發(fā)布時間：2018-02-28 01:30

  本文關鍵詞： 耳蝸毛細胞 氧化損傷 活性氧 微小RNA 表達譜 微陣列芯片　出處：《南方醫(yī)科大學》2010年博士論文　論文類型：學位論文

【摘要】： 背景 氧化損傷和活性氧(也稱氧自由基,Reactive Oxygen Species, ROS)與藥物性耳聾、噪聲性耳聾、以及老齡性耳聾密切相關。順鉑、氨基糖甙類藥物和持續(xù)的噪聲均可使耳蝸毛細胞產(chǎn)生高濃度的ROS而導致毛細胞損傷。此外,抗氧化劑有助于耳蝸毛細胞的存活及維持其正常功能。已有研究表明ROS可以調(diào)節(jié)基因轉(zhuǎn)錄水平的表達,慶大霉素和順鉑可通過產(chǎn)生高濃度的ROS而調(diào)節(jié)耳蝸毛細胞中基因的轉(zhuǎn)錄,以及氧化損傷相關的信號轉(zhuǎn)導通路。然而,轉(zhuǎn)錄后調(diào)控在基因表達和細胞存活中起著重要的作用,僅有研究發(fā)現(xiàn)在心肌細胞和血管平滑肌細胞中ROS可以通過轉(zhuǎn)錄后調(diào)控影響基因的表達,但未見有關耳蝸毛細胞氧化損傷相關的基因轉(zhuǎn)錄后調(diào)控方面的報道。因此,探尋ROS對耳蝸毛細胞基因轉(zhuǎn)錄后的調(diào)控及其調(diào)控機制,對于闡明耳蝸毛細胞氧化損傷的機制具有重要意義。 MicroRNAs(miRNAs)是一種內(nèi)源的、非編碼的小RNA,可通過對靶基因mRNAs的降解作用和翻譯抑制作用而負向調(diào)控基因的表達。一種miRNA可調(diào)控多個靶基因,而若干miRNAs也可協(xié)同作用于一個靶基因,從而調(diào)控細胞的分化、增殖/生長、遷移和凋亡。MiRNAs在小鼠內(nèi)耳感覺上皮的發(fā)育和成熟過程中具有重要作用,也是聽力損失的重要調(diào)控因素。最近的研究顯示miRNA異�？赡苁侨祟惡托∈筮M行性聽力損失的病因。然而未見有關耳蝸毛細胞氧化損傷相關的miRNA表達及其在基因調(diào)控方面的報道。因此,研究耳蝸毛細胞氧化損傷和高濃度ROS對miRNAs表達的影響,以及miRNAs在ROS介導的基因調(diào)控中的作用及其生物功能,將對深入了解耳蝸毛細胞損傷和聽力損失的機制具有重要意義。 研究目的 (1)利用有機氧化劑叔丁基過氧化氫(tert-Butyl Hydroperoxide, t-BHP)染毒耳蝸毛細胞(House Ear Institue-Organ of Corti 1, HEI-OC1),通過檢測t-BHP對細胞增殖、細胞凋亡和ROS產(chǎn)生的影響,建立耳蝸毛細胞氧化損傷模型； (2)通過miRNA表達芯片和全基因組mRNA表達芯片檢測,研究耳蝸毛細胞氧化損傷miRNA和mRNA表達譜； (3)通過生物信息學分析耳蝸毛細胞氧化損傷差異表達miRNA與mRNA,研究耳蝸毛細胞氧化損傷miRNA與mRNA表達調(diào)控網(wǎng)絡及其生物功能,為耳蝸毛細胞氧化損傷的基因表達及其轉(zhuǎn)錄后調(diào)節(jié)提供更直接的科學線索。 研究方法 (1)細胞增殖檢測HEI-OC1細胞經(jīng)t-BHP(0μM、25μM、50μM、100μM、200μM、400μM)染毒12h,以及100μM t-BHP染毒0h、3h、6h、12h、24h、48h后,分別用Cell Counting Kit-8 (CCK-8)檢測不同濃度和不同時間t-BHP染毒后細胞增殖能力的改變。 (2)細胞凋亡檢測HEI-OC1細胞經(jīng)t-BHP(0μM、25μM、50μM、100μM、200μM、400μM)染毒12h,用膜聯(lián)蛋白V(Annexin V)與碘化丙啶(Propidium Iodide, PI)雙標記后,通過流式細胞術檢測不同濃度t-BHP染毒對HEI-OC1細胞凋亡的影響。 (3)胞內(nèi)ROS檢測設置0μM、25μM、50μM、100μM、200μM、400μM6個濃度t-BHP染毒HEI-OC1細胞組,經(jīng)DCFH-DA探針標記后,通過熒光倒置顯微鏡觀察胞內(nèi)ROS生成情況,并用流式細胞術定量檢測胞內(nèi)ROS水平。 (4) microRNA芯片檢測HEI-OC1細胞經(jīng)0μM、50μM、100μM、200μMt-BHP染毒12h后,利用Exiqon LNA探針標記細胞總RNA,經(jīng)濃縮、雜交、圖像掃描后,分析耳蝸毛細胞氧化損傷miRNA表達譜。 (5)全基因組表達譜芯片檢測HEI-OC1細胞經(jīng)0μM、50μM、100μM、200μM t-BHP染毒12h后,利用Agilent 4×44K小鼠全基因組表達譜芯片檢測細胞氧化損傷后mRNA表達譜。 (6)實時定量RT-PCR檢測miRNA和mRNA表達t-BHP (0μM、50μM、100μM、200μM)染毒HEI-OC1細胞12h后,利用實時定量RT-PCR檢測各濃度t-BHP染毒組細胞中mmu-miR-29a、mmu-miR-203、CCND2、ATF7IP的表達水平,并分別以U6和GAPDH為內(nèi)參,用2-ΔΔCT法進行相對定量。 (7)生物信息學分析通過Targetscan 5.1預測耳蝸毛細胞氧化損傷差異表達miRNA的靶基因,并結(jié)合差異表達mRNA進行整合分析,通過Osprey 1.2.0構建miRNA與mRNA調(diào)控網(wǎng)絡；并利用DAVID對差異表達miRNA調(diào)控的表達上調(diào)(和下調(diào))的靶基因進行GO分析和Pathway分析。 (8)統(tǒng)計分析所有實驗結(jié)果以均數(shù)±標準差(x±SD)表示,并根據(jù)實驗數(shù)據(jù)的性質(zhì)利用SPSS16.0軟件進行方差分析。P0.05為有顯著性差異。 研究結(jié)果 (1) t-BHP對耳蝸毛細胞增殖能力的影響不同濃度t-BHP對HEI-OC1細胞染毒12h后各組細胞生長變化率有統(tǒng)計學差異(F=79.445,P0.001),且25μM以上濃度的t-BHP染毒12h后可抑制HEI-OC1細胞的增殖(P0.05)；100μMt-BHP染毒HEI-OC1細胞不同時間后各組細胞生長變化率有統(tǒng)計學差異(F=16.056,P0.001),且100μM t-BHP染毒HEI-OC1細胞6h以上可抑制細胞增殖(P0.01)。 (2) t-BHP對耳蝸毛細胞凋亡的影響各濃度t-BHP組早期細胞凋亡率無統(tǒng)計學差異(F=1.416,P=0.287)；各濃度t-BHP組細胞凋亡率有統(tǒng)計學差異(F=8.372,P=0.001),50μM以上濃度t-BHP致HEI-OC1細胞凋亡率增高(P0.05),且主要為晚期細胞凋亡(P0.05)的增加所致。 (3) t-BHP對耳蝸毛細胞胞內(nèi)ROS水平的影響熒光顯微鏡下可見ROS分布于胞內(nèi),不同濃度t-BHP染毒組間細胞內(nèi)熒光有極大差別。流式細胞術定量檢測胞內(nèi)ROS結(jié)果顯示,各濃度t-BHP組熒光細胞率有統(tǒng)計學差異(F=347.897,P0.001),未染毒對照組熒光細胞僅為3.45%,25μM t-BHP組熒光細胞為4.26%(P=0.651); 50μM t-BHP組熒光細胞為7.59%(P0.05)；100μM、200μM、400μM t-BHP組熒光細胞增多(P0.001),分別為17.26%、27.90%、59.85%；以上結(jié)果表明50μM以上濃度t-BHP可致HEI-OC1細胞氧化損傷,并致胞內(nèi)ROS生成量明顯增多。 (4)耳蝸毛細胞氧化損傷microRNA表達譜以0μM t-BHP作為對照組,50μM、100μM、200μM t-BHP染毒組共有40個miRNA表達上調(diào),35個miRNA表達下調(diào)。相比未染毒對照組,50μM t-BHP組有21個miRNA表達上調(diào)、30個miRNA表達下調(diào)；100μMt-BHP組有19個miRNA表達上調(diào)、17個miRNA表達下調(diào)；200μM t-BHP組有21個miRNA表達上調(diào)、33個miRNA表達下調(diào)。 (5)耳蝸毛細胞氧化損傷全基因組mRNA表達譜以0μM t-BHP作為對照組,50μM、100μM、200μM t-BHP染毒組共有2076個mRNA表達上調(diào),580個mRNA表達下調(diào)。相比未染毒對照組,50μM t-BHP組有62個mRNA表達上調(diào)、26個mRNA表達下調(diào)；100μM t-BHP組有1803個mRNA表達上調(diào)、298個mRNA表達下調(diào);200μM t-BHP組有533個mRNA表達上調(diào)、367個mRNA表達下調(diào)。 (6)實時定量RT-PCR驗證miRNA和mRNA芯片結(jié)果經(jīng)實時定量RT-PCR檢測,200μM t-BHP組mmu-miR-29a表達上調(diào)(P0.05),100μM、200μM t-BHP組mmu-miR-203表達下調(diào)(P0.05); 100μM、200μM t-BHP組ATF7IP表達上調(diào)(P0.01),50μM、100μM、200μM t-BHP組CCND2表達下調(diào)(P0.001)；與miRNA和mRNA表達芯片檢測結(jié)果基本一致,表明miRNA和mRNA芯片實驗結(jié)果可信。 (7)耳蝸毛細胞氧化損傷miRNA與mRNA調(diào)控網(wǎng)絡分析各濃度t-BHP組差異表達miRNA靶基因篩選結(jié)果表明,50μM t-BHP染毒HEI-OC1細胞組中3個表達上調(diào)的miRNA有5個表達下調(diào)的靶基因,4個表達下調(diào)的miRNA有4個表達上調(diào)的靶基因。100μM t-BHP染毒HEI-OC1細胞組中5個表達上調(diào)的miRNA有20個表達下調(diào)的靶基因,8個表達下調(diào)的miRNA有121個表達上調(diào)的靶基因。200μM t-BHP染毒HEI-OC1細胞組中8個表達上調(diào)的miRNA有63個表達下調(diào)的靶基因,11個表達下調(diào)的miRNA有65個表達上調(diào)的靶基因。整合3個濃度t-BHP染毒HEI-OC1細胞差異表達miRNA靶基因篩選結(jié)果,表明11個表達上調(diào)的miRNA有81個表達下調(diào)的靶基因,15個表達下調(diào)的miRNA有180個表達上調(diào)的靶基因。 (8)GO分析和Pathway分析GO分析結(jié)果顯示,受下調(diào)miRNA調(diào)控的表達上調(diào)的180個靶基因?qū)?7個GO (Biological Process)分類,其中"cellular process"分類中基因數(shù)最多,為105個基因；受上調(diào)miRNA調(diào)控的表達下調(diào)的81個靶基因?qū)?53個GO (Biological Process)分類,其中"regulation of biological process"和"biological regulation"分類中基因數(shù)最多,均為42個基因。Pathway分析結(jié)果顯示,受下調(diào)miRNA調(diào)控的表達上調(diào)的180個靶基因?qū)?個Pathway分類,受上調(diào)miRNA調(diào)控的表達下調(diào)的81個靶基因?qū)?4個Pathway分類。 結(jié)論 (1)50μM以上濃度的t-BHP染毒HEI-OC1細胞12h后可抑制細胞增殖、誘導細胞凋亡率增高、并致胞內(nèi)ROS生成增多,從而確定了通過50μM、100μM、200μM t-BHP染毒HEI-OC1細胞12h,建立輕、中、重度的耳蝸毛細胞氧化損傷模型。 (2)相比未染毒對照組,50μM、100μM、200μM t-BHP染毒HEI-OC1細胞組共有40個miRNA表達上調(diào)、35個miRNA表達下調(diào)；共有2076個mRNA表達上調(diào)、580個mRNA表達下調(diào),構建出耳蝸毛細胞氧化損傷miRNA和mRNA表達譜。 (3)生物信息學整合分析表明,耳蝸毛細胞氧化損傷中11個表達上調(diào)的miRNA有81個表達下調(diào)的靶基因,屬97個GO (Biological Process)分類、6個Pathway分類；15個表達下調(diào)的miRNA有180個表達上調(diào)的靶基因,屬153個GO (Biological Process)分類、14個Pathway分類；明確了耳蝸毛細胞氧化損傷miRNA與mRNA調(diào)控網(wǎng)絡及其生物功能。
[Abstract]:background
Oxidative damage and reactive oxygen species (also known as Reactive Oxygen, oxygen free radical, Species, ROS) and drug deafness, noise deafness, aging and deafness are closely related to cisplatin, aminoglycosides and continuous noise can make the cochlear hair cells to produce a high concentration of ROS caused by the damage of hair cells. In addition, antioxidants and survival to maintain its normal function in cochlear hair cells. Studies have shown that ROS can regulate the expression of gene transcription, gentamicin and cisplatin can regulate gene transcription in cochlear hair cells by high concentration of ROS, and the signal transduction pathways related to oxidative damage. However, in the post transcriptional regulation of gene expression and cell survival plays an important role, only found in myocardial cells and vascular smooth muscle cells through the regulation of ROS can affect gene expression after transcription, but not related to cochlear hair Therefore, exploring the regulation and regulation mechanism of ROS on cochlear hair cells after gene transcription is important for elucidating the mechanism of oxidative damage in cochlear hair cells.
