【作者】 王舒楠；

【导师】 张绍祥；

【作者基本信息】 第三军医大学 ， 计算医学， 2012， 博士

【摘要】 研究背景和目的：胶质瘤是脑内最常见原发性肿瘤，严重威胁人类健康。寻找胶质瘤靶向治疗的新方案、延长患者的生存期限一直是国内外学者努力的方向。研究表明，内皮祖细胞（EPCs）在胶质瘤血管新生以及肿瘤转移、复发中均起到重要作用。EPCs有望成为抗肿瘤治疗的新靶点或携带抗肿瘤治疗药物的载体，并在实验性动物模型中取得初步成功。但就目前来看， EPCs对胶质瘤血管生成的作用一直存在争议，不同文献报道的EPCs参与到实验性肿瘤血管形成的能力不一，定量研究发现含有EPCs的血管内皮占胶质瘤总血管比例从0%--50%不等。并且，目前缺乏较好的无创性活体示踪手段来评价EPCs在宿主组织中的存活、迁移及分布情况。这也限制了以EPCs作为载体或靶点的胶质瘤治疗新技术的发展。因此，需要用非侵入性的手段来对移植的EPCs进行活体动态观察，以评价移植EPCs在体内的分布、迁移及转归。随着分子影像技术的发展，特别是近期磁性纳米材料的出现，使得无创性活体示踪移植细胞成为可能，越来越受到研究人员的关注。磁共振成像具有空间分辨率高、清晰的解剖成像及强大的功能成像分析，可以成为显示磁性标记细胞在体内的生物分布的一个有效途径。将外源性移植细胞标记磁共振对比剂后，通过磁共振扫描能将细胞从其周围的组织中分辨出来。CT灌注成像（CT perfusion，CTP）脑功能成像方法之一，通过研究脑组织的血流灌注状态以及组织血管化程度来获取微循环信息。CT灌注成像技术能无创性活体评价肿瘤微血管功能和活性的影响，通过定量分析肿瘤区血流量（CBF）、脑血容量（CBV）、平均通过时间（MTT）、表面通透性（PS）等参数，反映肿瘤微血管的功能和活性变化。本研究针对EPCs参与胶质瘤血管形成领域中存在的一些不足，采用磁共振活体示踪及CT灌注成像技术开展对EPCs参与胶质瘤新生血管形成的定性、定量研究，将有助于了解EPCs与胶质瘤血管生成的时空关系，进一步明确EPCs对肿瘤血管生成的影响，为更好的运用以EPCs为靶点/载体的抗肿瘤治疗提供实验依据。研究方法：1、以大鼠骨髓作为EPCs的主要来源，利用密度梯度离心法分离出单个核细胞（Mononuclear cells, MNCs），再利用内皮系选择性培养基EGM-2MV筛选出高纯度的EPCs，并对培养的细胞进行了免疫表型检测、内皮功能分析和体外趋瘤性实验。2、以PLL和jetpet-FlouR分别介导两类磁性对比剂Resovist和Gd-DTPA标记EPCs，设置不同的标记浓度组，研究Resovist和Gd-DTPA标记效率和体外对细胞生物学特性的影响。进一步采用3.0-T磁共振对不同浓度Resovist和Gd-DTPA标记的细胞进行扫描，分析其影像信号特点。对比分析Resovist和Gd-DTPA标记细胞的特点和优劣性。3、建立大鼠脑胶质瘤原位模型，通过鼠尾静脉移植25ug/ml Resovist标记的EPCs。利用3.0-T磁共振活体示踪EPCs靶向胶质瘤的时空分布；利用CT灌注成像分析EPCs参与构建胶质瘤微血管血流动力学变化；通过病理检测和激光共聚焦进一步证实EPCs参与形成的胶质瘤微血管的形态结构和功能活性。研究结果:1、通过密度梯度离心法结合条件培养法能获得具有祖细胞自我更新的能力并能诱导分化为内皮细胞的EPCs。骨髓来源的EPCs培养初期细胞CD133、CD34和VEGFR2呈阳性表达，经诱导培养后，CD133表达逐渐降低、而CD34和VEGFR2表达增高，同时细胞开始表达内皮表型CD31和vWF。进一步对细胞内皮功能分析证实所培养的细胞具备内皮功能，能摄取低密度乙酰化脂蛋白和结合荆豆凝集素，具有体外形成血管的能力。EPCs在体外对胶质瘤具有明显趋向性，且同C6胶质瘤细胞数量存在相关性。2、采用Resovist和Gd-DTPA均能有效标记细胞， Resovist的最佳标记浓度范围为25-50ug/ml；Gd-DTPA的最适标记浓度为25ug/ml；磁共振T2序列为检测Resovist标记细胞的最佳序列，磁共振T1序列为检测Gd-DTPA标记的最佳序列。Resovist较Gd-DTPA更适合标记细胞，其操作方式简单便捷，具有较高的标记效率且对细胞的生物学特性影响较小。Gd-DTPA能由于具有T1正性对比效应，能有效弥补Resovist信号与某些疾病信号（如出血、金属蓄积、空气等）难以辨别的不足。3、Resovist标记的EPCs能向肿瘤定向迁移，并能引起磁共振T2信号变化。EPCs主要归巢至肿瘤边缘区，在磁共振T2WI像上呈低信号，随着时间延长，细胞归巢数量增多，相应信号改变的程度和范围均增大。普鲁士蓝检测结果同磁共振示踪具有良好的一致性，通过对比肿瘤组织和肾脏、脾脏、肺脏等组织蓝染细胞数，肿瘤边缘组织的阳性细胞数量明显高于非靶器官组织，亦证实循环性EPCs对胶质瘤具有明显的靶向性。采用CT灌注成像分析EPCs移植后肿瘤区不同部位的血流动力学变化，其结果可重复性高，并与病理组织学MVD检测具有良好的一致性。定量分析表明EPCs移植后，明显促进了肿瘤边缘区的血管生成，其综合贡献值约为15.09%-20.06%。激光共聚焦三维扫描能很好的显示EPCs参与形成肿瘤微血管的结构和形态。EPCs主要整合至血管的盲端或新生血管的分支，在肿瘤的血管构建中主要起支撑作用，通过参与形成新生血管的分支点，与母体血管形成相互连接且杂乱的血管网。研究结论：外源性EPCs主要归巢至肿瘤的边缘，整合至血管的分支点或盲端，参与构筑肿瘤微血管形成。定量分析表明EPCs移植后120小时明显促进了胶质瘤边缘区血管生成，其贡献度约为15.09%-20.06%。采用磁共振和CT灌注等影像学技术能无创性活体示踪EPCs移植后在宿主体内的迁移和转归，为今后以干细胞治疗为基础的临床应用提供强有力的监测手段。更多还原

【Abstract】 Background and ObjectiveGlioma is the most common form of primary brain tumor that seriously threatenshuman health. There is no cure for glioma so far. In recent years, researchers fromworldwide have made great efforts on glioma targeted therapy to prolong the survivalperiod of the patients who suffer from glioma. Accumulating evidence suggests thatendothelial progenitor cells (EPCs) play a key role in the development and infiltration ofgliomas. Thus, it has been considered that EPCs may be good vehicles for deliveringanti-angiogenesis genes for tumor therapy. Some