长链非编码RNA Sirt1-AS和RP11-618G20.1在抑制静脉桥血管内膜过度增生中的作用和机制研究

Vein graft failure seriously affects the long-term clinical success of coronary artery bypass grafting (CABG) as a regular therapy in coronary heart disease, which is the leading cause of death worldwide. Studies have shown shear stress changes from hemodynamic environment are essential in vein graft failure. Our previous studies have proved that high shear stress inactivats the MAPK signaling pathway, resulting in inhibition of venous intima hyperplasia. Previous studies found that long-chain non-coding RNA (LncRNA) under induced shear stress played an important role in the regulation of MAPK pathway. In addition, our pre-experiment verified the relationships between LncRNA expression and vein graft failure in clinical samples. . On the basis of preliminary study and pre-experiment results, We hypothesized that AP-1 could activate long LncRNA Sirt1-AS and RP11-618G20.1, which could up-regulate Sirt1 to inactivate MAPK signaling pathway to inhibit the proliferation of endothelial cells by genome-wide analysis, bioinformatics prediction, siRNA and luciferase reporter gene assay. The present study aims to further explore the effects of LncRNA on stress-cultured human umbilical vein endothelial cells, and to illuminate the molecular mechanism of LncRNA regulation of Sirt1 and AP-1 regulation of LncRNA in vivo and in vitroto, in order to provide a theoretical basis for clinical intervention target of vein graft failure, and mprove the therapeutic efficacy of CABG.

静脉桥再狭窄严重影响冠状动脉搭桥术疗效。内膜过度增生是静脉桥再狭窄的中心环节，血流动力学改变是内膜过度增生的启动因子。我们前期研究发现高切应力失活MAPK信号通路从而抑制静脉桥内膜过度增生。研究表明长链非编码RNA（LncRNA）在切应力诱导下对MAPK通路的调控起重要作用。在预实验中，我们通过临床样本证实LncRNA表达与静脉桥再狭窄的相关性，继而通过转录组测序、生信预测、小干扰RNA和荧光素酶报告基因检测等初步验证了下述机制：LncRNA Sirt1-AS和RP11-618G20.1被切应力诱导的转录因子AP-1激活，进而上调Sirt1，失活MAPK信号通路，抑制内膜过度增生。本项目拟进一步探讨LncRNA对切应力培养人脐静脉内皮细胞生物学行为的影响，完善LncRNA调控Sirt1以及AP-1调控LncRNA分子机制，并通过动物实验验证，为临床上寻找静脉桥再狭窄的干预靶点提供理论基础。

静脉桥再狭窄严重影响冠状动脉搭桥术疗效。内膜过度增生是静脉桥再狭窄的中心环节，血流动力学改变是内膜过度增生的启动因子。我们前期研究发现高切应力失活MAPK信号通路从而抑制静脉桥内膜过度增生。研究表明长链非编码RNA（LncRNA）在切应力诱导下对MAPK通路的调控起重要作用。在预实验中，我们通过临床样本证实LncRNA表达与静脉桥再狭窄的相关性，继而通过转录组测序、生信预测、小干扰RNA和荧光素酶报告基因检测等初步验证了下述机制：LncRNA Sirt1-AS和RP11-618G20.1被切应力诱导的转录因子AP-1激活，进而上调Sirt1，失活MAPK信号通路，抑制内膜过度增生。本项目进一步研究了切应力与MAPK通路对内皮细胞功能的影响，通过与血管平滑肌细胞共培养验证其内在机制。我们假设高切应力是通过抑制MAPK通路了抑制共培养条件下内皮细胞增殖与迁移，同时促进细胞凋亡。我们通过猪的大隐静脉分别提取原代内皮细胞与血管平滑肌细胞，通过在不同切应力条件下进行共培养，使用U0126或PD98059等不同MAPK通路抑制剂进行处理，通过MTT检测、BrdU检测、流式细胞术、细胞迁移实验等观察内皮细胞的增殖、细胞调亡与细胞迁移等细胞行为。我们发现，在高切应力共培养条件下，内皮细胞与血管平滑肌细胞增殖与细胞迁移受到抑制，而细胞凋亡被激活，通过MAPK通路抑制剂处理后，上述效应会进一步加重。于是我们得出重要发现，高切应力能够抑制静脉内皮细胞与血管平滑肌细胞共培养条件下的细胞增殖与迁移会受到抑制，细胞凋亡会受到促进，而这一效应是通过抑制ERK1/2和p38 MAPK通路完成的。这为临床上寻找静脉桥再狭窄的干预靶点提供了理论基础。

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