【作者】 李红；

【导师】 王雨生；

【作者基本信息】 第四军医大学 ， 眼科学， 2011， 博士

【摘要】 研究背景年龄相关性黄斑变性（age-related macular degeneration，AMD）在老年人群中，是最常见的、导致不可逆视力损害（包括法定盲）的主要原因之一。脉络膜新生血管（choroidal neovascularization，CNV）是AMD最严重的形式。目前，AMD的治疗方法包括激光治疗，光动力疗法（photodynamic therapy，PDT），玻璃体内注射皮类固醇类药物和抗血管生成因子的治疗。但这些方法都不能“治愈”AMD或者逆转它的病程，只是试图避免进一步视力损害。选择靶向骨髓来源细胞（bonemarrow–derived cells，BMCs）治疗AMD是一个有前景的治疗途径。通过体外培养和局部给药的方式，BMCs能有利于修复和再生视网膜和脉络膜组织的损伤或变性。然而，尽管靶向BMCs治疗AMD是一个令人振奋的途径，但BMCs在外周循环的数量有限，使这一治疗转入临床应用还面临很大障碍。目前的技术还达不到使体外扩增的BMCs能被常规用于细胞治疗。骨髓来源的内皮祖细胞（endothelial progenitor cells，EPCs)可能代表一个重要的内源性修复途径。体内动员和扩增骨髓来源的EPCs能对组织修复提供充足的自体细胞，因而可能解决目前细胞治疗的瓶颈问题。基质细胞衍生因子（stromal cell-derivedfactor1α，SDF-1α）及其受体CXCR4的相互作用是调控EPCs动员的关键。最近的发现提示肽酶CD26（dipeptidyl peptidase IV，DPP IV）能降解和失活骨髓内SDF-1α，最终导致阻断SDF-1/CXCR4axis，是EPCs动员一个重要的调控机制。而且，血管紧张素转换酶（angiotensin-converting enzyme，ACE）抑制剂能调控CD26/DPP IV从骨髓动员EPCs。尽管骨髓来源的EPCs参与了CNV的形成，但是否诱导体内EPCs动员对CNV是有益的仍然未被充分确定。EPCs能否在CNV损伤区域发挥保护作用还需要进一步被揭示。目的和内容本研究的目的是探讨调控CD26/DPP IV系统（CD26/DPP IVsystem）增加体内EPCs动员在CNV形成中的作用。确定是否在激光诱导的CNV，EPCs内源性的损伤修复机制和星形胶质细胞相关。本研究使用激光诱导的小鼠CNV模型1）观察ACE抑制剂（ACE inhibitor，ACEI），咪达普利（imidapril）在体内调控CD26/DPP IV system的作用，探讨在激光诱导的损伤条件下，ACEI对骨髓和外周血中肽酶活动的影响；2）观察ACEI调控CD26/DPP IV system降解SDF-1α，阻断SDF-1/CXCR4axis，动员体内EPCs参与激光诱导的CNV形成的作用；确定在损伤条件下，ACEI能否通过调控CD26肽酶活动诱发EPCs动员；3）观察动员的EPCs靶向视网膜神经纤维层（nerve filber layer，NFL）的星形胶质细胞（astrocytes）抑制CNV形成的作用。探讨EPCs在激光诱导的CNV可能通过保护星形胶质细胞，发挥内源性的损伤修复作用。方法532nm二极管激光器诱发C57BL/6J小鼠Bruch’膜的破裂建立CNV模型。小鼠被随机分为5组，在激光诱导CNV前5天，小鼠被分别预处理给予胃饲磷酸盐缓冲液（phosphate-buffered saline，PBS），胃饲咪达普利和/或皮下注射Diprotin-A（DPP IV抑制剂）治疗持续直到激光诱导后的14天。正常对照为无激光和无干预组。（1）荧光激活细胞分选（fluorescence-activated cell sorting，FACS）检测：在激光诱导CNV后12天，获得所有动物的外周血和骨髓样本。心脏穿刺获得外周血，同时股骨的骨髓细胞被提取。外周血细胞和骨髓细胞经鉴定使用单个核细胞部分。检测结合异硫氰酸荧光素(fluorescein isocyanat，FITC)-CD26单克隆抗体的CD26+细胞在外周血和骨髓中的数量。单个核细胞双标FITC-CD34单克隆抗体和藻红蛋白（phycoerythrin, PE）-Flk-1单克隆抗体被鉴定为EPCs。流式细胞仪计数循环EPCs（CD34+/VEGFR2+cells）的数量。（2）淋巴细胞丝状肌动蛋白（filamentous actin，F-actin）聚合作用检测。小鼠的全血样本收集来自激光诱导CNV后12天。细胞刺激或未刺激30nM的重组小鼠SDF-1蛋白和FITC-鬼笔环肽（FITC-phalloidin）染色。流式细胞仪检测细胞内平均荧光强度（mean fluorescence intensity，MFI）。（3）分别在不同干预的第1、5天和激光诱导CNV后3、7、14天尾静脉取血，外周血白细胞（white blood cells，WBCs）计数。（4）全自动酶标仪检测CD26肽酶活动，血浆和骨髓上清收集在激光诱导CNV后12天。肽酶活动通过检测生成对硝基苯胺（p-Nitroaniline，pNA）的水平，以酶的活性浓度单位（U/L）来表示。酶标仪读数405波长的吸光值。（5）酶联免疫吸附测定（enzyme linked immunosorbent assay，ELISA）SDF-1水平：激光诱导CNV后3、7、14天，全血离心收集血浆，同时来自股骨的骨髓上清被提取。ELISA检测血浆和骨髓上清的SDF-1α浓度。（6）荧光血管造影(fluorescein angiography，FA）评价激光诱导CNV后13天的荧光渗漏情况。FA图像被采集在注射造影剂后4-6分钟。按评分标准比较CNV的渗漏情况。（7）组织病理学观察CNV厚度、直径和面积。在激光诱导CNV后14天，苏木精和伊红（Hematoxylin-Eosin，HE）染色观察CNV的厚度和直径，FITC-植物凝集素（fluorescein isocyanate-Griffonia simplicifolia isolectin-B4，FITC-Isolectin B4）染色检测CNV表面积。（8）免疫荧光标记神经胶质酸性蛋白（glial fibrillary acidic protein，GFAP），激光诱导CNV后14天观察视网膜NFL星形胶质细胞的表达。（9）蛋白印记法（western blotting）检测激光诱导CNV后14天视网膜GFAP蛋白的表达。结果(1）ACEI能够调控CD26/DPP IV system打破骨髓和外周血之间的肽酶活动的平衡。激光诱导CNV后12天，咪达普利不能改变骨髓CD26+细胞数量，但增加了CD26/DPP IV肽酶活动。在外周血，咪达普利由于抗炎作用下降了WBCs数量，导致CD26+细胞数量明显减少，最终下降外周血总的CD26/DPP IV肽酶活动，而且Diprotin-A能完全阻断咪达普利调控CD26/DPP IV system的作用。