本文關(guān)鍵詞：急性中長波紫外線輻射“旁觀者效應(yīng)”相關(guān)分泌型miRNA的篩選及其功能預(yù)測　出處：《南京醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 紫外線旁觀者效應(yīng) 成纖維細(xì)胞 細(xì)胞外囊泡 氧化損傷 凋亡 分泌型 miRNA pre-miRNA

【摘要】：研究背景及目的輻射包括電離輻射和非電離輻射。紫外線(ultraviolet,UV)是波長為100-400 nm的非電離輻射。根據(jù)紫外線波長的不同,可將其分為短波紫外線UVC(200-280 nm)、中波紫外線 UVB(280-320 nm)和長波紫外線 UVA(320-400 nm)。而在透過大氣層時波長小于290 nm的紫外線會被大氣層中的臭氧吸收掉,能夠到達(dá)地面的主要是UVA和少量UVB。輻射誘導(dǎo)的旁觀者效應(yīng)(Bystander effects,BE)是指旁觀者細(xì)胞出現(xiàn)諸如細(xì)胞死亡、基因突變、染色體不穩(wěn)定等類似于細(xì)胞直接受到輻射作用后出現(xiàn)的反應(yīng)。旁觀者細(xì)胞指與受輻射細(xì)胞鄰近的細(xì)胞,也指用受輻射細(xì)胞的上清液培養(yǎng)的細(xì)胞。紫外線除了引起受輻射細(xì)胞的直接損傷外,還可以通過紫外線輻射旁效應(yīng)(Ultraviolet irradiation induced bystander effects,UV-BE)引起臨近細(xì)胞的間接損傷,這些旁觀者效應(yīng)包括氧化應(yīng)激反應(yīng),基因突變,凋亡,炎癥反應(yīng),免疫抑制,甚至腫瘤病變等。為了消除紫外線輻射所帶來的這些影響,維持染色體健全,細(xì)胞產(chǎn)生了一系列保護(hù)機制,包括DNA修復(fù),細(xì)胞周期阻滯以及凋亡。在旁觀者效應(yīng)中,受輻射細(xì)胞的DNA損傷反應(yīng)通過細(xì)胞間縫隙連接、細(xì)胞外可溶性因子與未受輻射細(xì)胞進(jìn)行細(xì)胞間通信。最近的研究表明小分子核糖核酸(microRNA,miRNA)在受輻射細(xì)胞和旁觀者細(xì)胞的信號通路中扮演重要的角色。以往人們都認(rèn)為miRNA只在細(xì)胞內(nèi)發(fā)揮作用,然而隨著研究的深入,研究者發(fā)現(xiàn)miRNA也會被分泌到細(xì)胞間隙中并發(fā)揮功能。大量的數(shù)據(jù)表明其實細(xì)胞外的miRNA十分穩(wěn)定,miRNA之所以能在細(xì)胞間隙中穩(wěn)定存在,是因其包裹于細(xì)胞外囊泡中,細(xì)胞外囊泡(Extracellular vesicles,EVs)是由磷脂雙分子層封閉組成不同大小的微粒(20～2000 nm),在體內(nèi)和體外幾乎所有類型的細(xì)胞都會釋放EVs到細(xì)胞外介質(zhì)中。到目前為止,根據(jù)不同的形成機制和生理特征分成三不同的EVs形式:外泌體、細(xì)胞膜微粒、調(diào)亡小體。外泌體產(chǎn)生于核內(nèi)體攜帶的多泡體,細(xì)胞膜微粒和調(diào)亡小體來源于細(xì)胞質(zhì)膜釋放的微囊泡(Microvesicles,MVs)。被包裹在EVs中的miRNA稱為"分泌型miRNA",EVs保護(hù)"分泌型miRNA",使其免受各種酶類的降解,從而被運輸?shù)较鄳?yīng)的受體細(xì)胞并調(diào)節(jié)受體細(xì)胞的生物功能。那么,在紫外線輻射"旁觀者效應(yīng)"中細(xì)胞的損傷是否由其中的miRNA介導(dǎo)或者由何種miRNA介導(dǎo)呢?本研究的目的在于篩選出有可能在急性中長波紫外線輻射"旁觀者效應(yīng)"中發(fā)揮重要作用的分泌型miRNA,并預(yù)測其可能的機制。方法1、紫外線輻射"旁觀者效應(yīng)"模型的建立及驗證分別采用不同劑量中長波紫外線(Ultraviolet A/Ultraviolet B,UVA/UVB)UVA 20J/cm2、UVB60mJ/cm2輻射人皮膚成纖維細(xì)胞(human skin fibroblasts,HSF),用特別配置的無胞外囊泡培養(yǎng)基孵育HSF,于照射后24h提取細(xì)胞上清液,與正常HSF共育,分為對照組(Ctr組)、UVA輻射組(UVA組)、UVB輻射組(UVB組)、旁觀者細(xì)胞對照組(BE-Ctr組)、旁觀者細(xì)胞UVA組(BE-UVA組)、旁觀者細(xì)胞UVB組(BE-UVB組),比較直接受輻射細(xì)胞和旁觀者細(xì)胞的變化,倒置顯微鏡下觀察細(xì)胞形態(tài),細(xì)胞計數(shù)CCK-8法檢測細(xì)胞增殖率,流式細(xì)胞儀檢測細(xì)胞凋亡率,DCFH-DA試劑盒檢測細(xì)胞內(nèi)的ROS量。2、miRNA芯片篩選差異表達(dá)的分泌型miRNA及生物信息學(xué)分析采用以上劑量中長波紫外線輻射人皮膚成纖維細(xì)胞,用無胞外囊泡培養(yǎng)基孵育24h,收集直接受輻射細(xì)胞各組上清液,進(jìn)行miRNA芯片分析,以歸一化強度的|log2(Ratio)|≥5為條件篩選上述模型中差異表達(dá)的上調(diào)miRNA、下調(diào)miRNA,利用qRT-PCR實驗法驗證UVA vs Ctr組、UVB vs Ctr組差異倍數(shù)均大于5倍的上調(diào)miRNA。通過TargetScan、microRNAorg、PITA數(shù)據(jù)庫對差異表達(dá)的miRNA進(jìn)行靶基因預(yù)測,并對所得差異miRNA的靶基因進(jìn)行GO分析以及pathway富集分析。3、qRT-PCR法深入驗證差異表達(dá)的分泌型miRNA利用qRT-PCR(Quantitative real time PCR,實時定量PCR)實驗法對上述差異表達(dá)的miRNA進(jìn)行深入驗證,分別檢驗直接受輻射細(xì)胞、直接受輻射細(xì)胞上清液和旁觀者細(xì)胞中miRNA的表達(dá)量,篩選出可能與紫外線"旁觀者效應(yīng)"相關(guān)的分泌型miRNA;繪制差異miRNA時效曲線圖;檢驗其前體RNA(pre-miRNA)在旁觀者細(xì)胞中的表達(dá)量。