摘要
以糖尿病性视网膜病变为代表的,包括早产儿视网膜病变、视网膜中央静脉阻塞等视网膜缺血性疾病,以新生血管形成为主要病理标志。视网膜循环障碍使血管内皮细胞与周细胞受损,从而导致毛细血管失去正常的屏障功能,渗透性增加,造成周围组织水肿、渗出,继而引起视网膜组织缺血缺氧,并代偿性生成组织结构不完整的新生血管,导致出血、增殖,甚至视网膜脱离,严重威胁患者的视力。目前临床上尚无有效的阻止新生血管形成的治疗方法,视网膜激光光凝、血管内皮生长因子抑制剂、光动力疗法、类固醇激素以及手术治疗能在一定程度上减少新生血管的形成,但不能从根本上消除新生血管形成因素,并且伴随着一系列的副损伤及复发的可能性。大量研究表明,血管内皮结构破坏和功能障碍在视网膜缺血性疾病和病理性新生血管形成和发展过程中发挥重要的作用,因此,控制视网膜缺血性疾病新生血管形成的关键在于修复受损伤的血管内皮,改善视网膜的缺血缺氧状态。内皮祖细胞(EPCs)是一类能增殖并分化为血管内皮细胞的前体细胞,具有修复血管内皮损伤和参与新生血管形成的功能。移植EPCs能够改善心、脑和肢体等局部缺血的损伤,增加缺血部位的血流量和毛细血管密度,提高对缺血性疾病的疗效。如果能将EPCs有效的移植到缺血缺氧的视网膜局部,就有可能修复受损的血管内皮,改善视网膜的血供,避免病理性新生血管的生成,这项研究不仅需要合适的细胞移植手段,还需要一种稳定、高效的示踪方法为实验提供客观的观察依据。目的
通过羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)标记EPCs、Dil标记的乙酰化低密度脂蛋白(DiI-AcLDL)标记EPCs以及慢病毒介导绿色荧光蛋白(GFP)转导EPCs,比较三种标记物在体外和体内对EPCs的示踪情况,并观察移植EPCs对视网膜血管损伤的修复,为今后细胞移植选择合适的标记物,更好的跟踪EPCs移植的疗效提供实验基础。
方法
(1)人脐带血EPCs的培养及鉴定：通过羟乙基淀粉沉降法和Percoll密度梯度离心法分离人脐带血中单个核细胞,在体外诱导分化成为EPCs,并通过观察细胞形态学、流式细胞术分析细胞表面标志、免疫荧光染色以及电镜等方法进行鉴定。(2)三种方法体外标记EPCs:分别用CFSE、DiI-AcLDL以及慢病毒介导GFP基因转导标记EPCs,通过倒置相差显微镜观察标记前后细胞形态的改变,台盼蓝拒染法和贴壁细胞计数法测定标记后EPCs的生存能力和粘附能力的变化,荧光显微镜下观察荧光强度以及标记荧光随培养时间的变化情况,流式细胞术测定标记阳性率,并综合比较三种方法体外标记细胞的优缺点。(3)标记的EPCs眼内移植示踪：利用多波长氪激光选择性损伤视网膜,建立C57BL/6N小鼠视网膜血管损伤模型。分别收集DiI-AcLDL、CFSE、慢病毒介导GFP基因转导三种方法标记的EPCs,手术显微镜下采用微量注射器移植入玻璃体腔内。分时段观察眼底照相、视网膜石蜡切片、冰冻切片和视网膜铺片,观察标记细胞在视网膜纵向切面和横向平面的分布、荧光强度和持续时间等情况,并比较EPCs移植前后视网膜血管损伤修复情况。
结果
(1)从人脐带血分离原代培养EPCs,在培养过程中表现为典型EPCs的形态变化特点,不同程度的表达CD34、CD133和’VEGFR-2等细胞表面标志,可吞噬DiI-AcLDL并同时结合FITC-UEA-I,电镜检查可见内皮细胞特有的W-P小体,证明所培养的细胞群体中大部分是正在分化中的EPCs。(2)CFSE标记后EPCs呈现绿色荧光,标记阳性率可达95%以上；DiI-AcLDL标记EPCs呈红色荧光,标记阳性率可达80%；CFSE和DiI-AcLDL标记荧光可持续4周,荧光强度随培养时间的延长逐渐下降。慢病毒介导GFP基因转导EPCs后4天,激光共聚焦显微镜下可观察到细胞发出绿色荧光,随后绿色荧光阳性细胞逐渐增多,荧光强度逐渐增强,转导后4周转染效率超过30%。三种标记方法细胞形态无明显改变,在标记2天和7天后,检测生存能力及粘附能力较未标记的细胞无明显变化。(3)体内试验中,通过视网膜激光光凝成功建立小鼠视网膜血管损伤模型。将标记的EPCs进行玻璃体腔注射,眼内移植4周后,眼底照相可见激光斑色素沉着及瘢痕形成较未移植者减轻。移植EPCs后4周眼球石蜡切片HE染色,可见神经纤维层血管周围有细胞聚集,视网膜各层结构比较规整,形成瘢痕较小。移植后视网膜冰冻切片显示,DiI-AcLDL和CFSE标记EPCs移植后2天于视网膜表面可见荧光细胞,1周时可见荧光细胞聚集于损伤部位,4周可见荧光细胞分布于视网膜各层,以视网膜血管富集的神经纤维层和内核层为主。慢病毒介导GFP基因转导EPCs移植后各时间点视网膜冰冻切片未见荧光细胞。伊文思蓝灌注血管造影可清晰显示视网膜毛细血管网的结构,视网膜血管损伤模型激光斑处可见荧光渗漏。移植CFSE标记的EPCs后2天行视网膜铺片,可见绿色荧光标记细胞群聚分布于视网膜上；移植后1周,绿色荧光标记细胞在激光损伤周围聚集；移植后4周,绿色荧光标记细胞形成类似管状结构,证实EPCs参与视网膜血管修复。
结论
