發(fā)布時(shí)間：2018-06-25 00:30

  本文選題：PIDD + PIDD-C��； 參考：《華中科技大學(xué)》2013年碩士論文

【摘要】：【背景與目的】：P53誘導(dǎo)含有死亡結(jié)構(gòu)域蛋白（PIDD,P53-inducedproteinwith a death domain）作為腫瘤抑制因子P53的靶蛋白之一，在調(diào)節(jié)細(xì)胞周期和細(xì)胞凋亡中發(fā)揮重要作用。但是PIDD在小膠質(zhì)細(xì)胞電離輻射所導(dǎo)致的炎癥反應(yīng)中作用與機(jī)制的研究未見報(bào)道，本研究旨在探討PIDD在小鼠小膠質(zhì)細(xì)胞（BV-2）中的表達(dá)及其在電離輻射后炎癥反應(yīng)中的作用與機(jī)制。 【方法】：1，針對(duì)小鼠來源的PIDD序列設(shè)計(jì)3條抗PIDD反義寡核苷酸（anti-PIDD-si-RNA，antisense oligonucleotides targeting PIDD mRNA）及其陰性對(duì)照反義寡核苷酸。2，使用帶有GFP熒光蛋白標(biāo)記的空白質(zhì)粒與siRNA共轉(zhuǎn)染BV-2細(xì)胞，轉(zhuǎn)染后，使用熒光顯微鏡觀察轉(zhuǎn)染效率；使用實(shí)時(shí)熒光定量PCR淘選出沉默效率最高的anti-PIDD-si-RNA。3，使用沉默效率最高的anti-PIDD-si-RNA及陰性對(duì)照siRNA分別轉(zhuǎn)染BV-2細(xì)胞后12小時(shí)，以0和32Gy兩個(gè)放射劑量照射BV-2細(xì)胞。4，實(shí)時(shí)熒光定量PCR檢測各組細(xì)胞照射后不同時(shí)間點(diǎn)(0h,3h,6h,12h,24h)PIDD及TNF-α，IL-1β（炎癥反應(yīng)因子）的mRNA變化。5，免疫蛋白印跡法（western blotting）檢測各組細(xì)胞PIDD蛋白，NF-κB蛋白的表達(dá)。6，激光共聚焦顯微鏡觀察各組細(xì)胞Iba-1及CD68（小膠質(zhì)細(xì)胞激活標(biāo)志蛋白）的表達(dá)。7，transwell小室實(shí)驗(yàn)觀察各組細(xì)胞遷移能力變化。8，電子顯微鏡觀察各組細(xì)胞照射后超微結(jié)構(gòu)變化。 【結(jié)果】：1，siRNA轉(zhuǎn)染效率可以達(dá)到50%至60%，anti-PIDD-si-RNA2沉默效率最高。2，BV-2細(xì)胞電離輻射后其PIDD，TNF-α及IL1-β的mRNA水平升高；轉(zhuǎn)染anti-PIDD-si-RNA2的BV-2細(xì)胞較轉(zhuǎn)染陰性對(duì)照組細(xì)胞，照射后PIDD，TNF-α及IL1-β的mRNA水平明顯下調(diào)。3，BV-2細(xì)胞電離輻射后PIDD蛋白表達(dá)隨著時(shí)間變化（24h內(nèi)）先升高后降低，NF-κB蛋白表達(dá)持續(xù)升高；轉(zhuǎn)染anti-PIDD-si-RNA2的BV-2細(xì)胞較轉(zhuǎn)染陰性對(duì)照組細(xì)胞PIDD的蛋白表達(dá)較對(duì)照組下降，NF-κB蛋白表達(dá)下降。4，BV-2細(xì)胞照射后，Iba-1，CD68表達(dá)明顯增加；轉(zhuǎn)染anti-PIDD-si-RNA2的BV-2細(xì)胞較單純對(duì)照組細(xì)胞，照射后Iba-1，CD68表達(dá)明顯減少。5，照射后BV-2細(xì)胞遷移能力增強(qiáng)；轉(zhuǎn)染anti-PIDD-si-RNA2的BV-2細(xì)胞較單純照射組細(xì)胞的遷移能力明顯減弱。6，BV-2細(xì)胞照射后次級(jí)溶酶體增多，可見高爾基體、內(nèi)質(zhì)網(wǎng)線粒體變型嚴(yán)重；轉(zhuǎn)染anti-PIDD-si-RNA2的BV-2細(xì)胞較單純照射組細(xì)胞次級(jí)溶酶體減少，，初級(jí)溶媒體比例增加，未見高爾基體，內(nèi)質(zhì)網(wǎng)及線粒體變性減輕。 【結(jié)論】：下調(diào)PIDD可能通過PIDD/NF-κB信號(hào)轉(zhuǎn)導(dǎo)途徑抑制小膠質(zhì)細(xì)胞激活及BV-2細(xì)胞炎癥因子表達(dá)。
[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- 偽 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- 偽 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- 偽 and IL-1- 尾 in PIDD- TNF- 偽 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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類號(hào)】：R818

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 田野,包仕堯,殷蔚伯;放射性腦損傷實(shí)驗(yàn)研究之近況[J];中華放射醫(yī)學(xué)與防護(hù)雜志;1998年06期

本文編號(hào)：2063678