（2）ACEI能够通过调控CD26/DPP IV system阻断了SDF-1/CXCR4axis，动员EPCs到外周循环。在激光诱导CNV后12天，咪达普利明显下降了骨髓SDF-1的水平，释放大量失活的SDF-1到外周循环，导致外周循环血的SDF-1水平增高和外周血中SDF-1诱导的F-actin聚合作用下降。咪达普利组的这个现象与骨髓内肽酶活动增高和外周血中肽酶活动的降低有关。最终，逆转的骨髓和外周血SDF-1浓度梯度导致显著增加了EPCs从骨髓动员到外周循环。而且，在激光诱导的CNV，咪达普利对EPCs的动员作用完全被Diprotin A阻断了。（3）咪达普利能够抑制CNV的形成与动员的EPCs对视网膜NFL星形胶质细胞有保护作用密切相关。在激光诱导CNV后13天行眼底FA检查，咪达普利比其它各组明显减少了CNV的渗漏。HE和FITC-Isolectin B4染色均显示咪达普利抑制了CNV，比其它各组明显减少了的病变的直径、厚度和面积。GFAP上调是星形胶质细胞活化的标志。GFAP免疫荧光染色显示咪达普利组视网膜NFL上GFAP免疫反应的细胞较其它各组明显增高。Western blot检测各组视网膜内GFAP蛋白表达比较，在激光诱导CNV后14天检测。咪达普利组比溶剂组明显增加了GFAP蛋白的表达。结论本研究证实（1）在激光诱导CNV损伤时，ACEI显著增加骨髓微环境内的CD26/DPP IV的肽酶活动，降低了外周循环的肽酶活动，破坏了骨髓和外周循环蛋白水解活动的平衡，有效的活化了CD26/DPPIV system。（2）CD26/DPP IV system通过增加骨髓内蛋白水解SDF-1的活动，能负向调节SDF-1/CXCR4axis，引起循环血中SDF-1水平增加，逆转骨髓和外周血的SDF-1浓度梯度，引起EPCs从骨髓动员到外周循环。（3）动员的骨髓来源的EPCs靶向NFL的星形胶质细胞，可能通过内源性修复途径抑制了激光诱导的CNV。动员骨髓来源的EPCs能抑制激光诱导的CNV。本研究首次明确了EPCs参与CNV的形成是有积极的作用。ACEI调控CD26/DPP IV system动员EPCs可能代表一个重要的内源性修复途径。这一研究结果不但为体内扩增骨髓来源EPCs治疗眼新生血管提供了新奇的靶向，而且为BMCs细胞治疗眼微血管疾病转入临床应用提供新的思路。目前和未来的治疗应靶向干预CNV发展的病程。相对与抗血管生成的治疗，EPCs可能通过促进血管修复和再生，探索一个新奇的治疗途径，这可能被认为是理想的治疗。更多还原

【Abstract】 Background Age-related macular degeneration (AMD) is one of the mostcommon irreversible causes of severe loss of vision, including legal blindness, in theelderly population. Choroidal neovascularization (CNV) is the most severe form of AMD.There are no current treatments that can “cure” AMD or reverse its course, including laserphotocoagulation, verteporfin photodynamic therapy (PDT) and intravitreal injections ofcorticosteroids and anti-angiogenic agents. Generally, these therapies seek to avoid furthervision loss rather than to improve existing vision. Selective targeting of bonemarrow–derived cells (BMCs) has been heralded as a promising avenue for age-relatedmacular degeneration (AMD) therapeutics. Cell therapy using BMCs by ex vivo isolation and local delivery could provide beneficial effects for repair and regeneration of injuredand degenerated retina and choroid tissues. Although BMC therapy with may be the mostexciting avenue for AMD therapeutics, a major barrier to transferring the use ofautologous BMCs into clinical practice is the limited quantity of BMCs in the peripheralcirculation. Technology has not yet reached a stage where ex vivo–expanded BMCs can beroutinely used for cell therapy.Bone marrow–derived endothelial progenitor cells (EPCs) may represent animportant endogenous repair mechanism. In vivo mobilization and expansion of EPCscould supply sufficient autologous cells for tissue repair, thereby circumventing theexisting issues of cells therapy. The interaction of stromal cell-derived factor1α (SDF-1α)and CXCR4has been identified as a principal axis in the regulation of EPC mobilization.A recent finding has shown that SDF-1α degradation and inactivation within the bonemarrow by the peptidase CD26(dipeptidyl peptidase IV, DPP IV) may ultimately result inthe abrogation of the