Id1和E2-2相互作用调节血管内皮祖细胞增殖的研究

发布时间：2018-06-11 09:51

本文选题：Idl + E2-2　；参考：《昆明医科大学》2016年硕士论文

【摘要】：[研究背景与研究目的]当前冠心病、高血压等慢性疾病发病率逐年升高,已经成为威胁人们健康、影响社会发展的重要元凶之一。血管性疾病的基本病理改变是多种原因造成内皮细胞损害、凋亡导致正常血管内膜结构和功能受损,进而启动一系列血管的病变包括血管狭窄、血栓形成。因此,如何实现损伤血管的再内皮化,恢复血管的正常功能和结构是血管损伤性疾病的研究热点。在此前的研究中人们发现血管内皮细胞可以通过增殖、迁移并合成和分泌多种生物活性物质修复受损的血管内膜。但由于内皮细胞为处于分化终末期的细胞,且病损部位的内皮细胞增殖能力有限,不足以完成受损内皮的修复,因此,研究者把关注点逐渐投向了内皮细胞的前体细胞,即血管内皮祖细胞(Endothelial progenitor cells,EPCs)。在缺血缺氧、细胞因子刺激、药物作用、血管损伤等状况下,EPCs可以从骨髓、脾脏处动员,“归巢”至受损血管部位并增殖、分化成为内皮细胞,还可以通过旁分泌等方式修复血管内膜。但是,EPCs如何增殖、迁移到靶血管实现再内皮化,其中的调控机制以及何种因子在起作用尚不十分明确。本课题组前期研究表明分化抑制因子1(Id-1)及转录因子E2-2在血管内皮细胞及内皮祖细胞增殖过程中起重要作用,但二者是否存在相互作用,及其对EPCs的影响效应不甚清楚。本课题拟探讨Id-1和E2-2相互作用对EPCs增殖的影响,并研究可能的调控机制。[方法]1.在课题组前期研究的基础上通过密度梯度离心法分离、培养EPCs并使用细胞形态学、免疫荧光及流式细胞仪对EPCs进行鉴定；2.购买慢病毒载体,通过PCR、酶切、测序进行鉴定并进行病毒的包装及滴度检测；3.在EPCs细胞模型中,采用免疫共沉淀检测Id-1及E2-2是否存在相互作用,并过表达及干扰Id-1及E2-2,采用CCK-8法检测细胞增殖情况,RT-PCR和Western blotting技术检测PI3K及Akt表达水平。[结果]1.小鼠骨髓源性单个核细胞可定向分化为EPCs,且EPCs同时具有干细胞与内皮细胞的双重特性；2.购买的Idl和E2-2过表达和干扰慢病毒载体构建成功,可用于后续实验；3.Id-1和E2-2之间存在相互作用,Id-1对PI3K/Akt起促进作用,而E2-2则发挥抑制效应,二者协同调控PI3K/Akt信号通路,进而影响EPCs的增殖。[结论]研究结果显示,Id-1和E2-2之间存在相互作用,并且两者通过PI3K/Akt信号通路调节内皮祖细胞EPCs的增殖。
[Abstract]:Background and objective: the incidence of coronary heart disease, hypertension and other chronic diseases has been increasing year by year, which has become one of the important culprits threatening people's health and affecting social development. The basic pathological changes of vascular diseases are caused by various causes of endothelial cell damage, apoptosis leading to normal intravascular membrane structure and function damage, and then start a series of vascular lesions, including vascular stenosis, thrombosis. Therefore, how to re-endothelialize the injured vessels and restore the normal function and structure of the vessels is a hot topic in the research of vascular injury diseases. In previous studies, it has been found that vascular endothelial cells can repair damaged vascular intima by proliferation, migration, synthesis and secretion of a variety of bioactive substances. But because the endothelial cells are at the end stage of differentiation, and the endothelial cells in the diseased site have limited proliferative ability, they are not enough to complete the repair of the damaged endothelial cells, so the researchers have gradually focused on the progenitor cells of the endothelial cells. That is Endothelial progenitor cells. Under the condition of ischemia and hypoxia, cytokine stimulation, drug action, vascular injury and so on, EPCs can be mobilized from bone marrow and spleen, "homing" to the injured vessel, proliferating and differentiating into endothelial cells. The vascular intima can also be repaired by paracrine. However, it is not clear how EPCs proliferate and migrate to target vessels for re-endothelialization. Our previous studies have shown that differentiation inhibitor 1- Id-1) and transcription factor E2-2 play an important role in the proliferation of vascular endothelial cells and endothelial progenitor cells, but whether there is interaction between them and their effects on EPCs is not clear. The purpose of this study was to investigate the effects of Id-1 and E2-2 interactions on the proliferation of EPCs and the possible regulatory mechanisms. [methods] 1. EPCs were isolated by density gradient centrifugation and identified by cell morphology, immunofluorescence and flow cytometry. The lentivirus vector was purchased and identified by PCR, enzyme digestion and sequencing, and the virus was packaged and titer detected. In EPCs cell model, immunoprecipitation was used to detect the interaction between Id-1 and E2-2, and overexpression and interference of Id-1 and E2-2. The expression of PI3K and Akt were detected by CCK-8 RT-PCR and Western blotting. [result] 1. Mouse bone marrow-derived mononuclear cells can differentiate into EPCs, and EPCs have the dual characteristics of stem cells and endothelial cells. The over expression of Idl and E2-2 and the construction of interfering lentivirus vectors were successfully constructed, which could be used to further study the interaction between Id-1 and E2-2. Id-1 promoted PI3K / Akt, while E2-2 played an inhibitory role. They coordinated the regulation of PI3K / Akt signaling pathway. And then affect the proliferation of EPCs. [conclusion] the results show that there is interaction between Id-1 and E2-2, and both of them regulate the proliferation of EPCs through PI3K / Akt signaling pathway.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R54

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