本文選題：輻射損傷　切入點(diǎn)：高爾基體　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：高爾基體是細(xì)胞內(nèi)膜系統(tǒng)中重要的細(xì)胞器,參與了細(xì)胞內(nèi)蛋白質(zhì)的加工、分泌和囊泡運(yùn)輸?shù)冗^(guò)程。近年發(fā)現(xiàn),DNA損傷可以引起高爾基體的重組,使其片段化并分散到整個(gè)細(xì)胞之中,并認(rèn)為這種高爾基體結(jié)構(gòu)的變化可能與受照細(xì)胞的存活有關(guān)。盡管此研究結(jié)果發(fā)表在著名的“細(xì)胞”雜志上,但相關(guān)領(lǐng)域的研究報(bào)道到目前為止仍然很少。高爾基體的結(jié)構(gòu)變化是否真正參與了細(xì)胞存活反應(yīng),或僅僅是細(xì)胞放射損傷后亞細(xì)胞結(jié)構(gòu)發(fā)生的一種伴隨反應(yīng),本文將以此為切入點(diǎn),探索放射損傷后高爾基體的彌散現(xiàn)象以及對(duì)其機(jī)制進(jìn)行探討。目的:通過(guò)觀察輻射對(duì)高爾基體形態(tài)結(jié)構(gòu)的影響,了解高爾基體彌散的機(jī)制以及對(duì)彌散效應(yīng)和生物學(xué)意義進(jìn)行初步探討。方法:采用免疫熒光技術(shù)檢測(cè)放射損傷細(xì)胞中高爾基體彌散現(xiàn)象并對(duì)彌散面積進(jìn)行統(tǒng)計(jì)分析,流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期變化,克隆形成實(shí)驗(yàn)分析細(xì)胞存活率,Western Blot檢測(cè)高爾基體蛋GOLPH3及DNA斷裂損傷標(biāo)志物γH2AX的表達(dá),采用RT-PCR檢測(cè)輻射細(xì)胞后miRNA分子miR-378e、miR-486-3p表達(dá)變化及其靶蛋白GOLPH3L、MYO18A表達(dá)變化。結(jié)果:免疫熒光檢測(cè)結(jié)果顯示,照射后高爾基體的彌散面積增加,具有一定的劑量效應(yīng)和時(shí)間效應(yīng)關(guān)系;具有激活DNA-PKcs的放射損傷防護(hù)劑香蘭素衍生物VND3207能夠減輕γ射線對(duì)高爾基體的損傷,其表現(xiàn)為與未加藥組相比,VND3207能抑制不同劑量照射后高爾基體彌散面積的增加,同時(shí)促進(jìn)了受照細(xì)胞的存活;細(xì)胞周期測(cè)定結(jié)果顯示,4Gyγ射線照射后G2/M期阻滯峰值出現(xiàn)在12h左右;免疫印跡結(jié)果顯示DNA損傷引起的G2/M期阻滯也在12h后解除;而高爾基體彌散在細(xì)胞周期阻滯解除后如照射后24h甚至更長(zhǎng)時(shí)間仍然存在。RT-PCR結(jié)果顯示,4Gy、10Gy照射后,人臍靜脈內(nèi)皮細(xì)胞(HUVEC)中miR378e、miR 486-3p和GOLPH3L及MYO18A m RNA分子有表達(dá)上調(diào)的趨勢(shì),12h趨勢(shì)明顯;免疫熒光檢測(cè)結(jié)果顯示,轉(zhuǎn)染miR 378e、miR 486-3p、si GOLPH3L后的細(xì)胞中,高爾基體呈現(xiàn)彌散的趨勢(shì);細(xì)胞周期結(jié)果顯示,轉(zhuǎn)染siGOLPH3L、siMYO18A能延長(zhǎng)細(xì)胞照射后G2/M期的阻滯;同時(shí)觀察到,轉(zhuǎn)染miR 378e、miR486-3p、siGOLPH3L、siMYO18A后,受照細(xì)胞的存活率降低。結(jié)論:1.γ射線照射后,細(xì)胞高爾基體出現(xiàn)彌散現(xiàn)象,且具有一定的劑量效應(yīng)和時(shí)間效應(yīng)。2.高爾基體的彌散現(xiàn)象與細(xì)胞周期G2/M期阻滯相關(guān),但在輻射損傷中的高爾基彌散與周期阻滯沒有因果關(guān)系。3.高爾基體的彌散與細(xì)胞所受損傷程度相關(guān),輻射防藥物VND3207能抑制彌散。4.DNA-PKcs抑制劑NU7441抑制了DNA損傷的修復(fù),也抑制了高爾基體彌散。5.高爾基體蛋白GOLPH3,在照射后表達(dá)下調(diào),可被DNA-PKcs敲低抑制下調(diào)。6.miRNA分子miR-378e、miR-486-3p在高爾基體彌散中起一定作用。
[Abstract]:Golgi apparatus is an important organelle in the endomembrane system, which is involved in the process of protein processing, secretion and vesicle transport. Recently, it has been found that DNA damage can lead to the recombination of Golgi body. It was fragmented and dispersed throughout the cell, and the changes in Golgi structure were thought to be related to the survival of irradiated cells, although the findings were published in the well-known journal Cell. However, there have been few reports about the changes in Golgi body structure, whether the changes in Golgi apparatus are really involved in cell survival response, or is it only a kind of accompanying reaction of subcellular structure after cell radiation injury. In this paper, we will explore the dispersion of Golgi apparatus after radiation injury and its mechanism. Objective: to observe the effect of radiation on the morphological structure of Golgi apparatus. To understand the mechanism of Golgi body dispersion and to explore the diffusion effect and biological significance. Methods: the dispersion of Golgi apparatus in radiation injured cells was detected by immunofluorescence technique and the dispersion area was analyzed