research groups have succeeded in usingthis new therapeutic strategy within animal models. However, the role of EPCs in gliomaangiogenesis has been controversial. The capability of EPCs involving in gliomamicrovascluar has also been confused. Quantitative study from different research groupreported that the proportion of glioma microvascular angiogenesis containing EPCs rangingfrom0%-50%. Additionally, limited of in vivo tracking means has restricted the researchersto evaluate the survival, migration and distribution of EPCs in the host tissues. Thesedrawbacks also limit the development of EPCs as a vector or target for glioma therapy.Presently, the advances in magnetic resonance imaging (MRI) equipment provide anew way to track transplanted cells. MRI is an ideal tool because it is a non-invasivetechnique, and can track and/or monitor the temporal and spatial migration of transplantedcells in vivo. To be visualized on the MRI, cells need to be magnetically labeled beforetransplantation. Magnetic iron-oxide nanoparticles (IONPs) have been widely used as MRIcontrast agents to label cells. These nanoparticles have a high relaxation rate, and they canshorten T2relaxation time, which results in a low signal in targeted tissue to visualize thelesion.CT perfusion (CTP) imaging has been wildely used to study the hemodynamic ofhuman brain function. CTP is able to obtain the microcirculation information through the perfusion and vascularlize level of brain tissue. CT perfusion imaging can be a non-invasivemethod to in vivo quantitative analysis of tumor volume blood flow (CBF), cerebral bloodvolume (CBV), mean transit time (MTT) and permeability surface (PS) parameters,reflecting the tumor microvascular function and activity.In this study, we intented to employ both MRI and CT perfusion technology to in vivoquantitative analysis the glioma angiogenises that EPCs involved in. Our work will help todemonstrate the relationship between EPCs and glioma angiogenesis.Our work may asloprovide useful support for the future application of EPCs as a vector or target foranti-angiogenic therapy for glioma.Materials and Methods1. EPCs were isolated from bone marrow obtained from healthy SD rats. Firstly,MNCs were isolated using Ficoll gradient centrifugation, and the cells were cultured inendothelial condition medium to harvest pure EPCs.2. EPCs were labeled with two classes of magnetic contrast agents Resovist andGd-DTPA, respectively. Both Resovist and Gd-DTPA were set up concertrations rangingfrom0-75ug/ml to study the efficiency and toxity to label EPCs. Next, the labeled cellswere scaned by3.0-T MRI to analysis the signal intensity on different sequences. At last,cross compared between Resovist and Gd-DTPA was employed to study the labelingcharacteristic and superiority-inferiority of each contrast agent.3. EPCs labeled with25ug/ml Resovist were transplanted into rat`s glioma models bytail vein injection.3.0-T MRI was employed to in vivo track the transplanted EPCs in thehost tissue at different stages. CT perfusion was used to study