MicroRNAs (miRNAs) is a kind of endogenous non encoding, small RNA, can be used to target gene mRNAs degradation and translational inhibition and negatively regulate gene expression. A miRNA can regulate the expression of multiple target genes, and several miRNAs can also synergistic effects on a target gene, regulating cell the proliferation / differentiation, growth, migration and apoptosis of.MiRNAs play an important role in the development and maturation of mouse inner ear sensory epithelium, hearing loss is also important regulatory factors. Recent studies have shown that abnormal miRNA may be the cause of hearing loss in humans and mice. However, expression of miRNA damage related cochlear hair cells and no oxidation in the report of gene regulation. Therefore, study on the effect of cochlear hair cell oxidative damage and high concentration of ROS on the expression of miRNAs, and the role of miRNAs in gene regulation mediated by ROS and its biological function in Yes, it will be of great significance in understanding the mechanism of cochlear hair cell damage and hearing loss.
research objective
(1) the use of organic oxidant tert butyl hydroperoxide (tert-Butyl, Hydroperoxide, t-BHP) in the cochlea hair cells (House Ear Institue-Organ of Corti 1, HEI-OC1), through the detection of t-BHP on cell proliferation, cell apoptosis and ROS, establish the model of cochlear hair cell oxidative damage;
(2) the expression of miRNA and mRNA in the oxidative damage of cochlear hair cells was studied by the miRNA expression chip and the whole genome mRNA expression chip.
(3) by bioinformatics analysis of differences in the cochlear hair cell oxidative damage and mRNA expression of miRNA, expression of cochlear hair cell oxidative damage miRNA and regulation of mRNA network and its biological function, to provide more direct scientific clues for gene expression in cochlear hair cell oxidative damage and post transcriptional regulation.
research method
(1) t-BHP cell proliferation was detected by HEI-OC1 cells (0 M, 25 M, 50 M, 100 M, 200 M, 400 M) were 12h, and 100 M t-BHP in 0h, 3h, 6h, 12h, 24h, 48h, Counting and Kit-8 respectively by Cell (CCK-8) detection of different concentrations and different time of exposure to t-BHP after the change of cell proliferation ability.
(2) t-BHP cell apoptosis was detected by HEI-OC1 (0 M, 25 M, 50 M, 100 M, 200 M, 400 M) at 12h, annexin V (Annexin V) and propidium iodide (Propidium Iodide, PI) by double labeling, the effect of flow cytometry in different dose t-BHP on the apoptosis of HEI-OC1 cells.