結(jié)果1、紫外線輻射人皮膚成纖維細(xì)胞后,可誘導(dǎo)紫外線"旁觀者效應(yīng)"的發(fā)生經(jīng)中長波紫外線UVA20J/cm2、UVB 60mJ/cm2輻射誘導(dǎo)生成的上清液與旁觀者細(xì)胞共孵育后24h,倒置顯微鏡下觀察直接受輻射細(xì)胞和旁觀者細(xì)胞,直接受輻射細(xì)胞和旁觀者細(xì)胞均出現(xiàn)較多的細(xì)胞碎片,細(xì)胞體積變大,過度伸展;CCK-8結(jié)果提示直接受輻射細(xì)胞和旁觀者細(xì)胞增殖率均減緩;流式細(xì)胞法結(jié)果提示直接受輻射細(xì)胞和旁觀者細(xì)胞表現(xiàn)出凋亡率升高;ROS結(jié)果提示,直接受輻射細(xì)胞和旁觀者細(xì)胞活性氧熒光強度均高于未輻射組。2、miRNA芯片篩選出差異表達(dá)miRNA紫外線輻射誘導(dǎo)生成的上清液中提取分泌型miRNA進(jìn)行質(zhì)量驗證并予芯片篩選,結(jié)果發(fā)現(xiàn),利用差異倍數(shù)Fold change值進(jìn)行篩選,標(biāo)準(zhǔn)為Fold change值=5.0,UVA vs Ctr組中有54個miRNA表達(dá)上調(diào)、11個miRNA表達(dá)下調(diào);UVBvsCtr組中有50個miRNA表達(dá)上調(diào),4個miRNA表達(dá)下調(diào)。其中,上調(diào)的 miRNA 中,UVA vs Ctr 組 Fold change 值=5.0 同時 UVB vs Ctr 組 Fold change值=5.0 的 miRNA 有 19 個,分別為 hsa-miR-8071、hsa-miR-769-5p、hsa-miR-758-3p、hsa-miR-7515、hsa-miR-6856-5p、hsa-miR-6837-5p、hsa-miR-6769a-5p、hsa-miR-6743-5p、hsa-miR-564、hsa-miR-4694-3p、hsa-miR-4655-3p、hsa-miR-4514、hsa-miR-4513、hsa-miR-4422、hsa-miR-432-5p、hsa-miR-3659、hsa-miR-3163、hsa-miR-22-5p、hsa-miR-1299。對差異表達(dá)的分泌型miRNA進(jìn)行生物信息學(xué)分析,預(yù)測靶基因功能,UVA組與UVB組顯著富集代謝通路大部分重疊,基因主要富集在能量代謝、轉(zhuǎn)錄調(diào)節(jié)、細(xì)胞增殖、跨膜信號轉(zhuǎn)導(dǎo)等生物過程中,并且這些基因主要參與Rap1信號通路、粘著斑激酶(FAK)相關(guān)信號通路、MAPK信號通路等重要代謝通路。3、差異表達(dá)的分泌型miRNA的進(jìn)一步驗證在直接受輻射細(xì)胞和旁觀者細(xì)胞中hsa-miR-4655-3p、hsa-miR-769-5p均呈現(xiàn)高表達(dá);hsa-miR-4655-3p、hsa-miR-769-5p在24h時間點表達(dá)量最高,上清液中miRNA變化趨勢與直接受輻射細(xì)胞中類似,但變化幅度更大;在旁觀者細(xì)胞中 pre-miR-4655-3p、pre-miR-769-5p 均呈現(xiàn)低表達(dá),結(jié)論急性中長波紫外線輻射人皮膚成纖維細(xì)胞可誘導(dǎo)出旁觀者效應(yīng);急性中長波紫外線輻射可介導(dǎo)分泌型miRNA的表達(dá),其中hsa-miR-4655-3p、hsa-miR-769-5p顯著上調(diào),極有可能參與調(diào)節(jié)紫外線"旁觀者效應(yīng)",值得進(jìn)一步研究。
[Abstract]:Background and objective: radiation including ionizing and non ionizing radiation. Ultraviolet (ultraviolet, UV) is a non ionizing radiation 100-400 nm wavelength. According to the different wavelengths of ultraviolet light, which can be divided into short wave ultraviolet UVC (200-280 nm), ultraviolet UVB (280-320 nm) and ultraviolet (320-400 UVA nm). And through the atmosphere below the wavelength of 290 nm ultraviolet ozone in the atmosphere will be absorbed, can reach the ground is mainly the bystander effect induced by UVA radiation and a small amount of UVB. (Bystander effects BE) refers to the bystander cells such as cell death, gene mutation, chromosome instability and other similar to the cells directly by the role of radiation after the reaction. The cells and bystander cells irradiated cells adjacent to the culture supernatant, also refers to the irradiated cells. In addition to causing ultraviolet irradiated cells directly Damage, but also through the ultraviolet radiation side effect (Ultraviolet irradiation induced bystander