miRNA-338-5p对肝热缺血损伤的调控作用研究

发布时间：2019-04-02 17:05

【摘要】：背景:肝移植是治疗终末期肝脏功能衰竭的重要方式,死亡后器官捐献是供体的主要来源。因捐献的器官热缺血损伤时间长短直接影响移植物的质量,甚至造成肝脏原发性无功使移植失败,所以了解热缺血损伤机制以及干预后减轻移植物损伤,对提高移植质量和成功率有重要意义。mi RNA是参与基因调控的一类新RNA分子,通过序列互补结合到mRNA 3,非编码区域,来降解mRNA或抑制翻译,从而调控靶基因的转录与表达。缺氧诱导因子(HIF)是细胞在基因转录水平时调节缺氧变化最重要的因子之一,其中HIF-1α与脏器热缺血关系密切,研究显示调控HIF-1α基因的表达可促进或抑制细胞凋亡。然而热缺血损伤后,miRNAs如何调控HIF-1α基因影响细胞凋亡的研究相对较少,这是本实验研究的重点内容。目的:建立小鼠肝脏热缺血模型,利用高通量测序法筛选有差异miRNAs,通过体外实验进一步对miRNA如何调控HIF-1α基因影响细胞凋亡进行探讨,以期阐明热缺血损伤后细胞凋亡的机制。方法:1.体内实验:1)通过建立小鼠肝脏热缺血损伤模型,利用高通量测序方法筛选出有明显差异miRNAs。2)从有明显差异miRNAs选取与HIF-1α相关联的某个miRNA进行研究,通过RT-qPCR检测该miRNA变化趋势。3)采用Western blot技术分析HIF-1α蛋白表达情况。4)采用HE染色检测不同时间段的肝组织病理情况。5)采用TUNEL法检测不同时间段肝组织凋亡情况。2.体外实验:1)建立小鼠肝细胞糖氧剥夺模型(模拟热缺血损伤)。2)采用RT-qPCR检测糖氧剥夺后该mi RNA表达情况。3)采用Western blot技术分析HIF-1α蛋白表达情况。4)采用流式细胞仪检测细胞凋亡情况。5)转染该miRNA模拟物(miRNA mimic)至细胞内,并用RT-qPCR验证转染效果。6)采用Western blot技术分析检测该miRNA过表达后HIF-1α、VEGFR2、Notch 1蛋白表达情况。7)采用流式细胞仪检测细胞凋亡情况。结果:1.通过建立小鼠热缺血模型利用高通量测序方法筛选出22个有明显表达差异的miRNAs,其中10个miRNAs表达上调,12个mi RNAs表达下调。2.通过生物信息学网站查询发现,mi R-338-5p的靶基因中包含与热缺血损伤相关联HIF-1α基因,故选取miR-338-5p进行研究,发现热缺血后miR-338-5p表达上调,与高通量测序结果表达趋势一致,并且随着热缺血时间延长miR-338-5p表达逐渐增多。3.在肝脏损伤方面,随着小鼠肝脏热缺血时间的延长,肝组织HE染色显示肝细胞炎性细胞浸润明显、细胞水肿明显、窦周隙减小、肝窦淤血增多、部分肝细胞出现凋亡特征、TUNEL法检测发现随着缺血时间的延长肝组织内的细胞凋亡逐渐增多。研究还发现缺血后HIF-1α蛋白表达增多。4.细胞糖氧剥夺1 h后miR-338-5p、HIF-1α表达明显升高,细胞凋亡增多。5.通过转染miRNA-338-5p mimic至细胞,miR-338-5p过表达后HIF-1α、VEGFR2、Notch 1蛋白表达减少。6.mi R-338-5p过表达后细胞凋亡增多。结论:1.通过构建肝脏的热缺血损伤模型,建立mi RNAs表达谱,发现了热缺血损伤后miRNAs有明显变化。2.miRNA-338-5p参与肝热缺血损伤并促进了细胞凋亡。3.本研究结果提示miRNA-338-5p通过调控HIF-VEGF-Notch信号通路调控肝热缺血损伤后细胞凋亡。
[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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R657.3

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