(1)CFSE和Dil-AcLDL适合短期示踪EPCs。CFSE标记EPCs效率最高,起始荧光最强,费用最低,操作最简便,短期示踪更有优势。CFSE标记EPCs联合视网膜冰冻切片与伊文思蓝灌注视网膜铺片为EPCs眼内移植的示踪建立了多角度的观察方法。慢病毒介导GFP基因转导EPCs在长期示踪方面更有潜力。(2)利用视网膜激光光凝建立了小鼠视网膜血管损伤模型,并通过玻璃体腔注射进行EPCs眼内移植。通过体内示踪,证实ECPs具有向损伤视网膜定向归巢的能力,并能够参与视网膜血管损伤的修复。
The majority of eye diseases that lead to vision loss result from retinal neovascularization, often in response to retinal ischemia, such as diabetic retinopathy, retinopathy of prematurity and central retinal vein occlusion. When ischemia takes place in organs such as the heart and brain, growth of collateral vessels may be beneficial. However, neovascularization secondary to ischemia in the eye is always harmful and causes bleeding, retinal edema, fibrovascular proliferation, and even retinal detachment. Currently, there is no satisfactory treatment to block the retinal neovascularization and vascular leakage. Laser photocoagulation, vascular endothelial growth factor (VEGF) inhibitors, photodynamic therapy, angiostatic steroids and vitrectomy have been reported to regress neovascularization to different degrees, but often fail to completely inhibit it, associating with recidivation or normal retinal injury, or other complications, and may ultimately aggravate the ischemia and hypoxia. Studies showed that the function of retinal vascular endothelium decreased in patients with retinal ischemic diseases. Therefore, the key to control the retinal neovascularization is to repair the damaged vascular endothelium and alleviate ischemia and hypoxia in the retina. Endothelial progenitor cells (EPCs) have the capacity to differentiate into mature endothelial cells, facilitate endothelial repair and angiogenesis in vivo. If EPCs can effectively migrate to the injured retina and maintain normal function, it is possible to repair the damaged vascular endothelium, give rise to the formation of functional retinal vessels, and ultimately alleviate ischemia and hypoxia. However, there is still no stable and efficient method for tracking transplanted EPCs in retina.
Objective:
The current study sought to establish a simple, reliable, fluorescent labeling method for tracking EPCs with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), 1,1'-dilinoleyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL) and green fluorescent protein (GFP)in laser-injured mouse retina, and observed the role of EPCs in retinal vascular repair.