statistically. Cell cycle changes were detected by flow cytometry, cell survival rate was analyzed by clone formation assay and the expression of Golgi body egg GOLPH3 and DNA break damage marker 緯 H 2AX was detected by Western Blot. RT-PCR was used to detect the expression of miR-378esimiR-486-3p and the target protein GOLPH3LnMYO18A after irradiation. Results: the results of immunofluorescence showed that the dispersion area of Golgi was increased after irradiation, and there was a dose-effect and time-effect relationship between the expression of MIR-486-3p and the target protein GOLPH3LnMYO18A. Vanillin derivative VND3207, a radiation-protective agent with activated DNA-PKcs, can attenuate the damage of Golgi body induced by 緯 -ray. VND3207 can inhibit the increase of Golgi dispersion area after different doses of irradiation compared with the control group. The cell cycle analysis showed that the peak of G _ 2 / M phase arrest appeared at about 12h after irradiation with 4Gy 緯 -ray, and the G _ 2 / M phase block induced by DNA damage was also released after 12h by Western blot. The results of RT-PCR showed that the expression of miR378eR486-3p, GOLPH3L and MYO18A m RNA in human umbilical vein endothelial cells (HUVECs) was up-regulated for 12 h after the release of Golgi apparatus cell cycle arrest, such as 24 h or longer after irradiation, and the results of RT-PCR showed that the expression of miR378eR486-3p and MYO18A m RNA in HUVECs was significantly up-regulated at 12 h. The results of immunofluorescence assay showed that after transfection of miR 378em miR486-3pnsiGOLPH3L, Golgi showed a diffusing tendency, cell cycle showed that transfection of siGOLPH3L siMYO18A could prolong the arrest of G _ 2 / M phase after irradiation, at the same time, it was observed that after transfection of miR 378eanmiR486-3psiGOLPH3LsiO18A, the cell cycle showed that transfection of siGOLPH3L siMYO18A could prolong the arrest of G _ 2 / M phase after irradiation. The survival rate of irradiated cells was decreased. Conclusion the Golgi body dispersion appears in the cells after irradiation by 1: 1. 緯-ray, and has a dose effect and a time effect. 2. The dispersion phenomenon of Golgi body is related to the arrest of G 2 / M phase of cell cycle. However, there was no causal relationship between Golgi dispersion and cycle arrest in radiation injury. The dispersion of Golgi apparatus was related to the degree of cell damage. Radiation inhibitor VND3207 could inhibit the dispersion. 4. DNA-PKcs inhibitor NU7441 inhibited the repair of DNA damage. Golgi body protein Golgi body protein GOLPH3 was down-regulated after irradiation and down-regulated by DNA-PKcs knockdown. 6. MiRNA molecule miR-378esimiR-486-3p played a certain role in Golgi dispersion.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.55

【參考文獻(xiàn)】

相關(guān)期刊論文 前3條

1 ;DNA Damage, Signaling and Repair: Protecting genomic integrity and reducing the risk of human disease[J];Chinese Science Bulletin;2011年30期

2 黃銳;賀性鵬;徐勤枝;王豫;黃波;周平坤;;香蘭素衍生物VND3207對(duì)γ射線照射人淋巴母細(xì)胞基因組損傷和凋亡的保護(hù)作用[J];輻射防護(hù);2009年04期

3 鄭紅;汪思應(yīng);嚴(yán)雨倩;王林;徐勤枝;叢建波;周平坤;;香蘭素衍生物對(duì)輻射損傷細(xì)胞保護(hù)作用的初步研究[J];輻射防護(hù);2008年04期

，

本文編號(hào)：1635442