【作者】 王文斌；

【导师】 李晓强；

【作者基本信息】 苏州大学 ， 胸心血管外科， 2014， 博士

【摘要】 深静脉血栓形成(Deep venous thrombosis DVT)是常见的外周血管疾病，可导致肢体肿胀、下肢缺血、血栓后综合症，甚至致死性肺栓塞的发生。现在对于DVT的治疗主要是各种抗凝溶栓药物的使用，但效果并不令人满意，同时抗凝药物的副作用可导致机体凝血功能下降、消化道出血、手术患者切口并发症发生。研究人员发现骨髓来源的内皮祖细胞(Bone marrow-derived endothelialprogenitor cells BMEPCs)可以分化为血管内皮细胞，分泌各种促血管生成因子从而促进缺血组织的血管新生。研究证实，EPCs可以归巢到深静脉血栓中促进血栓机化再通。我们实验组前期的研究也发现移植EPCs后可以改善血栓内的微环境，促进急性静脉血栓溶解及慢性血栓再通。但是由于EPCs增殖能力及迁移归巢能力较弱，EPCs的应用受到很大的限制。如何更好的改善EPCs的功能成为广大学者的研究方向。MicroRNAs(miRNAs)是一类小分子非编码RNAs，可在转录后水平调控基因表达。研究证明MicroRNAs在血管新生过程中扮演了重要角色。miR-150最初被发现于免疫相关细胞中，控制着淋巴细胞的生长分化。研究发现，miR-150可促进间充质干细胞迁移。单核细胞分泌的miR-150可以提高人微血管内皮细胞迁移及成血管能力。但是，miR-150对于EPCs的功能影响，特别在深静脉血栓再通中的作用却未见相关报道。本实验研究中，我们采用密度梯度离心法分离SD大鼠的骨髓单个核细胞（Bonemarrow-derived mononuclear cells, BMMNCs），EGM-2MV培养基诱导、培养、扩增骨髓源性EPCs并鉴定。体外实验中，我们将对照寡核苷酸和miR-150的模拟物或抑制物采用电转的方法，转染EPCs，探讨miR-150对EPCs的功能影响。为了进一步研究miR-150对EPCs在深静脉血栓机化再通中的作用，我们构建了携带miR-150的慢病毒表达载体并转染到大鼠EPCs中，将转染后的EPCs注入到大鼠深静脉血栓模型中。研究结果发现，过表达miR-150能够促进大鼠EPCs的迁移成管能力，促进EPCs归巢到静脉血栓中，加速血栓的机化再通。我们的研究揭示了miR-150对EPCs功能的影响，可能为深静脉血栓治疗带来新的希望。本实验研究，将分为4个部分。主要研究方法及结果如下。第一部分大鼠骨髓源性内皮祖细胞的分离、培养及鉴定目的：分离、培养并鉴定大鼠骨髓源性内皮祖细胞(endothelial progenitor cells，EPCs)。方法：采用密度梯度离心方法分离大鼠骨髓单个核细胞，采用内皮培养体系EGM-2培养剂进行诱导分化定向诱导培养14-21天，观察细胞的生长形态变化，结合免疫组化、免疫荧光、流式细胞仪技术检测内皮细胞表面标记及造血干细胞表面标记，采用Matrigel成管实验及DiI-ac-LDL摄取实验检测内皮细胞的功能学特性。结果：新分离的骨髓单个核细胞（BMMNCs）呈圆形，48h后部分细胞开始贴壁，贴壁细胞呈纺锤型形态，细胞集落形成，细胞培养至第10～12天逐渐融合，细胞形态呈现出鹅卵石样改变。DiI-acLDL摄取结果显示，细胞能够吸收DiI-ac-LDL。Matrigel成管实验显示培养的细胞能形成管状、网络状结构。流式细胞仪分析及免疫荧光检测结果显示，细胞主要表达内皮特异性标记物VEGFR、vWF；几乎不表达造血干细胞表面标记CD133。结论：本实验成功地建立了一套大鼠骨髓血管内皮祖细胞分离、培养、诱导分化及鉴定的方法体系。在特定培养体系诱导下，可从大鼠骨髓单个核细胞中获得内皮祖细胞，培养到第10～14天的内皮祖细胞呈现late-EPCs表型特征。第二部分MiR-150对内皮祖细胞功能影响的体外实验研究目的：探讨miR-150对大鼠内皮祖细胞(endothelial progenitor cells，EPCs)增殖、迁移、及成管能力的影响方法：将miR-150模拟物或抑制剂或阴性对照用电转的方法转染到EPCs中。采用MTT、流式细胞术检测miR-150对EPCs增殖及细胞周期的影响。采用划痕实验、穿膜实验检测miR-150对EPCs运动迁移能力的影响。采用Matrige管腔形成实验检测miR-150对EPCs成血管能力的影响。为了探查miR-150对EPCs功能影响的可能靶基因，我们使用在线microRNA数据库寻找miR-150调控EPCs的潜在靶基因。结果：miR-150对内皮组细胞的增殖及细胞周期没有影响。划痕实验及穿膜实验都证实了miR-150模拟物能够显著促进EPCs的迁移运动能力； miR-150抑制剂则能抑制EPCs的迁移运动能力。过表达miR-150能增强EPCs的成管能力。c-Myb的3’UTR区内有miR-150两个潜在的结合位点。结论：miR-150对细胞增殖及细胞周期没有作用。过表达miR-150促进大鼠EPCs迁移及运动及成管能力。c-Myb可能是miR-150的靶基因。第三部分构建miR-150慢病毒表达载体及其在内皮祖细胞有效性表达目的：构建miR-150慢病毒表达载体，感染大鼠EPCs后检测miR-150的表达，为研究体内条件下miR-150在EPCs中的生物学功能奠定基础。方法：将聚合酶链反应(PCR)扩增得到的miR-150前体序列pre-miR-150和慢病毒载体pLVX-IRES-ZsGreenl经酶切后连接产生pLVX-IRES-ZsGreenl-miR-150，经酶切及测序鉴定正确后在HEK293T细胞中包装病毒，收集病毒上清后感染EPCs。用实时荧光定量PCR(qPCR)方法检测感染后的EPCs中miR-150的表达。结果：酶切及测序结果示慢病毒表达载体pLVX-IRES-ZsGreenl-miR-150构建正确，病毒上清液感染EPCs后能有效提高miR-150的表达。结论：成功构建了pLVX-IRES-ZsGreenl-miR-150慢病毒表达载体及稳定表达miR-150的EPCs细胞系。第四部分miRNA-150对内皮祖细胞在深静脉血栓再通中的作用目的：探讨miRNA-150对大鼠内皮祖细胞(endothelial progenitor cells， EPCs)在血栓机化再通中的作用。方法：我们通过结扎大鼠下腔静脉构建深静脉血栓模型，将构建成功的慢病毒载体pLVX-IRES-ZsGreen1/pLVX-IRES-ZsGreen1-miR-150转染到EPCs中并移植到血栓模型中。在术后7天、14天收集标本称重。通过荧光示踪观察EPCs归巢到静脉血栓的情况。采用HE染色，CD34免疫组化染色观察转染miR-150后EPCs对静脉血栓机化再通的影响。为了探查miR-150促进血栓机化再通的可能机制，我们采用免疫组化染色检测血栓中MMP-2的表达情况。结果：移植的EPCs出现在静脉血栓中。EPCs/miR-150组中GFP阳性细胞数目较EPCs/vector组明显增多。EPCs移植后抑制静脉血栓形成，EPCs/miR-150组中静脉血栓的重量最低。HE染色和CD34免疫组化染色提示EPCs促进血栓的机化再通。相对于EPCs/vector组，EPCs/miR-150组中血栓机化再通最为明显。不论在术后7天还是14天，MMP-2在EPCs/miR-150组和EPCs/vecto组的表达皆高于空白对照组。其中，EPCs/miR-150组中MMP-2的表达最为丰富。结论：移植的EPCs可以归巢到静脉血栓中促进血栓溶解。EPCs显著抑制血栓形成，促进血栓的机化再通。miR-150增强了EPCs的归巢能力，促进了EPCs在血栓机化再通中的作用。提高EPCs中miR-150的表达水平可能为DVT的治疗带来希望。更多还原

