【摘要】：背景:肝移植是治療終末期肝臟功能衰竭的重要方式,死亡后器官捐獻(xiàn)是供體的主要來源。因捐獻(xiàn)的器官熱缺血損傷時(shí)間長短直接影響移植物的質(zhì)量,甚至造成肝臟原發(fā)性無功使移植失敗,所以了解熱缺血損傷機(jī)制以及干預(yù)后減輕移植物損傷,對提高移植質(zhì)量和成功率有重要意義。mi RNA是參與基因調(diào)控的一類新RNA分子,通過序列互補(bǔ)結(jié)合到mRNA 3,非編碼區(qū)域,來降解mRNA或抑制翻譯,從而調(diào)控靶基因的轉(zhuǎn)錄與表達(dá)。缺氧誘導(dǎo)因子(HIF)是細(xì)胞在基因轉(zhuǎn)錄水平時(shí)調(diào)節(jié)缺氧變化最重要的因子之一,其中HIF-1α與臟器熱缺血關(guān)系密切,研究顯示調(diào)控HIF-1α基因的表達(dá)可促進(jìn)或抑制細(xì)胞凋亡。然而熱缺血損傷后,miRNAs如何調(diào)控HIF-1α基因影響細(xì)胞凋亡的研究相對較少,這是本實(shí)驗(yàn)研究的重點(diǎn)內(nèi)容。目的:建立小鼠肝臟熱缺血模型,利用高通量測序法篩選有差異miRNAs,通過體外實(shí)驗(yàn)進(jìn)一步對miRNA如何調(diào)控HIF-1α基因影響細(xì)胞凋亡進(jìn)行探討,以期闡明熱缺血損傷后細(xì)胞凋亡的機(jī)制。方法:1.體內(nèi)實(shí)驗(yàn):1)通過建立小鼠肝臟熱缺血損傷模型,利用高通量測序方法篩選出有明顯差異miRNAs。2)從有明顯差異miRNAs選取與HIF-1α相關(guān)聯(lián)的某個(gè)miRNA進(jìn)行研究,通過RT-qPCR檢測該miRNA變化趨勢。3)采用Western blot技術(shù)分析HIF-1α蛋白表達(dá)情況。4)采用HE染色檢測不同時(shí)間段的肝組織病理情況。5)采用TUNEL法檢測不同時(shí)間段肝組織凋亡情況。2.體外實(shí)驗(yàn):1)建立小鼠肝細(xì)胞糖氧剝奪模型(模擬熱缺血損傷)。2)采用RT-qPCR檢測糖氧剝奪后該mi RNA表達(dá)情況。3)采用Western blot技術(shù)分析HIF-1α蛋白表達(dá)情況。4)采用流式細(xì)胞儀檢測細(xì)胞凋亡情況。5)轉(zhuǎn)染該miRNA模擬物(miRNA mimic)至細(xì)胞內(nèi),并用RT-qPCR驗(yàn)證轉(zhuǎn)染效果。6)采用Western blot技術(shù)分析檢測該miRNA過表達(dá)后HIF-1α、VEGFR2、Notch 1蛋白表達(dá)情況。7)采用流式細(xì)胞儀檢測細(xì)胞凋亡情況。結(jié)果:1.通過建立小鼠熱缺血模型利用高通量測序方法篩選出22個(gè)有明顯表達(dá)差異的miRNAs,其中10個(gè)miRNAs表達(dá)上調(diào),12個(gè)mi RNAs表達(dá)下調(diào)。2.通過生物信息學(xué)網(wǎng)站查詢發(fā)現(xiàn),mi R-338-5p的靶基因中包含與熱缺血損傷相關(guān)聯(lián)HIF-1α基因,故選取miR-338-5p進(jìn)行研究,發(fā)現(xiàn)熱缺血后miR-338-5p表達(dá)上調(diào),與高通量測序結(jié)果表達(dá)趨勢一致,并且隨著熱缺血時(shí)間延長miR-338-5p表達(dá)逐漸增多。3.在肝臟損傷方面,隨著小鼠肝臟熱缺血時(shí)間的延長,肝組織HE染色顯示肝細(xì)胞炎性細(xì)胞浸潤明顯、細(xì)胞水腫明顯、竇周隙減小、肝竇淤血增多、部分肝細(xì)胞出現(xiàn)凋亡特征、TUNEL法檢測發(fā)現(xiàn)隨著缺血時(shí)間的延長肝組織內(nèi)的細(xì)胞凋亡逐漸增多。研究還發(fā)現(xiàn)缺血后HIF-1α蛋白表達(dá)增多。4.細(xì)胞糖氧剝奪1 h后miR-338-5p、HIF-1α表達(dá)明顯升高,細(xì)胞凋亡增多。5.通過轉(zhuǎn)染miRNA-338-5p mimic至細(xì)胞,miR-338-5p過表達(dá)后HIF-1α、VEGFR2、Notch 1蛋白表達(dá)減少。6.mi R-338-5p過表達(dá)后細(xì)胞凋亡增多。結(jié)論:1.通過構(gòu)建肝臟的熱缺血損傷模型,建立mi RNAs表達(dá)譜,發(fā)現(xiàn)了熱缺血損傷后miRNAs有明顯變化。2.miRNA-338-5p參與肝熱缺血損傷并促進(jìn)了細(xì)胞凋亡。3.本研究結(jié)果提示miRNA-338-5p通過調(diào)控HIF-VEGF-Notch信號通路調(diào)控肝熱缺血損傷后細(xì)胞凋亡。
[Abstract]:Background: Liver transplantation is an important way to treat end-stage liver function failure, and organ donation after death is the main source of the donor. It is of great significance to understand the mechanism of the thermal ischemia injury and to reduce the graft damage after the intervention, so as to improve the quality of the transplantation and the success rate. Mi RNA is a new type of RNA molecule involved in gene regulation, and the transcription and expression of the target gene can be regulated by complementary binding of the sequence to the mRNA 3 and the non-coding region to degrade the mRNA or to inhibit the translation. The hypoxia-inducible factor (HIF) is one of the most important factors to regulate the change of hypoxia during the gene transcription level, in which the HIF-1 gene is closely related to the organ thermal ischemia, and the research shows that the regulation of the expression of the HIF-1 gene can promote or inhibit the apoptosis of the cells. However, after the thermal ischemia injury, how miRNAs regulate the HIF-1 gene to influence