RIPK1基因敲除細胞模型的構建及功能研究
發(fā)布時間:2018-06-16 03:27
本文選題:RIPK1 + CRISPR ; 參考:《河北大學》2017年碩士論文
【摘要】:RIP家族在介導細胞炎癥應答、氧化應激及死亡中扮演關鍵角色,其中RIPK1作為NF-κB通路上游重要調控因子,涉及炎癥和死亡的多條細胞通路。表皮細胞構成了人體最外層屏障,是環(huán)境刺激尤其是輻射的主要作用點。我們在前期工作中發(fā)現(xiàn),輻射誘導表皮細胞死亡、炎癥反應及氧化應激而造成損傷,因此我們以常用的表皮細胞模型Ha CaT為靶細胞,探究RIPK1在輻射誘導的表皮細胞炎癥反應和死亡中的調節(jié)作用。構建RIPK1基因穩(wěn)定敲除的細胞模型是研究其功能的基礎,而CRISPR/Cas系統(tǒng)作為新型基因編輯技術,可對基因信息進行快速精準修飾,具有明顯的優(yōu)勢。本研究中,我們利用CRISPR/Cas9技術構建了RIPK1穩(wěn)定敲除的Ha Ca T細胞株,在此基礎上對RIPK1缺失時HaCaT細胞生物學特性進行初步探究。主要分為以下兩部分:第一部分:完全敲除RIPK1基因的Ha CaT細胞模型的構建。首先針對GenBank中人RIPK1基因序列,設計靶向RIPK1不同外顯子的4條sg RNA,并分別將其克隆至SpCas9-2A-Puro V2.0(PX459)載體中,獲得4個PX459-sgRNA基因敲除載體。利用表達綠色熒光蛋白的pSpCas9n(BB)-2A-GFP(PX461)優(yōu)化細胞轉染條件,并通過野生型Ha CaT細胞確定嘌呤霉素篩選濃度。在上述實驗結果的基礎上,將重組載體PX459-sgRNA分別轉染Ha CaT細胞,嘌呤霉素篩選并提取混合克隆蛋白并通過免疫印跡鑒定,確定RIPK1敲除效率最高的重組載體。將上述敲除效率最高的混合克隆,通過有限稀釋法獲得單克隆細胞株,免疫印跡和DNA測序技術進一步鑒定RIPK1敲除效果最佳的單克隆。結果表明,成功構建了靶向人類RIPK1基因的高效敲除載體PX459-sgRNA2,并獲得RIPK1完全敲除的HaCa T細胞模型。第二部分:RIPK1缺失對HaCaT細胞生物學特性的影響。CCK-8實驗檢測RIPK1缺失對細胞增殖能力的影響。流式細胞術檢測RIPK1的缺失對細胞周期及細胞凋亡的影響。分別用TNF-α處理HaCaTWT細胞和HaCaTRIP1KO細胞,流式細胞儀檢測敲除RIPK1對TNF-α誘導細胞死亡的影響,進一步通過caspase抑制劑Z-VAD-FMK判斷細胞死亡類型,Western印跡檢測NF-κB通路和凋亡相關蛋白的活化。此外,分別用UVB照射HaCaTWT細胞和HaCa TRIP1KO細胞,CCK-8實驗檢測UVB照射對兩種細胞活力的影響,ELISA檢測炎性因子IL-1α的表達,Western印跡檢測NF-κB通路、P38 MAPK通路及凋亡相關蛋白的活化。結果顯示敲除RIPK1對靜息期Ha CaT細胞形態(tài)及細胞凋亡未產(chǎn)生明顯影響,但導致細胞增殖能力減慢及細胞周期G2M期的阻滯;與野生型細胞相比,HaCaTRIP1KO對TNF-α誘導的細胞凋亡異常敏感,可能與NF-κB通路的抑制相關;此外,敲除RIPK1導致UVB對細胞的生長抑制更加顯著,且上調炎性因子IL-1α的表達,P38 MAPK和NF-κB通路可能參與該過程。本研究成功構建了RIPK1穩(wěn)定敲除的HaCa T細胞模型,并對RIPK1的生物學功能進行了探究,不僅為進一步探明表皮細胞中RIPK1對促存活信號的調控作用,也為探究輻射損傷機制及相關藥物靶點開發(fā)奠定了工作基礎。
[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.
【學位授予單位】:河北大學
【學位級別】:碩士
【學位授予年份】:2017
【分類號】:R818
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相關期刊論文 前2條
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2 李莉;閆言;;受體相互作用蛋白激酶RIP1在細胞信號傳導途徑中作用的研究進展[J];基礎醫(yī)學與臨床;2011年10期
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