【Abstract】 Deep venous thrombosis (DVT) is a common peripheral vascular disease. DVTmay lead to the abnormal swelling and ulceration of lower limb, post-thromboticsyndrome (PTS) or even pulmonary embolism (PE) which can lead to the sudden deathof patients. Currently, the clinical treatment for DVT is using of anticoagulants to reducethe incidence of PE, PTS and recurrence of DVT. However,the usage of anticoagulantsis associated with the high risk of bleeding and wound complications.The discovery of marrow-derived circulating endothelial progenitor cells (EPCs)cause people’s great interest because of their plasticity to differentiate into endothelialcells (ECs) and as a source of paracrine proangiogenic factors. It has been proven thatEPCs are recruited into resolving venous thrombi. Our previous studies also showed thatEPCs significantly improved the microenvironment and promoted the resolution of acutevenous thrombus and the recanalization of chronic thrombus. However, studiesdemonstrated that EPCs recruitment to sites of neovascularition was limited due to theweak viability, migration and homing ability. Therefore, looking for a method toimprove the function of EPCs is being widely discussed.MicroRNAs are a class of~22nucleotides non-coding RNAs that suppress geneexpression at the posttranscriptional level by promoting mRNA degradation or inhibitingmRNA translation. Recent studies have shown that microRNAs are involved in theangiogenesis process. MiR-150, a key regulator for the development of immune cells,was originally detected in mature lymphocyte. It has been proven that miR-150regulatesthe migration of bone marrow stem cells. MiR-150, secreted by monocytes, enhanced the migration of human microvascular endothelial cells. However, the function ofmiR-150in rat EPCs, especially in the ischemia condition such as DVT, remainsunknown.In this study, rat bone marrow was obtained by rinsing bone marrow cavity, thenbone marrow mononuclearcells were isolated by gradient centrifugation using Ficollcentrifugate. mononuclearcells were then cultivated with EGM-2MV mediumcultivation. To investigate the role of miR-150in rat endothelial progenitor cells (EPCs)proliferation and migration, EPCs were transfected with control oligoes and miR-150mimics or inhibitor by electroporation. To further explore the role of miR-150in EPCsunder ischemia condition, EPCs stably expressing miR-150were achieved by lentivirusand delivered into rats with DVT. We demonstrated that ectopic expression of miR-150enhanced rat EPCs motility and tube formation in vitro. MiR-150promoted rat EPCshoming and venous thrombus