effects, UV-BE) caused by indirect injury to adjacent cells, the bystander effect including oxidative stress, gene mutation, apoptosis, inflammation, immunosuppression, and even tumors. In order to eliminate ultraviolet radiation caused by these effects, the maintenance of chromosomal sound the cell, resulting in a series of protection mechanisms, including DNA repair, cell cycle arrest and apoptosis in bystander effect in response to DNA damage by radiation cells connected by intercellular gap, extracellular soluble factor and non irradiated cells were intercellular communication. Recent studies show that microRNAs (microRNA, miRNA) in the play an important role in radiation cells and bystander cells. The signaling pathway is considered to play a role in intracellular miRNA However, with the deepening of the study, the researchers found that miRNA can be secreted into the intercellular space and function. A large number of data indicate that extracellular miRNA is very stable, miRNA can exist stably in the intercellular space, because it is wrapped in extracellular vesicles, extracellular vesicles (Extracellular vesicles, EVs) is a phospholipid bilayer enclosed particles of different sizes (20 ~ 2000 nm), in vitro and in vivo in almost all cell types will release EVs into the extracellular medium. So far, according to the different formation mechanism and physiological characteristics of EVs are divided into three different forms: exosomes. The cell membrane particles, apoptotic bodies. Exosomes originate in endosomes carrying multivesicular bodies, Microcystis cell membrane particles and apoptotic bodies comes from the release of cell membrane vesicles (Microvesicles, MVs). The EVs is wrapped in a miRNA called "secretory miRNA", EVs "Protection of secretory miRNA, degradation from the various enzymes, which are transported to the corresponding receptor cells and regulate receptor cell biological function. Then, in the ultraviolet radiation" bystander effect "in the cell injury by the miRNA mediated by miRNA mediated or what? The purpose of this study is to screening out possible in acute ultraviolet radiation" bystander effect "play an important role in the secretion of type miRNA, and forecast its possible mechanism. Methods 1, establishment and validation of ultraviolet radiation" bystander effect "model respectively with different doses of UVA (Ultraviolet A/Ultraviolet B, UVA 20J/cm2, UVA/UVB) UVB60mJ/cm2 radiation human skin fibroblasts (human skin, fibroblasts, HSF), with a special configuration of the no extracellular vesicles with HSF medium incubated in 24h cells after irradiation, extraction supernatant were incubated with normal HSF points For the control group (Ctr group), UVA