容性是导致供体器官短缺及器官移植失败的最主要原因。人类的胚胎干细胞特点是在体外培养条件下具有无限增殖及多向分化潜能,是一种理想的种子细胞,但是若直接将由胚胎干细胞分化的功能性细胞用于异体移植,却存在引发机体免疫排斥反应的风险,免疫排斥极大地阻碍了人类胚胎干细胞在临床上的应用[1]。本研究设计并构建了特异性识别人类胚胎干细胞系X2的HLA-A以及HLA-B5101基因的类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALENs)[2],从多对打靶TALENs中筛选出效率较高的TALENs在X2细胞系首先进行了HLA-A基因的敲除,建立了HLA-A在基因和蛋白水平均表达缺失的人类胚胎干细胞系X2#1,进而在该X2#1细胞系基础上,又利用靶向HLA-B5101基因的TALENs进行了第二轮基因敲除,在基因水平敲除了HLA-B5101基因。本研究在人类胚胎干细胞系中特异性敲除了人类白细胞抗原I类分子的HLA-A以及HLA-B5101基因,建立了HLA-A以及HLA-B5101表达缺失的人类胚胎干细胞系,该细胞系有望用作低免疫原性的种子细胞。本研究将有利于推动人类胚胎干细胞的临床应用,促进基于人类胚胎干细胞的移植治疗的应用发展。
[Abstract]:Organ or cell transplantation is the basic way to treat organ and organ terminal lesions. However, the incompatibility between Human leukocyte antigens (HLAs) and receptors is the main reason leading to donor organ shortage and failure of organ transplantation. Human embryonic stem cells in culture is characterized with unlimited proliferation and differentiation in vitro under, is a kind of ideal seed cells, but functional cells if directly by differentiation of embryonic stem cells for allogeneic transplantation, has raised the risk of immune rejection, immune rejection greatly hindered human embryonic stem the application of [1] cells in clinic. This study design and construct the specific recognition of human embryonic stem cell line HLA-A X2 and HLA-B5101 gene transcription activator effector nuclease (Transcription activator-like effector nuclease, TALENs [2]), from the targeting of TALENs screened high efficiency of TALENs in X2 cell line was the first knockout of HLA-A. A HLA-A expression in both gene and protein levels of human embryonic stem cell line X2#1 deletion, and then in the X2#1 cell line basis, and by targeting HLA-B5101 gene TALENs in the second round knockout, in the level of gene knock out the HLA-B5101 gene. In this study, we specifically knocked out the HLA-A and HLA-B5101 genes of human leukocyte antigen class I molecules in human embryonic stem cell lines, and established HLA-A and HLA-B5101 deficient human embryonic stem cell lines. This cell line is expected to be used as a low immunogenic seed cell. This study will help promote the clinical application of human embryonic stem cells and promote the application and development of transplantation therapy based on human embryonic stem cells.
【学位授予单位】：湖北工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R329.2

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