RIPK1基因敲除细胞模型的构建及功能研究

发布时间：2018-06-16 03:27

本文选题：RIPK1 + CRISPR　；参考：《河北大学》2017年硕士论文

【摘要】：RIP家族在介导细胞炎症应答、氧化应激及死亡中扮演关键角色,其中RIPK1作为NF-κB通路上游重要调控因子,涉及炎症和死亡的多条细胞通路。表皮细胞构成了人体最外层屏障,是环境刺激尤其是辐射的主要作用点。我们在前期工作中发现,辐射诱导表皮细胞死亡、炎症反应及氧化应激而造成损伤,因此我们以常用的表皮细胞模型Ha CaT为靶细胞,探究RIPK1在辐射诱导的表皮细胞炎症反应和死亡中的调节作用。构建RIPK1基因稳定敲除的细胞模型是研究其功能的基础,而CRISPR/Cas系统作为新型基因编辑技术,可对基因信息进行快速精准修饰,具有明显的优势。本研究中,我们利用CRISPR/Cas9技术构建了RIPK1稳定敲除的Ha Ca T细胞株,在此基础上对RIPK1缺失时HaCaT细胞生物学特性进行初步探究。主要分为以下两部分:第一部分:完全敲除RIPK1基因的Ha CaT细胞模型的构建。首先针对GenBank中人RIPK1基因序列,设计靶向RIPK1不同外显子的4条sg RNA,并分别将其克隆至SpCas9-2A-Puro V2.0(PX459)载体中,获得4个PX459-sgRNA基因敲除载体。利用表达绿色荧光蛋白的pSpCas9n(BB)-2A-GFP(PX461)优化细胞转染条件,并通过野生型Ha CaT细胞确定嘌呤霉素筛选浓度。在上述实验结果的基础上,将重组载体PX459-sgRNA分别转染Ha CaT细胞,嘌呤霉素筛选并提取混合克隆蛋白并通过免疫印迹鉴定,确定RIPK1敲除效率最高的重组载体。将上述敲除效率最高的混合克隆,通过有限稀释法获得单克隆细胞株,免疫印迹和DNA测序技术进一步鉴定RIPK1敲除效果最佳的单克隆。结果表明,成功构建了靶向人类RIPK1基因的高效敲除载体PX459-sgRNA2,并获得RIPK1完全敲除的HaCa T细胞模型。第二部分:RIPK1缺失对HaCaT细胞生物学特性的影响。CCK-8实验检测RIPK1缺失对细胞增殖能力的影响。流式细胞术检测RIPK1的缺失对细胞周期及细胞凋亡的影响。分别用TNF-α处理HaCaTWT细胞和HaCaTRIP1KO细胞,流式细胞仪检测敲除RIPK1对TNF-α诱导细胞死亡的影响,进一步通过caspase抑制剂Z-VAD-FMK判断细胞死亡类型,Western印迹检测NF-κB通路和凋亡相关蛋白的活化。此外,分别用UVB照射HaCaTWT细胞和HaCa TRIP1KO细胞,CCK-8实验检测UVB照射对两种细胞活力的影响,ELISA检测炎性因子IL-1α的表达,Western印迹检测NF-κB通路、P38 MAPK通路及凋亡相关蛋白的活化。结果显示敲除RIPK1对静息期Ha CaT细胞形态及细胞凋亡未产生明显影响,但导致细胞增殖能力减慢及细胞周期G2M期的阻滞;与野生型细胞相比,HaCaTRIP1KO对TNF-α诱导的细胞凋亡异常敏感,可能与NF-κB通路的抑制相关;此外,敲除RIPK1导致UVB对细胞的生长抑制更加显著,且上调炎性因子IL-1α的表达,P38 MAPK和NF-κB通路可能参与该过程。本研究成功构建了RIPK1稳定敲除的HaCa T细胞模型,并对RIPK1的生物学功能进行了探究,不仅为进一步探明表皮细胞中RIPK1对促存活信号的调控作用,也为探究辐射损伤机制及相关药物靶点开发奠定了工作基础。
[Abstract]:The RIP family plays a key role in mediating cellular inflammatory response, oxidative stress and death, in which RIPK1 is an important upstream regulator of the NF- kappa B pathway, involving multiple cellular pathways of inflammation and death. Epidermal cells constitute the outer barrier of the human body and are the main points of environmental stimulation, especially radiating. We found in earlier work Radiation induced epidermal cell death, inflammatory response and oxidative stress, resulting in damage. Therefore, we use the common epidermal cell model Ha CaT as the target cell to explore the regulatory role of RIPK1 in the inflammatory response and death of radiation induced epidermal cells. The construction of a cell model for the stable knockout of RIPK1 gene is the basis for the study of its function, and CRISPR/ As a new gene editing technique, Cas system can make rapid and accurate modification of gene information, which has obvious advantages. In this study, we constructed a Ha Ca T cell line with stable knockout of RIPK1 by using CRISPR/Cas9 technology. On this basis, the biological characteristics of HaCaT cells were preliminarily explored in the absence of RIPK1, and the following two parts were mainly divided into the following parts: The first part: the construction of the Ha CaT cell model that completely knocks out the RIPK1 gene. First, we