MiR-483-3p调控内皮祖细胞对静脉血栓溶解再通影响的研究

发布时间：2018-05-20 19:28

  本文选题：内皮祖细胞 + 分离　； 参考：《苏州大学》2016年博士论文

【摘要】：下肢深静脉血栓形成(Deep Venous Thrombosis,DVT)是常见的外周血管疾病,可引起血栓后综合征(Post-thrombotic syndrome,PTS)和致死性肺动脉栓塞(Pulmonary embolism,PE)。目前深静脉血栓治疗方法包括药物抗凝溶栓、手术取栓和导管溶栓,但都未能消除血栓后综合征发生、远期通畅率不高、易于复发等缺点,因此需要一种更加安全有效的方法治疗深静脉血栓。在缺血性疾病中,干细胞研究取得了积极进展,其中内皮祖细胞(Endothelial progenitor cells,EPCs)近年来研究较多,内皮祖细胞是来源于骨髓中的血管内皮前体细胞,在新生血管形成过程中起着重要作用。我们前期研究结果发现,在大鼠静脉血栓模型中,移植的大鼠EPCs可归巢到静脉血栓中,改善血栓微环境,促进急性血栓的溶解和慢性血栓的机化再通。但EPCs应用面临很多问题,移植的EPCs仅少量可分化为血管内皮细胞,如何改善EPCs功能,成为缺血性疾病重要的研究方向。MicroRNAs(miRNAs,miR)是一类长度约22nt的非编码RNA,miRNAs通过完全匹配或者不完全匹配的方式识别靶基因3’-UTR区域,抑制蛋白翻译或者影响mRNA稳定性,在转录后水平调控蛋白表达,发挥重要的生物学功能。近年研究证实miRNAs参与EPCs功能调节,在血管新生和血管生成中扮演重要角色。但miRNAs在深静脉血栓患者和正常患者外周血EPCs中表达有无差异,这些差异性的miRNAs能否调控EPCs功能和影响静脉血栓溶解再通,带着这些问题,设计了本课题。我们采集DVT患者和健康人外周血样本,通过密度梯度离心法分离外周血单个核细胞,体外诱导培养EPCs,利用基因芯片筛选DVT患者和健康人EPCs中miRNAs的表达谱差异,用实时荧光定量PCR(qRT-PCR)验证芯片结果,对和芯片结果一致的miRNAs,通过文献检索和生物信息学分析挑选认为值得深入研究的miRNAs做细胞功能实验(我们挑选了6个miRNAs),观察这些miRNAs对EPCs的迁移、成血管和凋亡等细胞功能是否有影响,细胞功能结果显示miR-483-3p对EPCs功能有影响,我们对miR-483-3p做更深入研究,通过生物信息学预测miR-483-3p可能的靶基因,通过荧光素酶报告基因实验、上调和下调miR-483-3p、共转染和基因沉默后用Western blot检测蛋白变化进一步确认靶基因,以及共转染和基因沉默后对EPCs功能的影响。体内实验通过建立大鼠血栓模型,共聚焦荧光显微镜、HE染色和DSA观察mi R-483-3p调控EPCs后对EPCs归巢和血栓溶解再通的影响。结果发现:miR-483-3p在DVT患者EPCs中高表达;下调EPCs中mi R-483-3p表达会促进EPCs迁移、成血管能力,抑制EPCs凋亡,促进EPCs归巢和EPCs对血栓的溶解再通。我们的研究为干细胞治疗静脉血栓探索新的思路。本实验研究,将分为5部分,主要研究方法及结果如下。第一部分人外周血内皮祖细胞和大鼠骨髓源性内皮祖细胞的培养和鉴定目的:建立人外周血内皮祖细胞(endothelial progenitor cells,EPCs)和大鼠骨髓源性内皮祖细胞的分离、培养及鉴定方法,为后续体外和体内实验奠定基础。方法:采用密度梯度离心法分离人外周血和大鼠骨髓单个核细胞,将单个核细胞悬于含20%胎牛血清(FBS)EGM-2培养基中,培养2~3周,显微镜下观察细胞形态特征,流式细胞仪检测细胞表面标记物CD34、CD133、VEGFR-2表达量,双荧光染色检测细胞摄取DiI-ac-LDL和FITC-UEA-1能力。结果:刚分离的人外周血单个核细胞(peripheral blood mononuclear cells,PBMC)和骨髓单个核细胞(Bone marrow-derived mononuclear cells,BMMNC)圆形体积小,2天后可见少数细胞贴壁,3天后细胞体积逐渐变大,贴壁细胞增多,5天后部分细胞呈纺锤形生长,出现细胞集落,四周细胞呈放射样排列,第10至14天,细胞呈现铺路石或鹅卵石样改变。流式细胞仪显示细胞表面主要表达内皮标记物VEGFR-2,CD34、CD133表达低。双荧光染色显示细胞能够吞噬DiI-ac-LDL和FITC-UEA-1。结论:在EGM-2培养基诱导下,成功的从人外周血单个核细胞和大鼠骨髓单个核细胞中培养出EPCs,2~3周培养后呈现晚期EPCs特征。第二部分下肢深静脉血栓患者EPCs中mi RNAs差异表达谱筛选及验证目的:利用基因芯片筛选DVT患者和健康人外周血EPCs中mi RNAs的表达谱差异,并用q RT-PCR验证芯片结果的可靠性。方法:采集DVT患者和健康人外周血样本,利用密度梯度离心法分离外周血单个核细胞进行EPCs培养,通过mi RNA基因芯片筛选DVT患者和健康人EPCs的mi RNAs表达谱差异,并用q RT-PCR验证芯片结果。结果:Mi RNA基因芯片显示mi R-483-3p等多个mi RNAs在DVT患者和健康人外周血EPCs中表达存在差异,q RT-PCR结果和芯片一致。结论:Mi R-483-3p等多个mi RNAs在DVT患者和健康人外周血EPCs中表达有差异。第三部分Mi R-483-3p对内皮祖细胞功能的影响及靶基因预测和验证目的:探讨mi R-483-3p对人外周血EPCs迁移、成血管能力和凋亡的影响,预测其靶基因并验证。方法:用Lipofectamine 3000将mi R-483-3p模拟物(agomir)、抑制物(antagomir)和阴性对照物转染到EPCs中,用transwell实验检测mi R-483-3p对EPCs迁移的影响,用matrigel管腔形成实验检测mi R-483-3p对EPCs成血管能力的影响,用流式细胞仪检测mi R-483-3p对EPCs凋亡的影响。利用生物信息学预测mi R-483-3p可能的靶基因,利用荧光素酶报告基因实验、q RT-PCR和western blots等实验确认靶基因。结果:上调EPCs中mi R-483-3p表达会抑制EPCs迁移和成血管能力,促进EPCs凋亡,下调EPCs中mi R-483-3p表达结果和上调相反,生物信息学预测血清反应因子(serum response factor,SRF)可能是靶基因,q RT-PCR和western blots等实验验证SRF就是mi R-483-3p的靶基因。结论:上调EPCs中mi R-483-3p表达会抑制EPCs迁移和成血管能力,促进EPCs凋亡;SRF是mi R-483-3p靶基因。第四部分Mi R-483-3p慢病毒载体的构建和表达目的:构建mi R-483-3p/mi R-483-3p sponge慢病毒表达载体,感染大鼠EPCs,并验证EPCs中mi R-483-3p表达情况,为后续体内实验奠定基础。方法:将mi R-483-3p前体序列和慢病毒载体经酶切连接产生p GLV3-H1-GFP-mi R-483-3p和p GLV3-H1-GFP-mi R-483-3p sponge,与辅助包装载体一起转染293T细胞,收集病毒上清感染EPCs,用荧光显微镜观察转染效率,用q RT-PCR检测感染后EPCs内mi R-483-3p表达情况。