the hemodynamic of gliomaangiogenesis that EPCs involved in. Immunohistochemistry and laser confocal microscopywere used to indentify the structure and fuction of glioma microvascular that EPCs involedin.Results1. EPCs isolated from bone marrow using gradient centrifugation combinedconditional cultured have the ability to self-renewal, and could be induced into ECs. EPCsexpressed the surface markes CD133, CD34and VEGFR2at the early stage. Afterconditional cultured, the intensity of CD133expression was change lower, while CD34and VEGFR2expression were enhanced. At this stage, EPCs expressed the endothelial markersCD31and vWF. EPCs have same fuction as ECs, which could uptake of DiI-ac-LDL andcombinding of FITC-UEA-1, and have the ability to angiogenesis in vitro. EPCs couldhome to glioma in vitro. The cell migration ability was related to the total amount of C6glioma cells.2. Both Resovist and Gd-DTPA could be used to label EPCs. The optimalconcentrations of Resovist labeling were25-50ug/ml. The optimal concentration ofGd-DTPA was25ug/ml. The T2sequence on MRI was the best way to detect Resovistlabeled EPCs, while T1sequence to detect the Gd-DTPA labeled cells. We suggest thatResovist should be the best for labeling EPCs due to its high labeling efficiency and lowtoxicity to the cells. Gd-DTPA contrast agents could be a secondary choice for labelingEPCs, especially in cell intra-foci administration and other hemorrhage model.3. EPCs specifically homed to gliomas and could be reliably tracked by1.5-T MR asearly as24hours after transplantation, causing a signal loss on T2-weighted images. Thelabeled cells were mainly located at the periphery of the tumor. Low signal intensitybecame more significant, and the dark areas were enlarged as time went on. The results ofPrussion blue stain showed that the number of blue stain cells at the rim of tumor washigher than that at other non-targeted tissue, such as spleen, kidney and lung tissues, whichwas consistent with MRI findings. Using CT perfusion imaging to analysis thehemodynamic of glioma angiogenesis demonstrated little variability, and the results of CTperfusion were consistent with MVD analysis. Quantitative analysis showed that EPCsenhancing the rim of tumor angiogenesis approximately15.09%to20.06%. The results ofLaser confocal microscopy demonstrated that EPCs mainly integrated into the caecum orbranch of microvacular. EPCs interconnected with tumor microvascular in situ to formdisorderly vascular network by involving in form new blood vessels to the branch spot.ConclusionsExogenous EPCs mainly homing to the rim of glioma, involving in tumor angiogenesisby integrated into the caecum and branch spot of tumor microvascular. Quantitativeanalysis showed that120hours after transplantation of EPCs，the hemodynamic of tumoredge was changed about15.09%to20.06%. MRI and CT perfusion imaging could be powerful tools to track the migration and fuction of EPCs in host tissues. MRI and CTperfusion aslo could be a powerful method for in vivo monitoring of clinical stem cellstherapy.更多还原