(3) intracellular ROS detection was set at 0 M, 25 M, 50 M, 100 M M, 200 M, 400 M6 M6 concentration t-BHP cell infected HEI-OC1 cell group, labeled by DCFH-DA probe, the intracellular ROS production was observed by fluorescence inverted microscope, and the level of intracellular HEI-OC1 was quantitatively detected by flow cytometry.
(4) microRNA chip was used to detect HEI-OC1 cells after 0 12h, 50 M, 100 M, 200 Mt-BHP Mt-BHP 12h exposure. Exiqon LNA probe was used to mark the total RNA of cells. After concentration, hybridization and image scanning, the expression profiles of oxidative damage in cochlear hair cells were analyzed.
(5) whole genome expression profiling chip was used to detect HEI-OC1 cells after 0 12h M, 50 M, 100 M, 200 t-BHP M t-BHP 12h exposure. Agilent expression was detected using Agilent Agilent Agilent expression chip.
(6) real time quantitative RT-PCR detection of miRNA and mRNA expression of t-BHP (0 M, 50 M, 100 M, 200 M) in HEI-OC1 cells after 12h, using real time quantitative RT-PCR detection of mmu-miR-29a, the concentration of t-BHP treated cells in mmu-miR-203, CCND2, the expression level of ATF7IP, and using U6 and GAPDH as a reference, the relative quantification of 2- Delta Delta CT method.
(7) the bioinformatics analysis of miRNA gene expression by Targetscan 5.1 prediction of cochlear hair cell oxidative damage and the integration of differences, combined with differential expression analysis of mRNA, 1.2.0 miRNA and mRNA Osprey through the construction of regulatory networks; and the use of DAVID up regulates the expression of miRNA on differentially expressed (and down) analysis of GO and Pathway. The target gene.
(8) statistical analysis showed that all the experimental results were expressed by mean + standard deviation (x + SD). According to the nature of the experimental data, SPSS16.0 software was used for variance analysis.P0.05, with significant difference.
Research results
(1) the effect of t-BHP on cochlear hair cell proliferation of HEI-OC1 cells exposed to different concentration of t-BHP groups after 12h cell growth rate had significant difference (F=79.445, P0.001), and more than 25 M concentration of t-BHP after 12h exposure can inhibit the proliferation of HEI-OC1 cells (P0.05); 100 Mt-BHP exposed HEI-OC1 cells at different time after the cell growth rate had significant difference (F=16.056, P0.001), and more than 100 M t-BHP in HEI-OC1 cells can inhibit the proliferation of 6h (P0.01).
(2) the effect of t-BHP on the cochlear hair cell apoptosis in different concentrations of t-BHP group early apoptosis rate showed no significant difference (F=1.416, P=0.287); the concentration of the apoptosis rate of t-BHP group had statistically significant difference (F=8.372, P=0.001), more than 50 mu M concentration t-BHP induced HEI-OC1 cell apoptosis rate increased (P0.05), and the main late apoptosis (P0.05) increase.
(3) the influence of t-BHP on the level of ROS in cochlear hair cells in cells under the fluorescence microscope ROS on intracellular distribution of different concentration of t-BHP treated group, there is a great difference between intracellular fluorescence and flow cytometry quantitative detection of intracellular ROS results showed that the concentration of t-BHP group of fluorescent cells was statistically significant (F=347.897, P0.001) no, the exposure control group of fluorescent cells was only 3.45%, 25 M t-BHP group of fluorescent cells was 4.26% (P=0.651); 50 M t-BHP group of fluorescent cells was 7.59% (P0.05); 100 M, 200 M, 400 M t-BHP group (P0.001), fluorescent cells were 17.26%, 27.90%, 59.85% the above results show that; more than 50 mu M concentration could induce t-BHP oxidative damage in HEI-OC1 cells, and induced intracellular ROS generation increased significantly.
(4) spectrum based on 0 M t-BHP as control group the expression of microRNA in cochlear hair cell oxidative damage, 50 M, 100 M, 200 M t-BHP treated group were upregulated 40 miRNA, 35 miRNA expression. Compared with non exposed control group, 50 M group t-BHP expression of 21 miRNA, 30 miRNA expression; 100 Mt-BHP group expression of 19 miRNA, 17 miRNA expression; 200 M group t-BHP expression of 21 miRNA, 33 miRNA expression.
