本文選題：RIPK1 + CRISPR��； 參考：《河北大學(xué)》2017年碩士論文

【摘要】：RIP家族在介導(dǎo)細(xì)胞炎癥應(yīng)答、氧化應(yīng)激及死亡中扮演關(guān)鍵角色,其中RIPK1作為NF-κB通路上游重要調(diào)控因子,涉及炎癥和死亡的多條細(xì)胞通路。表皮細(xì)胞構(gòu)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(diǎn)。我們?cè)谇捌诠ぷ髦邪l(fā)現(xiàn),輻射誘導(dǎo)表皮細(xì)胞死亡、炎癥反應(yīng)及氧化應(yīng)激而造成損傷,因此我們以常用的表皮細(xì)胞模型Ha CaT為靶細(xì)胞,探究RIPK1在輻射誘導(dǎo)的表皮細(xì)胞炎癥反應(yīng)和死亡中的調(diào)節(jié)作用。構(gòu)建RIPK1基因穩(wěn)定敲除的細(xì)胞模型是研究其功能的基礎(chǔ),而CRISPR/Cas系統(tǒng)作為新型基因編輯技術(shù),可對(duì)基因信息進(jìn)行快速精準(zhǔn)修飾,具有明顯的優(yōu)勢(shì)。本研究中,我們利用CRISPR/Cas9技術(shù)構(gòu)建了RIPK1穩(wěn)定敲除的Ha Ca T細(xì)胞株,在此基礎(chǔ)上對(duì)RIPK1缺失時(shí)HaCaT細(xì)胞生物學(xué)特性進(jìn)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(xì)胞模型的構(gòu)建。首先針對(duì)GenBank中人RIPK1基因序列,設(shè)計(jì)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(gè)PX459-sgRNA基因敲除載體。利用表達(dá)綠色熒光蛋白的pSpCas9n(BB)-2A-GFP(PX461)優(yōu)化細(xì)胞轉(zhuǎn)染條件,并通過野生型Ha CaT細(xì)胞確定嘌呤霉素篩選濃度。在上述實(shí)驗(yàn)結(jié)果的基礎(chǔ)上,將重組載體PX459-sgRNA分別轉(zhuǎn)染Ha CaT細(xì)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(xì)胞株,免疫印跡和DNA測(cè)序技術(shù)進(jìn)一步鑒定RIPK1敲除效果最佳的單克隆。結(jié)果表明,成功構(gòu)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(xì)胞模型。第二部分:RIPK1缺失對(duì)HaCaT細(xì)胞生物學(xué)特性的影響。CCK-8實(shí)驗(yàn)檢測(cè)RIPK1缺失對(duì)細(xì)胞增殖能力的影響。流式細(xì)胞術(shù)檢測(cè)RIPK1的缺失對(duì)細(xì)胞周期及細(xì)胞凋亡的影響。分別用TNF-α處理HaCaTWT細(xì)胞和HaCaTRIP1KO細(xì)胞,流式細(xì)胞儀檢測(cè)敲除RIPK1對(duì)TNF-α誘導(dǎo)細(xì)胞死亡的影響,進(jìn)一步通過caspase抑制劑Z-VAD-FMK判斷細(xì)胞死亡類型,Western印跡檢測(cè)NF-κB通路和凋亡相關(guān)蛋白的活化。此外,分別用UVB照射HaCaTWT細(xì)胞和HaCa TRIP1KO細(xì)胞,CCK-8實(shí)驗(yàn)檢測(cè)UVB照射對(duì)兩種細(xì)胞活力的影響,ELISA檢測(cè)炎性因子IL-1α的表達(dá),Western印跡檢測(cè)NF-κB通路、P38 MAPK通路及凋亡相關(guān)蛋白的活化。結(jié)果顯示敲除RIPK1對(duì)靜息期Ha CaT細(xì)胞形態(tài)及細(xì)胞凋亡未產(chǎn)生明顯影響,但導(dǎo)致細(xì)胞增殖能力減慢及細(xì)胞周期G2M期的阻滯;與野生型細(xì)胞相比,HaCaTRIP1KO對(duì)TNF-α誘導(dǎo)的細(xì)胞凋亡異常敏感,可能與NF-κB通路的抑制相關(guān);此外,敲除RIPK1導(dǎo)致UVB對(duì)細(xì)胞的生長(zhǎng)抑制更加顯著,且上調(diào)炎性因子IL-1α的表達(dá),P38 MAPK和NF-κB通路可能參與該過程。本研究成功構(gòu)建了RIPK1穩(wěn)定敲除的HaCa T細(xì)胞模型,并對(duì)RIPK1的生物學(xué)功能進(jìn)行了探究,不僅為進(jìn)一步探明表皮細(xì)胞中RIPK1對(duì)促存活信號(hào)的調(diào)控作用,也為探究輻射損傷機(jī)制及相關(guān)藥物靶點(diǎn)開發(fā)奠定了工作基礎(chǔ)。
[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(xué)位授予單位】：河北大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R818

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 常喜喜;成祥;王麗麗;王宇;張毅;陳國(guó)柱;于繼云;;RIP1介導(dǎo)腫瘤壞死因子α誘導(dǎo)的L929細(xì)胞凋亡與程序性壞死[J];中國(guó)細(xì)胞生物學(xué)學(xué)報(bào);2016年10期

2 李莉;閆言;;受體相互作用蛋白激酶RIP1在細(xì)胞信號(hào)傳導(dǎo)途徑中作用的研究進(jìn)展[J];基礎(chǔ)醫(yī)學(xué)與臨床;2011年10期

，

本文編號(hào)：2025061