the apoptosis of the cells is relatively small, which is the main content of this experiment. Objective: To establish a model of liver thermal ischemia in mice and to use high-throughput sequencing to screen differential miRNAs, and to further study how to control the apoptosis of HIF-1 gene by in vitro experiments, with a view to elucidating the mechanism of apoptosis after thermal ischemia. Method:1. in vivo experiment:1) by establishing a mouse liver thermal ischemia injury model, a high-throughput sequencing method is used to screen a miRNAs with obvious difference (miRNAs.2) a specific miRNA that is associated with the HIF-1 gene is selected from a significant difference miRNAs, The change trend of HIF-1 was detected by RT-qPCR.3) The expression of HIF-1 was analyzed by Western blot. in vitro experiment: 1) To establish a model of glucose deprivation in mouse hepatocytes (simulated thermal ischemia injury).2) The expression of mi-RNA was detected by RT-qPCR.3) The expression of HIF-1 was analyzed by Western blot.4) The expression of HIF-1 was detected by flow cytometry. (6) The expression of HIF-1, VEGFR2 and Notch 1 was detected by Western blot. Results:1. The expression of 10 miRNAs was up-regulated and the expression of 12 mi-RNAs was down-regulated by means of high-throughput sequencing using a high-throughput sequencing method. Through the inquiry of the bioinformatics website, the target gene of mi R-338-5p contains the HIF-1 gene which is associated with the thermal ischemia injury, so that the miR-338-5p is selected for research, the expression of the miR-338-5p is up-regulated after the thermal ischemia, and the expression trend of the miR-338-5p is consistent with the high-throughput sequencing result, And the expression of miR-338-5p is increased gradually with the time of thermal ischemia. In the aspect of liver injury, as the liver heat ischemia time of the mouse is prolonged, the liver tissue HE staining shows that the infiltration of the inflammatory cells of the liver cells is obvious, the edema of the cells is obvious, the peripheral clearance of the liver is reduced, the blood stasis in the liver is increased, and the apoptotic characteristics of the part of the liver cells occur, The TUNEL method was used to detect the increase of apoptosis in the liver tissue with the time of the ischemia. It was also found that the expression of HIF-1 was increased after ischemia. The expression of miR-338-5p and HIF-1 increased significantly after 1 h of cell glucose deprivation, and the apoptosis was increased. The expression of HIF-1, VEGFR2 and Notch 1 after overexpression of miR-338-5p decreased the apoptosis of the cells after overexpression of the .6.mi-338-5p by transfecting the miRNA-338-5p momic to the cells. Conclusion:1. 2. miRNA-338-5p was involved in the liver heat ischemia injury and the cell apoptosis was promoted. The results of this study suggest that the miRNA-338-5p can regulate the apoptosis of the cells after the hepatic thermal ischemia injury by regulating the HIF-VEGF-Notch signaling pathway.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R657.3

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