recanalization and resolution in vivo. Our study revealedthe function of miR-150in EPCs and presents a promising clinical therapy for ischemiadiseases.ChapterⅠ Isolation, culture and identification of endothelialprogenitor cellsObjectives：The goal of this study was to isolate, culture and identify endothelialprogenitor cells from rat bone marrow.Methods：Mononuclear cells (MNCs) from rat bone marrow isolated by densitygradient centrifugation were cultured in EGM-2medium. Morphological changes wereobserved with an inverted microscope. The EPCs were identified by the combinedDil-labeled acetylated low density lipoprotein(Dil-ac-LDL), in vitro tube formationassay, and the cell surface antigen was detected by immunofluorescence technology andflow cytometry.Results：Mononuclear cells isolated from rat bone marrow cultured in EGM-2Medium is round. Cells begin to adherel after48h.The adherent cells are fusiform shape,and cell colony appears. After10~12days culture, cell morphology presentscobblestone appearance. The cells uptake Dil-ac-LDL. Matrigel tubule test show thatcultured cells can form a tubular structure. The resultes of flow cytometry analysis andimmunofluorescence show that the main expression on the surface of cells is VEGFR、vWF; almost no expression of CD133.Conclusions： MNCs isolated from rat bone marrow cultured in EGM-2mediumcan proliferate and differentiate to late EPCs after10~12days culture. Chapter II Studies of miRNA-150effects on endothelialprogenitor cells in vitroObjectives: To investigate the role of miR-150in rat endothelial progenitor cells(EPCs) proliferation,migration and tube formation.Methods: EPCs were transfected with control oligoes and miR-150mimics orinhibitor by electroporation. MTT and flow cytometry analysis was performed toevaluate the growth of EPCs subjecting to miR-150. Cell migration analysis was doneby wound healing and transwell assay. To test whether miR-150affected the angiogenicactivity of EPCs in vitro, we performed matrigel tube formation assay. Bygain-of-function examination, we searched for its putative target genes using onlinesearch tool (TargetScan).Results: The EPCs growth and cell cycle was immue from miR-150application.Both the wound healing and transwell assay showed that miR-150promoted EPCsmigration and miR-150inhibitor inhibited EPCs migration in vitro. Ectopic expressionof miR-150enhanced EPCs tube formation in vitro. C-Myb was predicted to have twoputative miR-150binding sites within its3’UTR.Conclusions: MiR-150had no effect on the growth and cell cycle of EPCs.MiR-150enhanced the migration, tube formation ability of rat EPCs. C-Myb may be amiR-150target gene. Chapter III Construction of the miR-150Lentiviral ExpressionVector and its Expression in endothelial progenitor cellsObjectives: To construct the miR-150lentiviral vector, and to detect itseffectiveness in EPCs．