radiation group (UVA group), UVB group (group UVB), radiation bystander cells in the control group (BE-Ctr group), UVA group of bystander cells (BE-UVA group), UVB group of bystander cells (BE-UVB group), compared with directly irradiated cells and bystander cell changes were observed morphology under inverted microscope, cell proliferation was detected by cell counting CCK-8 assay, cell apoptosis was detected by flow cytometry, ROS.2 DCFH-DA kit to detect the intracellular and secreted miRNA and bioinformatics analysis of the dose of UVA irradiation of human skin fibroblasts miRNA chip to screen differentially expressed, with no extracellular vesicles with 24h medium were collected, directly irradiated cells supernatant, miRNA microarray analysis, using normalized intensity |log2 (Ratio) | more than 5 conditions are differentially expressed in the above model on miRNA, downregulation of miRNA, using qRT-PCR test method Validation of the UVA vs Ctr miRNA. UVB vs Ctr group, raised the group differences multiples are greater than 5 times by TargetScan, microRNAorg, PITA database of differentially expressed miRNA target gene prediction and target genes for income differences were analyzed by GO and miRNA pathway.3 qRT-PCR enrichment analysis method, in-depth verification of secretory miRNA expression by qRT-PCR (Quantitative real time PCR, real-time quantitative PCR) experimental method on the expression of the differences in the miRNA for further inspection, were directly irradiated cells, directly irradiated cells supernatant and the expression of miRNA in bystander cells, secretory miRNA may be screened with ultraviolet "bystander effect" related to the difference of miRNA time curve drawing; check the map; precursor RNA (pre-miRNA) expression in the bystander cells. Results 1, ultraviolet radiation in human fibroblasts can be induced by ultraviolet radiation, bystander "The occurrence of the effect of ultraviolet radiation UVA20J/cm2, UVB 60mJ/cm2 and the supernatant induced bystander cells were incubated with 24h, directly irradiated cells and bystander cells were observed under inverted microscope, directly irradiated cells and bystander cells showed more cell debris, cell size, excessive stretch; CCK-8 showed directly the slow rate of radiation and cell proliferation of bystander cells; flow cytometry showed that directly irradiated cells and bystander cells showed apoptosis rate increased; ROS results suggest that radiation directly affected by the activity of cells and the bystander cell oxygen fluorescence intensity were higher than the radiation group.2, miRNA microarray selected153differentially expressed supernatant miRNA ultraviolet radiation induced the extraction of