【作者】 刘珍珍；

【导师】 董宁征；

【作者基本信息】 苏州大学 ， 血液病学， 2013， 硕士

【摘要】 目的：周围血管疾病是威胁人类健康的世界难题，长期组织血流灌注不足可引起缺血性溃疡和坏疽，最终导致三分之一以上的病人截肢。自1997年以来，循环内皮祖细胞（endothelial progenitor cells, EPCs）参与的血管生成被认为是出生后血管发生的重要机制。基于这一认识，细胞基础的针对损伤血管的修复得到广泛的研究。EPCs定向归巢到缺血坏死组织的第一步是EPCs在缺血部位的滚动粘附，这是由P-选择素（P-selectin）和E-选择素（E-selectin）与其共同的配体P-选择素糖蛋白配体（P-selectinglycoprotein ligand-1, PSGL-1）的相互作用完成的。研究发现，大约25%的脐血CD34+造血干细胞表面的PSGL-1不能有效地与P-选择素以及E-选择素结合。由于EPCs与CD34+造血干细胞来源于同一前体细胞-成血管细胞，因此，我们假设EPCs具有同样的不足，如果我们能够修正这一功能缺陷，将会促进EPCs归巢至血管形成部位，从而为增强干细胞的治疗效果提供了一种新的有效的治疗策略。方法：1.通过密度梯度离心法从脐血中分离出单核细胞层（mononuclear cells, MNCs），通过免疫磁珠方法分离纯化CD34+细胞，将纯化的CD34+细胞接种于含EGM-2培养基的人纤维连接蛋白（fibronectin, FN）包被的培养皿中进行培养及体外扩增。2.通过流式细胞术检测脐血EPCs表面分子的表达，如CD133、CD31、CD34、CD144、VEGFR2、CD105、CD146、CD14和CD45。3.通过逆转录聚合酶链反应（reverse transcription polymerase chain reaction,RT-PCR）分析脐血EPCs特异基因的表达。4.通过免疫荧光技术分析血管性血友病因子（von Willebrand factor, vWF）、CD31、CD144在细胞中的表达。5.分析所培养细胞对乙酰化低密度脂蛋白（Acetylated LDL, Ac-LDL）的摄取能力及体外成血管能力。6.将岩藻糖基转移酶V（Iα1,3-fucosyltransferase VI, FucT VI）质粒转染至EPCs中，并通过Western blot方法对岩藻糖基转移酶蛋白的表达进行鉴定。7.使用特异性识别人唾液酸化的路易斯寡糖（sialyl Lewis X, sLex）结构的抗体HECA-452，通过流式细胞术分析转染FucT VI表达质粒及转染空载体质粒的EPCs表面sLex的表达变化。8.流式细胞仪检测转染FucT VI表达质粒及对照质粒的EPCs与P-选择素和E-选择素的结合能力变化。9.检测FucT VI质粒转染组及空载体质粒转染组的EPCs与经肿瘤坏死因子α（tumour necrosis factor-alpha, TNF-α）处理的人脐静脉内皮细胞株（human umbilicalvein endothelial cell, HUVEC）的粘附能力。10.建立联合免疫缺陷小鼠下肢缺血模型，将体外标记的FucT VI质粒转染组EPCs及空载体质粒转染组EPCs通过尾静脉注射至小鼠体内，并检测其向缺血部位的归巢。11.建立联合免疫缺陷小鼠下肢缺血模型，将试验小鼠随机分为3组：生理盐水组、EPCs未经过糖化处理组、EPCs糖基化处理组。将细胞或生理盐水通过尾静脉注射到小鼠体内，通过多普勒超声检测三组小鼠的血流恢复情况，通过H&E染色评价小鼠的微脉管结构，通过免疫荧光技术检测小鼠的下肢缺血部位毛细血管密度。结果：1.通过密度梯度离心法和免疫磁珠法筛选出的CD34+细胞，接种于FN包被的培养皿中，静置48小时后，可见数个散在的细胞集落，鹅卵石样集落于一周左右出现。2.通过流式细胞术对所培养的EPCs进行鉴定，结果CD133、CD31、CD34、CD144、VEGFR2、CD105和CD146的表达均为阳性，CD14及CD45的表达为阴性，符合EPCs的表型特征。3.通过RT-PCR对所培养的细胞进行鉴定，结果EPCs的特异基因，如CD31、CD34、CD144、VEGFR-2和vWF，均有表达。4.通过免疫荧光技术检测所培养EPCs的表型，结果CD31、CD144及vWF的表达均为阳性，符合EPCs的表达特征。5.所培养细胞具有EPCs的特征，具有摄取DiI标记的Ac-LDL的能力和体外成血管能力。6.将FucT VI质粒转染至EPCs中，48小时后通过Western blot检测到FucT VI蛋白在EPCs中表达。7.使用特异性识别人sLex结构的抗体HECA-452，通过流式细胞术比较转染FucT VI表达质粒及转染空载体质粒的EPCs表面sLex的表达差异，结果FucT VI质粒转染组sLex的阳性表达率约89.93%±2.13，空载体质粒转染组sLex的阳性表达率约5.29%±1.25，两组比较p <0.01。8.通过流式细胞术比较FucT VI质粒转染组与空载体质粒转染组与P-选择素和E-选择素的结合能力差异，结果FucT VI质粒转染组与P-选择素结合率为87.61%±2.37，与E-选择素结合率为83.44%±3.11；空载体质粒转染组与P-选择素结合率为8.56%±1.17，与E-选择素结合率为3.11%±0.89。由此可见，FucT VI质粒转染组与P-选择素和E-选择素的结合能力较空载体质粒转染组明显增强。9. FucT VI质粒转染组EPCs与TNF-α刺激后的人脐静脉内皮细胞（HUVEC）的粘附能力是空载体质粒转染组粘附能力的1.3倍。10.建立联合免疫缺陷小鼠下肢缺血模型，将体外标记的FucT VI质粒转染组及空载体质粒转染组的EPCs通过尾静脉注射入小鼠体内，三天后收集下肢缺血侧肌肉，结果在荧光显微镜下观察到缺血部位EPCs的粘附，且在缺血部位EPCs的粘附FucTVI质粒转染组为空载体质粒转染组的转染组的2.44倍，p <0.01。11.在缺血后14天通过激光多普勒超声检测三组小鼠下肢血流情况，结果显示糖基化处理组缺血侧/对照侧的血流灌注比值较未糖基化处理组升高34%，未糖基化处理组较生理盐水组升高42%，糖基化处理组较生理盐水组升高90%，三组之间的差异均有统计学意义。缺血侧下肢肌肉组织H&E染色显示糖基化处理组微脉管结构再生强于未糖基化处理组，未糖基化处理组强于生理盐水组。免疫荧光检测缺血侧下肢肌肉毛细血管密度，结果糖基化处理组毛细血管密度为未糖基化处理组1.57倍，未糖基化处理组毛细血管密度为生理盐水组1.75倍，三组之间的差异均有统计学意义（p<0.01）。结论：首先我们从脐血中分离和培养出了EPCs，研究发现脐血来源的EPCs存在PSGL-1糖基化缺陷，通过体外糖基化修饰恢复了EPCs的PSGL-1功能后，经体外实验和体内动物模型证实了糖基化修饰后EPCs的治疗效果。此研究发现为促进EPCs归巢至血管形成部位从而增强干细胞的治疗效果提供了一种新的有效的治疗策略。更多还原

