mir-142-5p在动脉粥样硬化斑块中的表达及通过TGF-β2调控人巨噬细胞凋亡功能的作用

  本文关键词：mir-142-5p在动脉粥样硬化斑块中的表达及通过TGF-β2调控人巨噬细胞凋亡功能的作用，由笔耕文化传播整理发布。

        研究背景冠状动脉粥样硬化性心脏病(冠心病,coronary heart disease, CHD)近年的发病率和死亡率逐年上升,成为威胁人类健康的重大主要原因。动脉粥样硬化是心血管疾病的一个主要的危险因素,近年研究表明microRNAs在动脉粥样硬化斑块的形成及发展过程中参与并起着重要的基因调控作用。动脉粥样硬化和心血管疾病的进展中,microRNAs参与了包括内皮功能的变化、血管平滑肌细胞增殖和迁移、巨噬细胞的功能以及泡沫细胞的形成。巨噬细胞参与先天免疫系统,帮助清除凋亡细胞和去除细胞碎片产生的组织重构和细胞坏死；巨噬细胞可以释放炎性物质,影响动脉粥样硬化。在冠心病的发生及发展过程中,炎症起着重要的作用。研究发现microRNAs不但参与了炎症细胞的发育、分化,还参与调控炎症细胞的活化以及各种炎症因子的相互作用。mir-142-5p目前在肿瘤、免疫疾病、淋巴组织及红细胞胚胎干细胞方面都有研究,但在动脉粥样硬化方面的研究尚未见报道。研究目的(1)探讨mir-142-5p在动脉粥样硬化斑块中表达的变化；(2)进一步探讨mir-142-5p在人巨噬细胞中的表达；(3)探讨mir-142-5p的靶基因并观察其对人巨噬细胞凋亡功能的影响。方法1.1动物模型的构建实验选用8周龄的雄性ApoE-/-小鼠,在普食喂养3天后转为高脂喂养；2周后给予右侧颈动脉套管术；给予套管高脂饮食8周,根据干预措施不同分为3组(12只/组)：空白对照组；稳定斑块组；易损斑块组。易损斑块组给予限制性应激及噪音干预4周(前两周4h/天,后2周6h/天,5天/周)后,行麻醉处死小鼠采集血样、收集样本入液氮罐中。1.2microRNA芯片检测将取材后的颈动脉血管在冰上清理后送康城生物公司进行microRNA芯片检测,筛查在易损斑块表达变化较明显的microRNA,明确mir-142-5p在动脉粥样硬化斑块中的表达变化。1.3观察mir-142-5p在血管壁不同细胞中的表达,选择变化明显的细胞进一步研究在人平滑肌细胞、内皮细胞、巨噬细胞进行培养,用ox-LDL刺激,应用RT-PCR技术比较mir-142-5p在不同细胞中的表达变化。1.4寻找mir-142-5p的靶基因通过数据库及预测软件预测mir-142-5p的靶基因：TGF-β2;通过脂质体2000将mir-142-5p的inhibitor及mimics转染进入人巨噬细胞中,应用Western-bolt及RT-PCR技术对靶基因TGF-β2进行验证。