SDF-1/CXCR4axis, which is an important regulatory mechanism formobilization of EPCs. Moreover, CD26/dipeptidylpeptidase IV (DPP IV) modification byangiotensin-converting enzyme (ACE) inhibitor plays a critical role in mobilizing EPCsfrom bone marrow. In spite of bone marrow-derived EPCs have been shown to contributeto CNV, whether the beneficial effect of induced EPCs mobilization has yet to be fullydetermined. In addition, whether homing of EPCs to the site requiring repair may exertspositive effectsf for laser-induced CNV remains to be answered.Objectives The purpose of the present study was to investigate the role of theincreased EPC mobilization by modulation CD26/DPP IV system in the development ofCNV. To determine whether the endogenous repair mechanism of EPCs may beassociated with reactive astrocytes in the laser-induced CNV.This study, using a murine model of laser-induced CNV,(1) observe that imidapril, anACE inhibitor (ACEI), regulate the activity of CD26/DPP IV system in vivo andinvestigate the effects of ACEI on proteolytic activation between bone marrow andperipheral circulation under laser-induced injure conditions;(2) observe that ACEImodulate the function of SDF-1and influence the level of EPC mobilization throughmanipulation of the CD26system in the development of CNV and confirm the underlyingmechanism under injury stress conditions;(3) observe mobilization of EPCs target astrocytes at the retina nerve filber layer (NFL) to intervene development of CNV. Weinvestigate that EPCs target activated astrocytes by manipulation of endogenous repairmechanisms in the laser-induced CNV.Methods CNV in C57BL/6J mice was generated by focal rupture of Bruch’smembrane with a532-nm diode laser. Animals were randomized to5treatment groups.Animals were pretreated intragastrically with phosphate-buffered saline (PBS),intragastrically with imidapril (ACEI) and/or subcutaneously with diprotin-A (a DPP IVantagonist) for days5before photocoagulation and the treatments were continued dailyuntil days14after laser induction. Normal control group is nontreated and nonlasered.(1)Fluorescence-activated cell sorting (FACS) Analysis: Peripheral blood and bone marrowsamples were obtained from all groups after laser-induced CNV day12. Blood was wasobtained by cardiac puncture, while bone marrow cells were extracted from femurscirculating cells and bone marrow cells were identified using a nucleated cell fraction. Thequantity of CD26+cells in the bone marrow and peripheral blood was estimated using afluorescein isocyanate (FITC)-conjugated anti-mouse CD26antibody. The nucleated cellswere double labelled with FITC-conjugated anti-CD34monoclonal antibody andphycoerythrin (PE)-conjugated anti-Flk-1antibody. Circulating EPCs were quantified byenumerating CD34+and VEGFR2+cells. The cells were examined by flow cytometry.