designed 4 SG RNA to target different exons of RIPK1 in GenBank, and cloned them into the SpCas9-2A-Puro V2.0 (PX459) carrier, and obtained 4 PX459-sgRNA gene knockout carriers. B) -2A-GFP (PX461) optimized the cell transfection conditions and determined the concentration of purinamycin through the wild Ha CaT cells. On the basis of the above experimental results, the recombinant vector PX459-sgRNA was transfected to Ha CaT cells respectively. The mixed cloned protein was screened and extracted by purinamycin, and the highest efficiency of the recombination was determined by immunoblotting. The most efficient hybrid clones, using the finite dilution method to obtain the monoclonal cell lines, the immunoblotting and DNA sequencing technology to further identify the RIPK1 knockout effect is the best monoclonal. The results show that the highly efficient knockout carrier PX459-sgRNA2 target to the human RIPK1 gene is successfully constructed and the HaCa T completely knockout of RIPK1 is obtained. Cell model. The second part: the effect of RIPK1 deletion on the biological characteristics of HaCaT cells.CCK-8 test was used to detect the effect of RIPK1 deletion on cell proliferation. Flow cytometry detected the effect of the deletion of RIPK1 on cell cycle and apoptosis. TNF- alpha was used to treat HaCaTWT and HaCaTRIP1KO cells, and the flow cytometry was used to detect RIPK1 The effect of TNF- - alpha induced cell death was further evaluated by caspase inhibitor Z-VAD-FMK, and Western blot was used to detect the activation of NF- kappa B pathway and apoptosis related protein. In addition, HaCaTWT cells and HaCa TRIP1KO cells were irradiated with UVB, and the effect of UVB irradiation on the activity of two cells was detected by CCK-8 experiment. The expression of factor IL-1 alpha and Western blot to detect the activation of NF- kappa B pathway, P38 MAPK pathway and apoptosis related protein. The results showed that knockout RIPK1 had no obvious effect on the morphology and apoptosis of Ha CaT cells in resting stage, but resulted in the decrease of cell proliferation and the retardation of the G2M period of cell cycle. Compared with wild type cells, HaCaTRIP1KO on TNF- alpha. The induced apoptosis is abnormal and may be associated with the inhibition of NF- kappa B pathway; besides, knockout RIPK1 leads to more significant inhibition of the growth of UVB cells, and up regulation of the expression of inflammatory factor IL-1 a. P38 MAPK and NF- kappa B pathway may be involved in this process. The function of physical culture is explored, not only to further explore the regulatory role of RIPK1 in epidermal cells to promote the survival signal, but also to explore the mechanism of radiation damage and the development of related drug targets.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R818

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