结果:慢病毒载体p GLV3-H1-GFP-mi R-483-3p/p GLV3-H1-GFP-mi R-483-3p sponge构建成功,转染EPCs后能有效上调和下调EPCs内的mi R-483-3p表达。结论:成功构建了携带目的基因mi R-483-3p的慢病毒载体p GLV3-H1-GFP-mi R-483-3p/p GLV3-H1-GFP-mi R-483-3p sponge。第五部分Mi RNA-483-3p调控内皮祖细胞对静脉血栓溶解再通的影响目的:研究mi R-483-3p调控大鼠EPCs后对EPCs归巢和EPCs对静脉血栓溶解再通的影响。方法:将慢病毒载体p GLV3-H1-GFP vector、p GLV3-H1-GFP-mi R-483-3p和p GLV3-H1-GFP-mi R-483-3p sponge转染至EPCs中,采用结扎左肾静脉下方的下腔静脉构建大鼠深静脉血栓模型,再将转染的EPCs通过大鼠尾静脉移植到血栓模型中。分四组:A组(10只),空白对照组,经尾静脉注入1 ml PBS;B组(10只),EPCs/p GLV3-H1-GFP vector(EPCs/vector组),经尾静脉注入1 ml含有1.0×106EPCs/vector的PBS细胞悬液;C组(10只),EPCs/p GLV3-H1-GFP-mi R-483-3p(EPCs/mi R-483-3p组),经尾静脉注入1 ml含有1.0×106 EPCs/mi R-483-3p的PBS细胞悬液;D组(10只),EPCs/p GLV3-H1-GFP-mi R-483-3p sponge(EPCs/mi R-483-3p sponge组),经尾静脉注入1 ml含有1.0×106EPCs/mi R-483-3p sponge的PBS细胞悬液。术后7天收集标本,经荧光显微镜观察EPCs在血栓中的归巢,经HE染色、数字减影血管造影(digital subtract angiography,DSA)观察静脉血栓溶解再通情况。结果:经GFP荧光标记的EPCs出现在静脉血栓中,不同实验组的阳性细胞数比较:EPCs/mi R-483-3p sponge组EPCs/vector组EPCs/mi R-483-3p组,提示mi R-483-3p抑制EPCs归巢至静脉血栓中。各实验组血栓重量比较:blank control组EPCs/mi R-483-3p组EPCs/vector组EPCs/mi R-483-3p sponge组,提示mi R-483-3p抑制EPCs的溶栓能力。HE染色观察各实验组血栓溶解再通情况比较:EPCs/mi R-483-3p sponge组EPCs/vector组EPCs/mi R-483-3p组blank control组;DSA观察各实验组血栓溶解再通情况比较:EPCs/mi R-483-3p sponge组EPCs/vector组EPCs/mi R-483-3p组blank control组;提示过表达mi R-483-3p会抑制EPCs对静脉血栓的溶解再通,下调mi R-483-3p能促进EPCs的溶栓能力。结论:移植转染后的EPCs能够归巢至静脉血栓中,上调mi R-483-3p抑制了EPCs归巢能力和EPCs对静脉血栓的溶解再通,下调mi R-483-3p能促进EPCs归巢和溶栓能力。
[Abstract]:Deep Venous Thrombosis (DVT) is a common peripheral vascular disease, which can cause Post-thrombotic syndrome (PTS) and fatal pulmonary embolism (Pulmonary embolism, PE). At present, the methods of deep venous thrombosis include anticoagulant thrombolysis, surgical thrombolysis and catheter thrombolysis, but they have not been eliminated. After thrombus syndrome, the long-term patency rate is not high and it is easy to relapse. Therefore, it needs a more safe and effective method for the treatment of deep vein thrombosis. In the ischemic disease, the stem cell research has made positive progress, in which the Endothelial progenitor cells (EPCs) has been studied more in recent years, the endothelial progenitor cell is the source. The vascular endothelial progenitor cells in the bone marrow play an important role in the formation of neovascularization. In our previous study, we found that in the rat venous thrombosis model, the transplanted rat EPCs could return to the venous thrombosis, improve the microenvironment of thrombus, promote the dissolution of acute thrombus and the pathogenesis of chronic thrombus. But the application of EPCs is facing Many problems, only a small number of transplanted EPCs can differentiate into vascular endothelial cells, how to improve the function of EPCs, and become an important research direction of ischemic disease.MicroRNAs (miRNAs, miR) is a class of non coded RNA with a length of about 22nt, miRNAs can identify the target gene 3 '-UTR region by fully matched or incomplete matching, and inhibit the translation of protein or protein translation. Influence the stability of mRNA and regulate protein expression at post transcriptional level and play an important biological function. In