【摘要】：背景:肝移植是治療終末期肝臟功能衰竭的重要方式,死亡后器官捐獻是供體的主要來源。因捐獻的器官熱缺血損傷時間長短直接影響移植物的質量,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制以及干預后減輕移植物損傷,對提高移植質量和成功率有重要意義。mi RNA是參與基因調控的一類新RNA分子,通過序列互補結合到mRNA 3,非編碼區(qū)域,來降解mRNA或抑制翻譯,從而調控靶基因的轉錄與表達。缺氧誘導因子(HIF)是細胞在基因轉錄水平時調節(jié)缺氧變化最重要的因子之一,其中HIF-1α與臟器熱缺血關系密切,研究顯示調控HIF-1α基因的表達可促進或抑制細胞凋亡。然而熱缺血損傷后,miRNAs如何調控HIF-1α基因影響細胞凋亡的研究相對較少,這是本實驗研究的重點內容。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,通過體外實驗進一步對miRNA如何調控HIF-1α基因影響細胞凋亡進行探討,以期闡明熱缺血損傷后細胞凋亡的機制。方法:1.體內實驗: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相關聯(lián)的某個miRNA進行研究,通過RT-qPCR檢測該miRNA變化趨勢。3)采用Western blot技術分析HIF-1α蛋白表達情況。4)采用HE染色檢測不同時間段的肝組織病理情況。5)采用TUNEL法檢測不同時間段肝組織凋亡情況。2.體外實驗:1)建立小鼠肝細胞糖氧剝奪模型(模擬熱缺血損傷)。2)采用RT-qPCR檢測糖氧剝奪后該mi RNA表達情況。3)采用Western blot技術分析HIF-1α蛋白表達情況。4)采用流式細胞儀檢測細胞凋亡情況。5)轉染該miRNA模擬物(miRNA mimic)至細胞內,并用RT-qPCR驗證轉染效果。6)采用Western blot技術分析檢測該miRNA過表達后HIF-1α、VEGFR2、Notch 1蛋白表達情況。7)采用流式細胞儀檢測細胞凋亡情況。結果: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個有明顯表達差異的miRNAs,其中10個miRNAs表達上調,12個mi RNAs表達下調。2.通過生物信息學網(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聯(lián)HIF-1α基因,故選取miR-338-5p進行研究,發(fā)現(xiàn)熱缺血后miR-338-5p表達上調,與高通量測序結果表達趨勢一致,并且隨著熱缺血時間延長miR-338-5p表達逐漸增多。3.在肝臟損傷方面,隨著小鼠肝臟熱缺血時間的延長,肝組織HE染色顯示肝細胞炎性細胞浸潤明顯、細胞水腫明顯、竇周隙減小、肝竇淤血增多、部分肝細胞出現(xiàn)凋亡特征、TUNEL法檢測發(fā)現(xiàn)隨著缺血時間的延長肝組織內的細胞凋亡逐漸增多。研究還發(fā)現(xiàn)缺血后HIF-1α蛋白表達增多。4.細胞糖氧剝奪1 h后miR-338-5p、HIF-1α表達明顯升高,細胞凋亡增多。5.通過轉染miRNA-338-5p mimic至細胞,miR-338-5p過表達后HIF-1α、VEGFR2、Notch 1蛋白表達減少。6.mi R-338-5p過表達后細胞凋亡增多。結論:1.通過構建肝臟的熱缺血損傷模型,建立mi RNAs表達譜,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了細胞凋亡。3.本研究結果提示miRNA-338-5p通過調控HIF-VEGF-Notch信號通路調控肝熱缺血損傷后細胞凋亡。
[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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R657.3

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