PIDD对电离辐射所致BV-2细胞炎症反应的调节作用及其机制的初步研究

发布时间：2018-06-25 00:30

本文选题：PIDD + PIDD-C　；参考：《华中科技大学》2013年硕士论文

【摘要】：【背景与目的】：P53诱导含有死亡结构域蛋白（PIDD,P53-inducedproteinwith a death domain）作为肿瘤抑制因子P53的靶蛋白之一，在调节细胞周期和细胞凋亡中发挥重要作用。但是PIDD在小胶质细胞电离辐射所导致的炎症反应中作用与机制的研究未见报道，本研究旨在探讨PIDD在小鼠小胶质细胞（BV-2）中的表达及其在电离辐射后炎症反应中的作用与机制。【方法】：1，针对小鼠来源的PIDD序列设计3条抗PIDD反义寡核苷酸（anti-PIDD-si-RNA，antisense oligonucleotides targeting PIDD mRNA）及其阴性对照反义寡核苷酸。2，使用带有GFP荧光蛋白标记的空白质粒与siRNA共转染BV-2细胞，转染后，使用荧光显微镜观察转染效率；使用实时荧光定量PCR淘选出沉默效率最高的anti-PIDD-si-RNA。3，使用沉默效率最高的anti-PIDD-si-RNA及阴性对照siRNA分别转染BV-2细胞后12小时，以0和32Gy两个放射剂量照射BV-2细胞。4，实时荧光定量PCR检测各组细胞照射后不同时间点(0h,3h,6h,12h,24h)PIDD及TNF-α，IL-1β（炎症反应因子）的mRNA变化。5，免疫蛋白印迹法（western blotting）检测各组细胞PIDD蛋白，NF-κB蛋白的表达。6，激光共聚焦显微镜观察各组细胞Iba-1及CD68（小胶质细胞激活标志蛋白）的表达。7，transwell小室实验观察各组细胞迁移能力变化。8，电子显微镜观察各组细胞照射后超微结构变化。【结果】：1，siRNA转染效率可以达到50%至60%，anti-PIDD-si-RNA2沉默效率最高。2，BV-2细胞电离辐射后其PIDD，TNF-α及IL1-β的mRNA水平升高；转染anti-PIDD-si-RNA2的BV-2细胞较转染阴性对照组细胞，照射后PIDD，TNF-α及IL1-β的mRNA水平明显下调。3，BV-2细胞电离辐射后PIDD蛋白表达随着时间变化（24h内）先升高后降低，NF-κB蛋白表达持续升高；转染anti-PIDD-si-RNA2的BV-2细胞较转染阴性对照组细胞PIDD的蛋白表达较对照组下降，NF-κB蛋白表达下降。4，BV-2细胞照射后，Iba-1，CD68表达明显增加；转染anti-PIDD-si-RNA2的BV-2细胞较单纯对照组细胞，照射后Iba-1，CD68表达明显减少。5，照射后BV-2细胞迁移能力增强；转染anti-PIDD-si-RNA2的BV-2细胞较单纯照射组细胞的迁移能力明显减弱。6，BV-2细胞照射后次级溶酶体增多，可见高尔基体、内质网线粒体变型严重；转染anti-PIDD-si-RNA2的BV-2细胞较单纯照射组细胞次级溶酶体减少，，初级溶媒体比例增加，未见高尔基体，内质网及线粒体变性减轻。【结论】：下调PIDD可能通过PIDD/NF-κB信号转导途径抑制小胶质细胞激活及BV-2细胞炎症因子表达。
[Abstract]:Background & AIM: PIDD P53-induced protein with a death domain), as one of the target proteins of tumor suppressor p53, plays an important role in regulating cell cycle and apoptosis. However, the role and mechanism of PIDD in inflammatory response induced by ionizing radiation of microglia have not been reported. The purpose of this study was to investigate the expression of PIDD in mouse microglia (BV-2) and the role and mechanism of PIDD in inflammatory response after ionizing radiation. Anti-PIDD-si-RNAantisense oligonucleotides targeting PIDD mRNA and its negative control antisense oligodeoxynucleotides (AODN) were co-transfected into BV-2 cells using a blank plasmid labeled with GFP fluorescent protein and siRNA. After transfection, the transfection efficiency was observed by fluorescence microscope, the anti-PIDD-si-RNA.3was selected by real-time fluorescent quantitative PCR, the anti-PIDD-si-RNA with the highest silencing efficiency and the negative control siRNA were transfected into BV-2 cells 12 hours after transfection. BV-2 cells were irradiated with 0 and 32 Gy radiation doses. The changes of PIDD and TNF- 伪 IL-1 尾 (inflammatory response factor) mRNA at different time points (0 h ~ 3 h ~ 6 h ~ 12 h ~ 24 h) were detected by real-time fluorescence quantitative PCR. Western blot (western blotting) was used to detect PIDD protein in each group. Expression of NF- 魏 B protein .6. the expression of Iba-1 and CD68 (microglial activation marker protein) was observed by laser confocal microscopy. [results] the transfection efficiency of 1: 1 siRNA could reach 50% to 60% and the silencing efficiency of anti-PIDD-si-RNA2 was the highest. The mRNA levels of PIDD- TNF- 伪 and IL1- 尾 in BV-2 cells were increased after ionizing radiation. In BV-2 cells transfected with anti-PIDD-si-RNA2, the mRNA levels of TNF- 伪 and IL-1- 尾 in PIDD- TNF- 伪 and IL-1- 尾 decreased significantly after irradiation, and the expression of PIDD protein increased first (within 24 hours) and then increased continuously after irradiation. The protein expression of anti-PIDD-si-RNA2 transfected BV-2 cells was significantly higher than that of PIDD cells transfected with anti-PIDD-si-RNA2.The expression of anti-PIDD-si-RNA2 transfected BV-2 cells was significantly higher than that of control cells, and the expression of anti-PIDD-si-RNA2 transfected BV-2 cells was significantly higher than that of control cells. The migration ability of BV-2 cells transfected with anti-PIDD-si-RNA2 was significantly decreased after irradiation, and the secondary lysosomes of BV-2 cells transfected with anti-PIDD-si-RNA2 were significantly decreased after irradiation, and Golgi body could be seen in the BV-2 cells transfected with anti-PIDD-si-RNA2, and the migration ability of BV-2 cells transfected with anti-PIDD-si-RNA2 was significantly decreased. The endoplasmic reticulum mitochondrial aberration was severe, and the secondary lysosomes of BV-2 cells transfected with anti-PIDD-si-RNA2 were lower than those of the control group, and the proportion of primary lytic media was increased, and Golgi apparatus was not found in BV-2 cells transfected with anti-PIDD-si-RNA2. Endoplasmic reticulum and mitochondrial degeneration were alleviated. [conclusion] down-regulation of PIDD may inhibit the activation of microglia and the expression of inflammatory factors in BV-2 cells through PIDD / NF- 魏 B signal transduction pathway.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R818

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