recent years, studies have shown that miRNAs plays an important role in the regulation of EPCs function and plays an important role in angiogenesis and angiogenesis. However, there is no difference in the expression of miRNAs in the peripheral blood of patients with deep venous thrombosis and normal patients, and these differences of miRNAs can be found. The EPCs function and the effect of venous thrombosis and recanalization were not regulated. With these problems, we designed this topic. We collected peripheral blood samples from DVT patients and healthy people, isolated peripheral blood mononuclear cells by density gradient centrifugation, induced EPCs in vitro, and screened the difference of miRNAs expression profiles in DVT patients and healthy people EPCs by gene chip. Using real time fluorescence quantitative PCR (qRT-PCR) to verify the results of the chip, the miRNAs, which is consistent with the results of the chip, is selected by literature search and bioinformatics analysis to select the miRNAs to do cell function experiments (we select 6 miRNAs), observe the migration of these miRNAs to EPCs, the function of cell formation and apoptosis and so on. The results of cell function show that miR-483-3p has an impact on the function of EPCs. We do a more thorough study of miR-483-3p, predict the possible target gene of miR-483-3p through bioinformatics, up and down miR-483-3p through the luciferase reporter gene experiment, CO transfection and gene silencing, and further confirm the target gene with the change of Western blot detection protein. And the effect of CO transfection and gene silencing on the function of EPCs. In vivo, the effect of rat thrombus model, confocal fluorescence microscopy, HE staining and DSA observation on the effect of MI R-483-3p on EPCs homing and thrombolytic recanalization after EPCs were observed in vivo. The results showed that miR-483-3p was highly expressed in EPCs of DVT suffering from EPCs; Promoting EPCs migration, vascular capacity, inhibiting EPCs apoptosis, promoting EPCs homing and dissolution of thrombus by EPCs. Our study will explore new ideas for the treatment of venous thrombosis by stem cells. This experimental study will be divided into 5 parts. The main research methods and results are as follows. Cell culture and identification Objective: to establish the isolation, culture and identification of human peripheral blood endothelial progenitor cells (endothelial progenitor cells (EPCs) and rat bone marrow derived endothelial progenitor cells. Methods: the separation of human peripheral blood and rat bone marrow mononuclear cells by density gradient centrifugation method will be established by the method of density gradient centrifugation. The nuclear cells were suspended in the EGM-2 medium containing 20% fetal bovine serum (FBS), cultured for 2~3 weeks and observed the morphological characteristics of the cells under microscope. Flow cytometry was used to detect the cell surface markers CD34, CD133, VEGFR-2 expression, and double fluorescence staining was used to detect the uptake of DiI-ac-LDL and FITC-UEA-1 in cells. Results: isolated human peripheral blood mononuclear cells (peripheral) Blood mononuclear cells, PBMC) and bone marrow mononuclear cells (Bone marrow-derived mononuclear cells, BMMNC) have small round volume. After 2 days, a few cells are adhered to the wall. After 3 days, the cell volume becomes larger and the adherent cells increase. After 5 days, the cells are spindle shaped, cell colonies appear, and the surrounding cells are arranged radiated from tenth to 14 days. The cells showed the paving stone or cobblestone like changes. Flow cytometry showed that the cell surface mainly expressed the endothelial markers VEGFR-2, CD34 and CD133. The double fluorescence staining showed that the cells were able to swallow DiI-ac-LDL and FITC-UEA-1. conclusion: under the inducement of EGM-2 medium, the cells were successfully derived from human peripheral blood mononuclear cells and rat bone marrow mononuclear cells. EPCs and 2~3 weeks were cultured for advanced EPCs. The screening and verification of MI RNAs differential expression profiles in EPCs in second patients with deep vein thrombosis in the lower extremities were screened and verified by using gene chip to screen the difference of MI RNAs expression in the peripheral blood EPCs of DVT patients and healthy people, and the reliability of the result of Q RT-PCR test chip. Using the density gradient centrifugation method, the peripheral blood mononuclear cells were isolated from the peripheral blood samples from the healthy people for EPCs culture. The MI RNA gene chip was used to screen the MI RNAs expression profiles of the DVT patients and the healthy people EPCs, and the Q RT-PCR was used to verify the results of the chip. The expression of EPCs in peripheral blood was different, and the results of Q RT-PCR were consistent with the chip. Conclusion: the expression of multiple mi RNAs, such as Mi R-483-3p, is different in the peripheral blood EPCs of DVT patients and healthy people. Third part Mi R-483-3p on the function of endothelial progenitor cells and target gene prediction and verification purpose: To explore the migration of human peripheral blood and angiogenesis. The effect of ability and apoptosis was predicted and the target gene was predicted. Methods: Mi R-483-3p mimics (agomir), inhibitor (antagomir) and negative control were transfected into EPCs with Lipofectamine 3000. The effect of MI R-483-3p on EPCs migration was detected by Transwell test, and Matrigel lumen formation was used to detect the capacity of MI. The effect of MI R-483-3p on EPCs apoptosis was detected by flow cytometry. Using bioinformatics to predict the possible target genes of MI R-483-3p, the target genes were confirmed by luciferase reporter gene experiment, Q RT-PCR and Western blots. The result: up regulation mi R-483-3p expression in EPCs inhibits migration and angiogenesis, and promotes apoptosis. The result and up regulation of MI R-483-3p expression in EPCs were down, and bioinformatics predicted that the serum reaction factor (serum response factor, SRF) might be the target gene, and Q RT-PCR and Western blots showed that SRF was the target gene. SRF is the target gene of MI R-483-3p. The construction and expression of the fourth part of the