(2)SDF-1filamentous actin (F-actin) polymerization of lymphocytes by is quantified bymeans of flow cytometry. Whole blood samples collected from the animals afterlaser-induced CNV day12. The cells were stimulated with or without30nM of SDF-1and stained with FITC phalloidin. The intracellular fluorescence was determined by FACSanalysis. The lymphocyte population was gated, and median fluorescence was measured.(3) Blood samples were obtained by via a tail vein on pretreated with different drug dailyon days1,5and after laser-induced CNV day3, day7, day14. The total number of whiteblood cells (WBCs) was enumerated with a Neubauer hematocytometer.(4) CD26proteolytic activity was examined by a microplate reader. plasma and bone marrowextracellular fluids were obtained after laser-induced CNV day12. Proteolytic activity wasdetermined by measuring the amount of p-Nitroaniline (pNA) formed and the DPP IVactivity in units/liter (U/L) calculated in the supernatant at405nm.(5) Enzyme linkedimmunosorbent assay (ELISA) measurement of cytokines: peripheral blood murine samples and bone marrow were obtained from all groups after laser-induced CNV day3,7,14. Blood was centrifuged to collect plasma, while bone marrow extracellular fluids wereextracted from femurs as previously described. Plasma and bone marrow SDF-1αconcentrations were measured with a mouse SDF-1α ELISA kit.(6) Fluoresceinangiography (FA) was performed after laser photocoagulation day13and fluoresceinleakage was evaluated. Late-stage FA images were taken at4-6minutes post injection. Thegrading protocol used to compare leakage in experimentally induced CNV.(7)Histopathology study evaluated CNV Lesion size by hematoxylin-eosin (HE) staining andfluorescein isocyanate-Griffonia simplicifolia isolectin-B4(FITC-isolectin B4) Stainingafter laser-induced CNV day14.(8) Glial fibrillary acidic protein (GFAP) immunoreactivecells were identified as astrocytes cells by immunofluorescence labelling within the retinaNFL after laser-induced CNV day14.(9) The protein expression of GFAP was measuredin the retinal by western blotting after laser-induced CNV day14.Results (1) ACEI disrupted the balance of the proteolytic activity of bone marrowand peripheral blood by manipulation of CD26/DPP IV system. In the bone marrow,imidapril was primarily through the upregulation of CD26/DPP IV activity on bonemarrow cell rather than through altering the number of CD26+cells after laser-inducedCNV day12. In the blood, imidapril significantly decreased CD26+cell numbers, leadingto a decrease in total CD26/DPP IV activity, because decrease the number of WBCsthrough an anti-inflammatory effect. Furthermore, Diprotin A can completely blockedCD26/DPP IV activity caused by imidapril intervention.