优化的小鼠STO细胞系重编程多潜能干细胞的建立

发布时间：2018-01-05 10:27

本文关键词：优化的小鼠STO细胞系重编程多潜能干细胞的建立　出处：《西北农林科技大学》2012年硕士论文　论文类型：学位论文

更多相关文章： 慢病毒载体 STO细胞 iPSCs 干细胞

【摘要】：诱导多功能干细胞（induced pluripotent stem cells，iPSCs）是指通过外源导入一些与维持胚胎干细胞（embryonic stem cell，ES细胞）多能性相关的基因，，使终末分化的成体细胞重编程为多能性干细胞，获得多向分化潜能和自我更新能力。iPS细胞目前可以用来替代干细胞治疗人类疾病，在分子细胞生物学、发育生物学、新药物的筛选和研发、临床细胞替代治疗等方面有着广阔的发展空间，但是在诱导效率和安全性方面存在一定不足，研究人员在不断寻找高效、安全的方法来弥补iPS细胞的缺陷。本研究采用连接有Oct4，Sox2，Klf4，c-Myc四个转录因子的慢病毒载体FUW-OSKM的诱导方法将鼠胚成纤维细胞系STO (SIM-6-thiogunanie-oualiain)重编程为STO-iPSCs。首先用磷酸钙法转染293FT细胞来包装慢病毒，然后用慢病毒感染STO细胞，以干细胞培养条件培养，诱导过程中加入了小分子物质丙戊酸（VPA，组蛋白去乙酰化抑制剂）。诱导15d后，挑取阳性细胞克隆并在无饲养层的培养体系中培养。对获取的STO-iPSCs进行生物学特性分析，包括形态学观察、碱性磷酸酶染色、免疫荧光染色、蛋白印迹检测、实时定量PCR和维甲酸定向诱导分化等，同时比较了STO-iPSCs冷冻复苏后在明胶和基质胶包被的培养皿中分别饲养时形态上的差异。研究结果：STO-iPSCs具有典型胚胎干细胞的形态特征；碱性磷酸酶染色呈阳性；实时定量PCR结果表明STO-iPSCs有很高的内源性Nanog，Oct4基因表达；免疫细胞染色进一步证明表达胚胎干细胞特异性标志Nanog和Oct4，并且能够体外诱导分化为神经细胞；基质胶包被的培养皿更适合STO-iPSCs的生长。综上所述表明，实验获得了STO-iPSCs，建立了STO-iPSCs无饲养层培养体系。本实验首次采用细胞系作为重编程研究的供体细胞，并且成功诱导鼠胚成纤维STO细胞系为STO-iPSCs，由于细胞系可以长期在体外培养传代和冻存复苏，在研究iPS细胞信号通路和药物检测等试验中，采用细胞系作为供体细胞，可以减少每次制备供体细胞的步骤，不但丰富了供体细胞种类，还有利于iPS细胞的产生，有助于实验研究的进展。
[Abstract]:Multifunctional stem cell induced pluripotent stem cells was induced. Pics is the introduction of some genes related to the maintenance of embryonic stem cells (es cells) by exogenous introduction. The terminal differentiation of adult cells is reprogrammed as pluripotent stem cells to obtain multidirectional differentiation potential and self-renewal ability. IPS cells can be used to replace stem cells in the treatment of human diseases in molecular cell biology. Developmental biology, screening and development of new drugs, clinical cell replacement therapy and other aspects have a broad development space, but there are certain deficiencies in the efficiency and safety of induction, researchers are constantly looking for high efficiency. A safe way to compensate for defects in iPS cells. In this study, Oct4 was connected with Sox2 and Klf4. Induction of lentivirus vector FUW-OSKM from four transcription factors of c-Myc the mouse embryo fibroblast cell line STO (. SIM-6-thiogunanie-oualiain (SIM-6-thiogunanie-oualiain) was reprogrammed as STO-iPSCs.Firstly, calcium phosphate was transfected into 293FT cells to package lentivirus. Then STO cells were infected with lentivirus and cultured in stem cell culture condition. The small molecule valproate was added in the induction process and histone deacetylation inhibitor was added after 15 days of induction. The positive cells were cloned and cultured in a culture system without feeder layer. The biological characteristics of the obtained STO-iPSCs were analyzed, including morphological observation, alkaline phosphatase staining and immunofluorescence staining. Western blotting, real-time quantitative PCR and retinoic acid induced differentiation. At the same time, the morphological differences of STO-iPSCs in culture dishes coated with gelatin and matrix were compared after freezing and resuscitation. Results\\\. Alkaline phosphatase staining was positive. The results of real-time quantitative PCR showed that STO-iPSCs had high endogenous Nanog-Oct4 gene expression. The expression of Nanog and Oct4, the specific markers of embryonic stem cells, was further demonstrated by immunocytochemistry, and differentiation into neural cells could be induced in vitro. The results showed that STO-i SCS was obtained and the STO-iPSCs culture system without feeding layer was established. In this study, the mouse embryo fibroblast STO cell line was induced to be STO-iPSCs for the first time. Because cell lines can be cultured and resuscitated in vitro for a long time, cell lines are used as donor cells in the study of iPS cell signaling pathway and drug detection. It can reduce the steps of preparing donor cells each time, which not only enriches the donor cell types, but also contributes to the production of iPS cells, which is helpful to the progress of experimental research.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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