It is the bases of functional study of miR-150effects on ratendothelial progenitor cells (EPCs) in vivo.Methods: MiR-150was amplified from the genomic DNA and inserted intopLVX-IRES-ZsGreenl vector after double digestion to generate pLVX-IRES-ZsGreenl-miR-150．The vector was then confirmed by PCR and DNA sequencing. EPCs wasinfected by the concentrated lentivirus293T produced. MiR-150were assessed byqPCR．Results: The recombinant lentiviral vector was Successfully established andconfirmed by PCR and DNA sequencing. MiR-150was integrated into the genome ofEPCs and miR-150expression was enhanced effectivcly by qPCR．Conclusions: The miR-150lentiviral expression vector was successfullyconstructed, and the miR-150overexpressed in EPCs． Chapter IV Studies of miRNA-150effects on endothelialprogenitor cells in deep venous thrombosisObjectives: To investigate the functional role of miR-150in rat endothelialprogenitor cells (EPCs) and its potential application in deep venous thrombosis.Methods: We ligated the inferior vena cava(IVC) of rats and developed the DVTmodels. EPCs with stable expression of miR-150or empty vector viapLVX-IRES-ZsGreen1lentivirus were transplanted into these DVT models. On the dayof7and14after the transplantation, the rats were sacrificed and the thrombi wereweighed. The homing of EPC was shown by GFP expression and observed using fluorescence microscope. To observe the thrombus organization and recanalization, thesections were performed with HE staining and immunohistochemicalstaining for CD34.To investigate the molecular mechanisms underlying the increased thrombus resolvingability of EPCs overexpressing miR-150, the intrathrombotic expression of MMP-2wasdetected using immunohistochemical staining.Results: Compared with control group, transplanted EPCs were situated around thethrombus in both EPCs/vector and EPCs/miR-150group. The larger number of GFPpositive cells appeared in EPCs/miR-150group at day7and14compared withEPCs/vector group. Compared with control group, significant decrease of thrombusweight was observed in EPCs/vector and EPCs/miR-150groups. The thrombus weightof EPCs/miR-150group was even lower than that of EPCs/vector group. HE stainingand CD34immunohistochemical analysis showed that thrombus organization andrecanalization were better in EPCs/vector and EPCs/miR-150group than control group.Larger and more channels were seen in EPCs/miR-150group in comparison withEPCs/vector group. Compared at the indicated time points, there were moreMMP-2-positive cells in EPCs/vector and EPCs/miR-150group in contrast with controlgroup. Furthermore, there were more MMP-2-positive cells in EPCs/miR-150groupcompared with EPCs/vector group.Conclusions: The exogenous EPCs are recruited into resolving venous thrombi.EPCs transplantation significantly inhibited thrombus formation and improved thrombusorganization and recanalization. MiR-150enhanced the homing, thrombusrecanalization and resolution ability of rat EPCs. Restoring miR-150in EPCs presents apromising clinical therapy for DVT therapy.更多还原