(5) expression profiles based on 0 M t-BHP as control group the whole genome mRNA oxidative damaged hair cells in the cochlea, 50 M, 100 M, 200 M t-BHP treated group were upregulated 2076 mRNA, 580 mRNA expression. Compared with non exposed control group, 50 M group t-BHP expression 62 mRNA, 26 mRNA expression; 100 M group t-BHP expression of 1803 mRNA, 298 mRNA expression; 200 M group t-BHP expression of 533 mRNA, 367 mRNA expression.
(6) the validation of real-time RT-PCR chip miRNA and mRNA results by quantitative real-time RT-PCR assay, expression of 200 M t-BHP mmu-miR-29a group (P0.05), 100 M, 200 M in group t-BHP decreased expression of mmu-miR-203 (P0.05); 100 M, 200 M t-BHP group up-regulated expression of ATF7IP (P0.01), 50 M, 100 M, 200 M in group t-BHP decreased expression of CCND2 (P0.001); chip detection results are basically consistent with the miRNA and mRNA expression, indicating that miRNA and mRNA chip experimental results are reliable.
(7) cochlear oxidative damage in miRNA cells and mRNA regulatory network analysis of the concentration of t-BHP group miRNA target gene expression screening showed that 3 up-regulated miRNA 50 M t-BHP HEI-OC1 exposure in groups of cells 5 target gene expression down regulated and 4 down regulated miRNA 4 expression 5 upregulation of miRNA expression of the target gene of.100 M t-BHP in HEI-OC1 cell group in the 20 target gene expression down regulated, downregulation of 8 of the 121 miRNA 8 up-regulated the expression of miRNA target genes.200 M t-BHP in HEI-OC1 cell group 63 target gene expression by 11. Downregulation of miRNA 65 expression of target genes. The integration of differences between the 3 dose t-BHP HEI-OC1 cells expression of miRNA target gene screening results showed that 11 up-regulated miRNA 81 gene expression down regulated and 15 down regulated miRNA 180 A target gene for up-regulated expression.
(8) GO analysis and Pathway GO analysis showed that 180 genes up-regulated by down-regulation of miRNA expression regulated by the 97 GO (Biological Process) classification, the "gene cellular process" classification number, 105 genes; 81 target genes expression is up-regulated by miRNA regulation of the genus 153 GO (Biological Process) classification, including "of biological gene regulation process" and "biological regulation" in the classification of the largest number of all 42 genes.Pathway analysis showed that 180 genes up-regulated by down-regulation of miRNA expression regulated the 6 Pathway classification, 81 target genes expression is up-regulated by miRNA the regulation of 14 Pathway classification.
conclusion
(1) t-BHP in HEI-OC1 cells 12h more than 50 mu M concentration can inhibit cell proliferation, induce cell apoptosis rate increased, and caused the generation of intracellular ROS increased, confirming by 50 M, 100 M, 200 M t-BHP in HEI-OC1 cells, 12h, a light, cochlear hair cell oxidation severe injury model.
(2) compared with the unexposed control group, 50 miRNA M, 100 M and 200 M t-BHP exposure group had 40 miRNA expression up-regulated, 35 miRNA down regulated, 2076 mRNA expression up-regulated, 580 mRNA down regulated, and cochlear hair cell oxidative damage miRNA and mRNA expression profiles were constructed.
(3) bioinformatics integration analysis shows that there are 11 up-regulated miRNA oxidative damaged hair cells in the cochlea of 81 target gene expression is down regulated, 97 GO (Biological Process), 6 Pathway; 15 expression decreased miRNA 180 expression of the target gene is 153 GO (Biological Process) classification, 14 Pathway classification; clear cochlear oxidative damage in miRNA cells and mRNA regulatory network and its biological function.

【學位授予單位】：南方醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R764

【引證文獻】

相關期刊論文 前2條

1 劉海洋;范靈凱;吳尚榮;謝樂云;章海寧;段斌勇;郭銳;;鉻及其化合物與噪聲聯(lián)合作用對聽力影響的分析[J];實用預防醫(yī)學;2011年06期

2 王艷玲;;噪聲與其他職業(yè)病危害因素的聯(lián)合作用[J];職業(yè)與健康;2014年07期

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本文編號：1545182