1.5mir-142-5p及TGF-32干预对人巨噬细胞凋亡功能的影响应用转染技术将mir-142-5p的inhibitor、mimics及TGF-p2的inhibitor转染进入人巨噬细胞中,通过Annexin V-PE (Annexin V-PE Apoptosis Detection Kit)细胞凋亡检测试剂盒在激光共聚焦显微镜下观察其对人巨噬细胞功能的影响。1.5统计学分析两组之间的差异比较采用独立样本t检验；多组之间差异的比较采用了方差分析(ANOVA)。以上结果应用了SPSS17.0软件进行统计分析。以P<0.05作为具有统计学意义。结果2.1mir-142-5p在斑块中的表达MicroRNA芯片检测结果显示,mir-142-5p在稳定斑块组中的表达比空白对照组高6.8倍；mir-142-5p在易损斑块组中的表达比空白对照组高2.7倍。2.2mir-142-5p在三种细胞中的表达培养人内皮细胞、平滑肌细胞及巨噬细胞,并用ox-LDL50ng/ml刺激24h后提取(?)microRNA并应用RT-PCR技术检测mir-142-5p在三种细胞中的表达,结果显示内皮及平滑肌细胞中mir-142-5p的表达在对照组与ox-LDL干预组间差异均低于1.5倍(P>0.05),而mir-142-5p在巨噬细胞ox-LDL干预组中的表达高于对照组5.4倍(P<0.05),与芯片结果趋势符合。2.3mir-142-5p的靶基因预测应用数据库靶基因预测软件预测mir-142-5p的靶基因可能为：TGF-β2;通过转染技术将(?)nir-142-5p的inhibitor及rnimics转染进入人巨噬细胞中,提取蛋白及RNA, Western-bolt及RT-PCR的结果均提示TGF-β2可能为mir-142-5p的靶基因。2.5Mir-142-5p及TGF-β2干预对人巨噬细胞凋亡功能的影响control组细胞基本无凋亡；与control组比较,ox-LDL组细胞凋亡增加(凋亡细胞率：6.83±0.17%,P<0.05)；mir-142-5p mimics+ox-LDL组(凋亡细胞率：5.27±0.19%)和TGF-β2inhibitor+mir-142-5p inhibitor+ox-LDL组(凋亡细胞率：1.38±0.27%)及TGF-β2inhibitor+ox-LDL组(凋亡细胞率：2.74±0.21%)细胞凋亡也较ox-LDL组少(P<0.05)；与其它组比较mir-142-5p inhibitor+ox-LDL组凋亡细胞最多(凋亡细胞率：12.31±0.22%,P<0.05)。凋亡细胞率=凋亡细胞数/总细胞数%。结论1mir142-5p在动脉粥样硬化斑块中表达上调。2细胞学检测显示mir142-5p在人巨噬细胞中表达上调,易损斑块中表达增加明显与芯片检测的结果趋势相符,具有抑制ox-LDL诱导的巨噬细胞凋亡作用。3TGF-β2可能是mirl42-5p的靶基因并参与调控人巨噬细胞凋亡。