Mi R-483-3p lentivirus vector: construct mi R-483-3p/mi R-483-3p sponge Lentivirus Expression Vector, infect rat EPCs, and verify the MI expression in EPCs. P GLV3-H1-GFP-mi R-483-3p and P GLV3-H1-GFP-mi R-483-3p sponge were connected by the enzyme, and 293T cells were transfected with the auxiliary package carrier, and the infection EPCs in the virus supernatant was collected. The transfection efficiency was observed by the fluorescence microscope. The expression of the infection was detected by Q RT-PCR. 3-H1-GFP-mi R-483-3p sponge was successfully constructed. After transfection of EPCs, the expression of MI R-483-3p in EPCs was up and down effectively. Conclusion: a lentiviral vector carrying the target gene mi R-483-3p P GLV3-H1-GFP-mi R-483-3p/p, which regulates the thrombolysis of venous thrombosis, is regulated by the fifth part. Objective: To study the effect of MI R-483-3p on EPCs homing and EPCs on the dissolving and recanalization of venous thrombosis after EPCs in rats. Methods: transfection of the lentivirus carrier P GLV3-H1-GFP vector, P GLV3-H1-GFP-mi R-483-3p and P GLV3-H1-GFP-mi into the inferior vena cava under the left renal vein to construct the rat Deep vein thrombosis model, and then transfected EPCs through rat tail vein to thrombus model, divided into four groups: A group (10), blank control group, 1 ml PBS via tail vein, B group (10 rats), EPCs/p GLV3-H1-GFP vector (EPCs/vector group), 1 ml containing 1 x 106EPCs/vector PBS cell suspension through tail vein injection; 10 group (10) GFP-mi R-483-3p (group EPCs/mi R-483-3p) was injected into the tail vein for 1 ml PBS cell suspension containing 1 x 106 EPCs/mi R-483-3p; D group (10), EPCs/p GLV3-H1-GFP-mi R-483-3p, and injected into the tail vein for 1 * 7 cells suspension. The specimens were collected on 7 days after the operation. A microscope was used to observe the homing of EPCs in thrombus, HE staining, and digital subtract angiography (DSA) to observe the dissolving and recanalization of venous thrombosis. Results: the GFP labeled EPCs appeared in the venous thrombosis, and the number of positive cells in the different experimental groups was compared with the EPCs/mi R-483-3p sponge group EPCs/vector group Group 3P, suggesting that MI R-483-3p inhibited EPCs homing to venous thrombosis. Compared with the experimental group, the thrombus weight of the experimental group was compared with the EPCs/vector group EPCs/mi R-483-3p sponge group in group blank control EPCs/mi R-483-3p, and the thrombolytic ability of the experimental groups was compared. Tor group EPCs/mi R-483-3p group blank control group; DSA observation of thrombolytic recanalization in each experimental group: EPCs/mi R-483-3p sponge group EPCs/vector group EPCs/mi R-483-3p group. The transfected EPCs can be returned to the venous thrombosis. Up regulation of MI R-483-3p inhibits the EPCs homing ability and the dissolution and repassage of EPCs to venous thrombosis, and the downregulation of MI R-483-3p can promote the homing and thrombolytic ability of EPCs.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R543.6
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本文编号：1915938