【Abstract】 Objective:Peripheral vascular disease is a major health problem in the world. In the late stagesof this disease, progression of tissue hypoperfusion results in ischemic ulceration andgangrene, leading to amputation in more than a third of patients. Since1997, postnatalvasculogenesis has been purported to be an important mechanism for angiogenesis viacirculating endothelial progenitor cells (EPCs). Based on this paradigm, EPCs have beenextensively studied as a cell-based therapy for repair of damaged blood vessels. P-selectin,E-selectin, and P-selectin glycoprotein ligand-1(PSGL-1) cooperatively mediate EPCsrolling, the first step in EPCs home to the lesion. Recent experiments suggest thatapproximately25%of cord blood CD34+hematopoietic stem cells failed to bind toP-selectin and E-selectin. EPCs and hematopoietic stem cells are considered to be derivedfrom common mesodermal precursor cells-hemangioblasts, we hypothesize that a similardefect may occur to EPCs and partial EPCs may failed to bind to P-selectin and E-selectin,and repair this defect may provide an effective and innovative therapeutic strategy toimprove the recruitment of injected cells to sites of neovascularization and thereby theefficiency of stem cell therapy.Methods:1. Human mononuclear cells (MNCs) were isolated from cord blood by densitygradient centrifugation with Histopaque1077, then CD34+cells were separated fromMNCs by a magnetic bead separation method. Cells were plated on culture dishes coatedwith human fibronectin and maintained in EGM-2. 2. Human cord blood-derived endothelial progenitor cells were subjected to flowcytometric analysis to examine surface expression of CD133, CD31, CD34, CD144,VEGFR2, CD105, CD146, CD14and CD45.3. Expression of specific genes in human cord blood-derived endothelial progenitorcells were analyzed by RT-PCR.4. Expression of von Willebrand factor (vWF), CD31and CD144of human cordblood-derived endothelial progenitor cells were determined by immunostaining.5. The abilities of human cord blood-derived endothelial progenitor cells toincorporate DiI-acetylated LDL (DiI-Ac-LDL) and to form capillary-like structures onMatrigel were investigated.6. Expression of recombinant FucT VI protein in transfected human cordblood-derived endothelial progenitor cells were analyzed by Western blot.7. Cell-surface sLexdeterminants were measured by flow cytometric analysis withanti-sLex mAb HECA-452.8. The P-and E-selectin-binding acticities of human cord blood-derived endothelialprogenitor cells, treated or not with the FucT VI, were analyzed by using recombinant P-and E-selectin and flow cytometric analysis.9. HUVEC was treated with EPCs TNF-α, and used to detect the EPCs adhesionability by crystal violet staining assay.10. Athymic Nude mice underwent surgery to induce unilateral hind limb ischemia,EPCs were intravenously administered6hours after induction of hind limb ischemia todemonstrate homing to ischemic muscles.11. Laser Doppler perfusion imaging was used to provide functional evidence forischemia-induced changes in vascularization. Capillary density in the gastrocnemiusmuscle was assessed by immunohistochemistry. Microvessels