(2) ACEI has the ability tonegatively regulate the SDF-1/CXCR4axis by manipulation of CD26/DPP IV system.Imidapril-treated animals after laser-induced CNV demonstrated significant increases inplasma–SDF-1compared with other groups after laser-induced CNV day12. Meanwhile,SDF-1concentrations in the bone marrow were significantly lower in the imidapril groupcompared to the other groups. Imidapril also caused a significant decrease SDF-1inducedactin polymerization in whole blood. These phenomena were associated with a lowerCD26activity in bone marrow but higher in the blood in the imidapril group, compared tothe other groups. The inversing SDF-1concentration gradient between the bone marrowand the peripheral blood significantly mobilized EPCs from the bone marrow into thecirculation. The effect of imidapril on EPC mobilization in laser-inducd CNV was significantly blocked by Diprotin A.(3) The mobilization of EPC significantly inhibitedthe laser-induced CNV and reactive astrocytes. FA was conducted after laserphotocoagulation day13. Treatment with imidapril significantly decreased CNV leakagecompared to the other groups after laser-induced CNV. HE and FITC-Isolectin B4-stainedalso showed mice treated with imidapril suppressed CNV volume versus other groups.GFAP up-regulation is a hallmark of reactive astrocytes. Immunofluorescence taining ofGFAP showed mice treated with imidapril GFAP immunoreactive cell were significantlyincreased in the retina NFL versus the other groups. Imidapril group was significantlyincreased the protein expression of GFAP compare with other groups after thelaser-induced CNV day14.Conclusions (1) ACE inhibitor effectively regulated the activity of CD26/DPPIV system in laser-induced CNV. In the bone marrow, imidapril did not alter CD26+cellnumbers; however, it did amplify CD26/DPP IV activity. In the blood, through theanti-inflammatory effect, imidapril significantly decreased CD26+cell numbers, leading toa decrease in total CD26/DPP IV activity.(2) CD26/DPP IV has the ability to negativelyregulate the SDF-1/CXCR4axis by proteolytic degradation of SDF-1α in bone marrow,which significantly reduced the concentration of SDF-1α and increases in plasma–SDF-1α.The inversing SDF-1α concentration gradient between the bone marrow and the peripheralblood significantly mobilized EPCs from the bone marrow into the circulation.(3) Themobilization of bone marrow-derived EPCs target reactive astrocytes at the retina NFLandinhibits the laser-induced CNV in term of manipulation of endogenous repair avenue.Bone marrow-derived EPCs mobilization can inhibit the laser-induced CNV. Thebeneficial effect of induced EPCs mobilization in the development of CNV wasdetermined firstly. CD26/DPP IV modification by ACEI activate EPCs mobilization mayrepresent an important endogenous repair avenue. The therapeutic strategy may providethe novel target of EPC mobilization to treat CNV and overcome the existing problems ofBMC-based therapy in clinical application. Present and future therapies will be aimed atmodifying the course of CNV development. EPC mobilization may provide a novelavenue of therapeutic exploitation to promote repair and re-generation，then it may betimely to consider therapies other than antiangiogenesis.更多还原