    BackgroundMorbidity and mortality rate of coronary heart disease have been continous increasing in recent years and become serious threaten to human health. Atherosclerosis is a major risk factor of cardiovascular disease. Previous research have illustrated that microRNA play a key role as gene regulator in formation and development of atherosclerotic plaques. In development of atherosclerotic plaques and cardiovascular disease, microRNA participate in dysfunction of endothelium and macrophage, proliferation and migration of vascular smooth muscle cells and formation of foam cell.Research on expression status of mir-142-5p in atherosclerotic plaques has not been reported. Macrophage was involved in several progresses of innate immunity, including clearing apoptotic cell, avoiding cell apoptosis and tissue restructuring and release of inflammatory factors. Inflammatory play an important role in pathological process of CHD. MicroRNAs have been reported to participate in phylogenesis,differentiation and activiation of inflammatory cells. Previous research of mir-142-5p have focused on tumors, immunity diseases and stem cells. Research of mir-142-5p in CHD have not been reported at present.Research objectives(1) To discuss the changes of mir-142-5p expression level.(2)To investigate expression of mir-142-5p in macrophage further.(3)To explore target gene of mir-142-5p and the affect to apoptosis of macrophage.Methods1.1Construction of animal model8weeks old male ApoE-/-mice was used in present research. The mice was changed to high-fat diet after3days’basal diet for2weeks.Then we performed right common carotid artery tubulization. After high-fat diet for8weeks, the mice were divided to three groups(12mice each groups):control group, stabilized plague group, vulnerable plague group. The vulnerable plague group was given Pisaj syndrome noise interference for4weeks. Then all the mice were anesthesia and executed to obtain blood sample.1.2MicroRNA biochip detectionSend the flash-frozen carotid sample to Kangcheng-bio for a microRNA chip detection to identity the change of of mir-142-5p expression in atherosclerotic plaques.1.3The change of mir-142-5p expression in human macrophage.The expression level of mir-142-5p in ox-LDL stimulated cells was detected with RT-PCR technique.1.4Tracing for target gene of mir-142-5p.TGF-β2was predicted to be target gene of mir-142-5p. Inhibitor of mir-142-5p and mimics were transfected into human macrophage and detect the influence on TGF-β2by Western-bolt and RT-PCR.1.5mir-142-5p influences apoptosis of macrophage.Inhibitor of mir-142-5p mimics and inhibitor of TGF-β2were transfected into human macrophage. Function of human macrophage was observed by Annexin V-PE Apoptosis Detection Kit.1.6Statistics analysisData analysis was conducted with SPSS17.0. All data were presented as mean6standard error of the mean, and statistical comparisons were made with a paired t test and ANOVA tests. Differences were considered significant if P<0.05.Results2.1Expression of mir-142-5in plagueBiochip detection of microRNA results show expression level of mir-142-5p in stabilized plague group is6.8folds higher than blank control; expression level of mir-142-5p is2.7folds higher than vulnerable plagues group.2.2Expression of mir-142-5p in macrophage Human endothelial cells, smooth muscle cells and macrophage was cultured, then stimulated with ox-LDL50ng/ml for24hours before microRNA was extracted. RT-PCR was used to analyze expression of the3cell lines. In endothelial cells and smooth muscle cells, mir-142-5p expression of ox-LDL stimulated group decrease1.5folds compared with non-stimulated group. According to biochip detection result, mir-142-5p expression in macrophage cells is5.4folds higher than blank control.2.3Prediction of mir-142-5p target geneAs processed with database-based target gene prediction software, TGF-β2is the most probable target gene of mir-142-5p. Inhibitor of mir-142-5p and mimics was transfected into human macrophage cells. Protein and RNA were extracted and analyzed by western blot. Both results show TGF-β2is target gene of mir-142-5p.2.4mir-142-5p affects apoptosis of human macrophage cellsApoptosis of the blank control group was not observed. Cell apoptosis of ox-LDL group was increased(Apoptosis cell percentage:6.83±0.17%).Cell apoptosis of mir-142-5p mimics+ox-LDL group(Apoptosis cell percentage:5.27±0.19%);TGF-β2inhibitor mir-142-5p inhibitor+ox-LDL group (Apoptosis cell percentage:1.38±0.27%) and TGF-02inhibitor+ox-LDL group (Apoptosis cell percentage:2.74±0.21%) was also more slight than the other two groups. Cell apoptosis of N.C+ox-LDL group(Apoptosis cell percentage:6.49±0.25%) was also increased. Mir-142-5p restrains apoptosis of marque cells partly through TGF-β2.(P<0.05)Conclusions1. Expression of mir142-5p is at a high level in atherosclerotic plaques.2.mir142-5p is over expressed in human macrophage cells in CHD; Accords with the trend of the testing results of the chip, has the inhibitory effect of ox-LDL induced apoptosis.3. TGF-P2is target gene of mir142-5p and regultates apoptosis of human macrophage cells.

mir-142-5p在动脉粥样硬化斑块中的表达及通过TGF-β2调控人巨噬细胞凋亡功能的作用

中文摘要6-9英文摘要9-11符号说明12-13前言13-151 资料与方法15-262 结果26-283 讨论28-354 结论35-365 附表附图36-42参考文献42-46综述46-56    参考文献53-56致谢56-57攻读学位期间发表的学位论文目录57-58附表58

  本文关键词：mir-142-5p在动脉粥样硬化斑块中的表达及通过TGF-β2调控人巨噬细胞凋亡功能的作用，，由笔耕文化传播整理发布。