were detected by theevaluation of H&E-stained sections taken from the gastrocnemius muscle.Results:1. In order to isolate EPCs from human cord blood, MNCs were separated by density gradient centrifugation with Histopaque1077, and CD34+cells were separated from MNCsby a magnetic bead separation method. When CD34+cells were plated on culture dishescoated with human fibronectin, a number of cell clusters appeared within48hours, andcobblestone-like clusters start to appear around1week.2. The results of flow cytometric analyses showed that EPCs were positive forCD133, CD31, CD34, CD144, VEGFR2, CD105, and CD146, but not for CD14, andCD45.3. Specific genes in EPCs, such as CD31, CD34, CD144, VEGFR-2, and vWF，were detected by RT-PCR.4. To examine whether cobblestone-like cells have endothelial phenotypes, cellswere subjected to immunocytochemistry and positively stained for CD31, CD144, andvWF.5. EPCs have ability to incorporate DiI-acetylated LDL and form capillary-likestructures on Matrigel.6. Western blot showed that FucT VI protein was expressed in transfected human cord blood-derived endothelial progenitor cells.7. Cell-surface sLexdeterminants were measured by flow cytometric analysis withanti-sLexmAb HECA-452. The results showed that sLexexpression was more in EPCstreated with FucT VI than that in non-treated.8. Human cord blood-derived endothelial progenitor cells, treated or not with theFucT VI, were subjected to flow cytometric analysis for the P-and E-selectin-bindingassay. We found that FucT VI treatment dramatically increased the percentage of EPCsbinding P-and E-selectin.9. FucT VI treatment resulted in an significant increase of the ability of EPCs toadhere to TNF-α acitivated HUVEC monolayer.10. Athymic Nude mice underwent surgery to induce unilateral hind limb ischemia,single-fluorescence labeling was used to detect injected EPCs in sections of thegastrocnemius muscle. Our results indicated that transplanted EPCs were detected in sections of the gastrocnemius muscle. In addition, the number of incorporated EPCs, asdetected with DiI labeling, was higher in FucT VI treatment group.11. Laser Doppler perfusion imaging was used to provide functional evidence forischemia-induced changes in vascularization14days after injection. Capillary density inthe gastrocnemius muscle was assessed by immunohistochemistry. Microvessels weredetected by the evaluation of H&E-stained sections taken from the gastrocnemius muscle.Blood flow ratio, capillary density, and microvessels regeneration were higher in micetransplanted with FucT VI-treated EPCs compared with that of PBS-and EPC-injectedmice, respectively.Conclusions:First, we isolated and differentiated EPCs from human cord blood. Flow cytometricanalysis demonstrated that PSGL-1lacks key fucosylation in EPCs. Treatment of EPCswith recombinant FucT could restore the impaired fucosylation and improve their ability tobinding with P-and E-selectin. These approaches may improve the recruitment of injectedcells to sites of neovascularization and thereby the efficiency of stem cell therapy.更多还原