TAT蛋白转运结构域介导的重组蛋白诱导成体细胞重编程

发布时间：2018-03-15 11:18

本文选题：诱导多能干细胞　切入点：重编程　出处：《华东师范大学》2013年博士论文　论文类型：学位论文

【摘要】：诱导干细胞(induced pluripotent stem cell,iPS或iPSCs)的建立在再生医学领域有着极大的应用潜力。但是,在该技术应用于临床之前,安全与效率是其必须应对的重要问题。众多研究致力于推进以上问题的解决：介导重编程因子转染供体细胞的方法由最初的整合型病毒(逆转录病毒、慢病毒)到非整合型病毒(腺病毒),继而发展到无病毒(如PB转座子系统、episome),直至穿膜肽介导的目的因子融合蛋白直接转运至细胞内。09年4月,Zhou等人利用11精氨酸穿膜肽介导四因子融合蛋白建立了小鼠蛋白iPS细胞系(protin-iPS, p-iPS),同年5月Kim等人利用同方法建立了人p-iPS。由于蛋白诱导iPS彻底排除了外源遗传物质,被认为是目前为止最安全的iPS诱导方法。但是,以上的非整合型的诱导方法效率低、耗时长、不稳定,增加了细胞变异的可能,限制了iPS在临床和研究方面的应用。 TAT-PTD是迄今为止应用最为广泛的转膜肽,大量研究表明该系统能够介导多种融合蛋白高效转染不同种类的细胞且重折叠后的外源蛋白多具有生物学活性。我们尝试检测TAT-PTD转导系统是否可以促进蛋白iPS细胞系的建立。在本工作中,我们运用TAT蛋白转导结构域构建了TAT-PTD介导的蛋白转运系统。通过原核表达His-TAT-Sox2/Oct4/Klf4/c-Myc/Nanog等多能因子融合蛋白；从TAT融合蛋白转染条件、工作浓度、孵育时间等方面优化转染效果,并检测融合蛋白在细胞内的稳定性；在多细胞系上测试TAT-目的蛋白的转染效果以测定所得优化条件的稳定性和通用性。在所得条件下,于成体细胞重编程系统中比较TAT和11精氨酸(11R)蛋白转导结构域的异同,并探索基于蛋白转导结构域的重编程因子(reprogramming factor, RF)重组蛋白诱导iPS的条件。我们发现：TAT和11R融合重编程因子具有转录活性,在荧光素报告系统中能够激活其对应的下游靶基因的报告基因。TAT-RFs在转录活性上要优于相应的11R-RFs,但弱于相应的病毒因子。在我们构建的“3病毒+1蛋白”重编程系统中,TAT-RF能够一定程度上替代相应病毒因子的作用,同时利用该实验系统最终确定各TAT-转录因子在重编程过程中的工作条件,确定了TAT-转录因子融合蛋白(4因子)诱导iPS的诱导策略和具体条件：四个TAT-RFs因子(各50nM工作浓度,每次孵育2h)每48h孵育HFFs细胞至第17天。遗憾的是,在以上实验条件下,我们并没有获得iPS状克隆(OCT4, NANOG和AP阳性)。这个结果暗示TAT-RFs的转录活性或许相较对应病毒因子较弱。我们又进行了进一步的探索：增加TAT-mNanog和联合使用小分子组蛋白去乙酰化酶抑制剂VPA来增强重组蛋白的重编程活性。在此方法下,两周即可出现与iPS形态相似的克隆。经检测,这些克隆具iPS形态学特征,呈AP染色阳性,在转录水平和蛋白水平上表达多能相关基因。这说明TAT介导的融合蛋白转录因子在较短时间内起始了人成体细胞HFFs的重编程过程,且AP阳性克隆得率与病毒诱导iPS的效率相当。我们的工作的兴趣点在于：利用不同系统中(如从荧光素报告系统,“3病毒+1蛋白”重编程系统)摸索PTD-融合蛋白转染条件、工作浓度、孵育时间等方面的合适条件,探索优化蛋白iPS诱导策略。针对蛋白转录因子转录活性可能低于相应病毒转录因子这一制约蛋白-iPS应用的瓶颈,我们增加TAT-mNanog和联合使用小分子组蛋白去乙酰化酶抑制剂VPA来增强重组蛋白的重编程活性,从而在细胞水平上(AP染色,多能因子在转录水平和蛋白水平的表达)证明说明TAT介导的融合蛋白转录因子在较短时间内起始了人成体细胞HFFs的重编程过程,且AP阳性克隆得率与病毒诱导iPS的效率相当。我们的工作对TAT融合重编程因子在重编程过程中的系统检测,可以为建立具临床应用价值和不含外源遗传物质的人iPSC的获得提供依据。
[Abstract]:The establishment of induced pluripotent stem cell (iPS or iPSCs) has great potential in the field of regenerative medicine. However, before the technology is applied to clinic, safety and efficiency are important issues that it must deal with.
To solve many studies dedicated to advancing the above problems: mediated method of reprogramming factor transfected donor cells is integrated by virus first (retroviral, lentiviral) to the non integrated virus (adenovirus), and then to no virus (such as PB transposon system, episome), until the transmembrane peptide mediated the objective factor fusion protein is directly transported to the cell.09 in April, Zhou et al use 11 arginine peptide mediated four factor fusion protein was established iPS cell mouse protein (protin-iPS, p-iPS) system, Kim et al in May of the same year is established by using p-iPS. as the protein iPS induced by eliminating the exogenous genetic material with method is considered to be by far the most secure iPS induced method. However, the non integrated induction method above the low efficiency, time-consuming, instability, increased cell variation may limit the application of iPS in clinical and research.
TAT-PTD is by far the most widely used transmembrane peptides, a large number of studies show that the system can mediate a variety of exogenous protein fusion cell transfection efficiency of different types and protein after refolding of biologically active. We try to establish whether the system can promote the detection of TAT-PTD transduction protein iPS cell line. In this work, we the use of TAT protein transduction domain constructs protein delivery system mediated by TAT-PTD. The prokaryotic expression of His-TAT-Sox2/Oct4/Klf4/c-Myc/Nanog multi factor fusion protein fusion protein; transfection