secretory miRNA quality verification and to the chip screening results showed that using the difference of multiple Fold change value selection Fold change, the standard value of =5.0, expression of 54 miRNA UVA vs in the Ctr group, 11 miRNA expression; expression of 50 miRNA in the UVBvsCtr group, 4 miRNA expression. The upregulation of miRNA, UVA vs Ctr Fold change =5.0 and the UVB value of group vs group Ctr Fold change the value of =5.0 19 in miRNA, respectively hsa-miR-8071, hsa-miR-769-5p, hsa-miR-758-3p, hsa-miR-7515, hsa-miR-6856-5p, hsa-miR-6837-5p, hsa-miR-6769a-5p, hsa-miR-6743-5p, hsa-miR-564, hsa-miR-4694-3p, hsa-miR-4655-3p, hsa-miR-4514, hsa-miR-4513, hsa-miR-4422, hsa-miR-432-5p, hsa-miR-3659, hsa-miR-3163, hsa-miR-22-5p, hsa-miR-1299. on the differential expression of secreted miRNA by bioinformatic analysis, the predicted target gene function, UVA group and UVB group significantly enriched the metabolic pathways of most overlapping genes mainly enriched in energy metabolism, transcription regulation, fine The process of cell proliferation, transmembrane signal transduction in organisms, and these genes mainly involved in Rap1 signaling pathway, focal adhesion kinase (FAK) signaling pathway, MAPK signaling pathway and other important metabolic pathways of.3, differential expression of secretory miRNA to further validate the directly irradiated hsa-miR-4655-3p cells and bystander cells, showed a high hsa-miR-769-5p the expression of hsa-miR-4655-3p, hsa-miR-769-5p in 24h; time expression was the highest, and directly affected by the trend of similar changes in miRNA in the supernatant of cell radiation, but larger change; pre-miR-4655-3p in bystander cells, pre-miR-769-5p showed low expression, long wave ultraviolet radiation in acute human skin fibroblasts can induce bystander effect in the long wave; acute ultraviolet radiation can mediate the expression of secretory miRNA in hsa-miR-4655-3p, hsa-miR-769-5p was down regulated, is likely to. It is worth further study to adjust the "bystander effect" of ultraviolet light.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R758.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 Kurt Fisher;Jingmei Lin;;Micro RNA in inflammatory bowel disease: Translational research and clinical implication[J];World Journal of Gastroenterology;2015年43期

2 王申;周炳榮;劉娟;張麗超;吳紅巾;易飛;胡燕燕;張家安;馬立文;駱丹;;中波紫外線誘導(dǎo)的提前衰老成纖維細(xì)胞培養(yǎng)液對正常人真皮成纖維細(xì)胞的氧化損害[J];中國中西醫(yī)結(jié)合皮膚性病學(xué)雜志;2014年05期

3 朱海濤;董瓊珠;賈戶亮;欽倫秀;;miRNA-769-5p在乙型肝炎相關(guān)肝細(xì)胞癌中的表達(dá)[J];貴陽醫(yī)學(xué)院學(xué)報;2012年01期

4 YANG ChengHsun;;The miRNA expression profile of the uveal melanoma[J];Science China(Life Sciences);2011年04期

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