conditions, from the concentration of TAT, optimization of transfection effect of incubation time, stability and detection of fusion protein in cells; to determine the stability of the optimal conditions and the general test TAT- protein transfection effect in multicellular system. In the condition, in a somatic cell reprogramming system In comparison with TAT and 11 arginine (11R) and the protein transduction domain, and to explore the reprogramming factors based on protein transduction domain (reprogramming factor RF) iPS recombinant protein induced conditions. We found that TAT and 11R fusion of reprogramming factors with transcription activity, reporter gene.TAT-RFs can activate downstream target genes the corresponding report in fluorescein in the system is superior to the corresponding 11R-RFs in transcriptional activity, but weaker than the corresponding viral factors. In our construction of the "3 virus +1 protein" TAT-RF programming system, to a certain extent for the generation of the corresponding virus factors, at the same time using the experimental system to determine the final TAT- transcription factor working conditions in the reprogramming process, determine the TAT- transcription factor fusion protein (factor 4) induction strategy and specific conditions induced by iPS: four TAT-RFs factors (each 50nM work each concentration. 2H incubation of HFFs cells incubated with 48h) each to seventeenth days. Unfortunately, in the above experimental conditions, we did not get a iPS like clone (OCT4, NANOG and AP positive). The results suggest that the transcriptional activity of TAT-RFs may be compared with corresponding virus factor is weak. We have made further exploration: the increase of TAT-mNanog and the combined use of small molecule inhibitors of histone deacetylase VPA to enhance recombinant protein reprogramming activity. In this way, two weeks can appear similar to the iPS clone form. After testing, these clones with iPS morphology, AP staining was positive, multiple related gene expression at the transcriptional level and protein level this shows that the reprogramming process. Transcription factor TAT mediated fusion in a relatively short period of time starting the adult human HFFs cells, and the efficiency of AP positive clones yield and virus induced iPS.
Our work points of interest lies in using different systems (such as fluorescein from reporting system, "3 virus +1 protein reprogramming system) explore PTD- fusion protein transfection conditions, concentration, incubation time and suitable conditions, explore the optimal strategy for protein protein iPS induced transcriptional activity may be lower than the bottleneck the corresponding virus transcription factor that restricts the application of the -iPS protein, we added TAT-mNanog and combined use of small molecule inhibitors of histone deacetylase VPA to enhance recombinant protein reprogramming activity, resulting in cell level (AP staining, multi factor expression at the transcriptional level and protein level) that reprogramming process transcription factor TAT mediated fusion in a relatively short period of time starting the adult human HFFs cells, and the efficiency of AP positive clones yield and virus induced iPS.
Our work on systematic detection of TAT fusion reprogramming factor in reprogramming process can provide a basis for establishing clinical application value and human iPSC without exogenous genetic material.

【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R318.0

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