Methods:
(1) EPCs were isolated by hydroxyethyl starch sedimentation and density gradient centrifugation from human umbilical cord blood mononuclear cells, and cultivated. Then EPCs were identified by cytomorphology, specific markers detected by flow cytometry, immunofluorescence observed by confocal microscope and electron microscope. (2) EPCs were labeled with CFSE, Dil-AcLDL and GFP. The morphology of labeled EPCs were observed by inverted optical microscope. The survival capability and the adhesion ability of labeled EPCs were measured by Trypan blue staining and adherent cells count respectively. Fluorescence microscopy was used to observe the label stability and the fluorescence intensity during the extended culture period. The proportion of labeled cells was detected by flow cytometry. (3) The mouse retinal vascular injured model was estabalished by injured the retinal veins and capillarity network around the optic disc with retinal laser photocoagulation. Labeled EPCs were transplanted into the vitreous cavity of pigmented mouse model. Fundus photography, paraffin section, retinal frozen section, angiography and flat mounted were used to obeserved the distribution, fluorescence duration and intensity change of labeled cells, and the repair of retinal vascular injury after the transplantation of EPCs.
Results:
(1) EPCs were isolated from human umbilical cord blood and the cells presented numberous cell clusters and cobblestone morphology. Most adherent cells were double stained by Dil-AcLDL and FITC-UEA-I, and the flow cytometry showed that the cells were positive for CD34, CD 133 and VEGFR-2. W-P body was observed by electron microscope. (2) Our data indicated that EPCs labeled with CFSE presented intense green fluorescence and the positive rate was above 95%; while EPCs labeled with Dil-AcLDL presented red fluorescence and the positive rate was about 80%. The fluorescence intensity gradually decreased in the cells at the end of 4 weeks. GFP transduced EPCs showed efficiency more than 30% at the end of 4 weeks, and fluorescence intensity gradually strengthened. EPCs labeled with the three methods maintained normal morphology. The survival capability and the adhesion ability were not statistically different between labeled and unlabeled EPCs. (3) The mouse retinal vascular injured model was estabalished by retinal laser photocoagulation. The paraffin section and HE staining of retinal displayed the injury of laser photocoagulation and the repair by EPCs transplantation for 4 weeks. The retinal frozen section displayed distribution of labeled cells in the retinal layers in 4 weeks. Evans blue angiography of the retina displayed the retinal capillarity network clearly and fluorescence leakage was observed around photocoagulated spots in the laser-injured mouse model. One week after transplantation of CFSE labeled EPCs, the fluorescent cells were identified around the photocoagulated lesions. Four weeks after transplantation, fluorescent tube-like structures were observed in the retinal vascular networks. The results indicated that EPCs could participate to the repair of injured retinal vessels.
Conclusions:
(1) EPCs could be labeled by CFSE and Dil-AcLDL, be monitored in vivo for at least 4 weeks. CFSE has more advantages for short time tracing. Combining CFSE with retinal frozen section and Evans blue angiography contributite a multi-angle observation to tracking EPCs. GFP could transduce EPCs by lentivirus and has the potential for long time tracing. (2) The mouse retinal vascular injured model could be estabalished by retinal laser photocoagulation. EPCs could be transplanted into eyes by intravitreal injection. EPCs could localize directly to retinal injured sites and participate to the repair of injured retinal vessels.
引文
[1]MECHOULAM H, PIERCE E A. Retinopathy of prematurity:molecular pathology and therapeutic strategies [J]. Am J Pharmacogenomics,2003,3(4):261-77.
[2]MADHUSUDHANA K C, NEWSOM R S. Central retinal vein occlusion:the therapeutic options [J]. Can J Ophthalmol,2007,42(2):193-5.
[3]AICHER A, HEESCHEN C, MILDNER-RIHM C, et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells [J]. Nat Med,2003,9(11): 1370-6.
[4]KIM B O, TIAN H, PRASONGSUKARN K, et al. Cell transplantation improves ventricular function after a myocardial infarction:a preclinical study of human unrestricted somatic stem cells in a porcine model [J]. Circulation,2005,112(9 Suppl):196-104.
[5]SHIBATA T, NARUSE K, KAMIYA H, et al. Transplantation of bone marrow-derived mesenchymal stem cells improves diabetic polyneuropathy in rats [J]. Diabetes,2008,57(11): 3099-107.
[6]DAS A, MCGUIRE P G. Retinal and choroidal angiogenesis:pathophysiology and strategies for inhibition [J]. Prog Retin Eye Res,2003,22(6):721-48.
[7]ADAMIS A P, ALTAWEEL M, BRESSLER N M, et al. Changes in retinal neovascularization after pegaptanib (Macugen) therapy in diabetic individuals [J]. Ophthalmology,2006,113(1):23-8.
[8]JONAS J B, KREISSIG I, DEGENRING R. Intravitreal triamcinolone acetonide for treatment of intraocular proliferative, exudative, and neovascular diseases [J]. Prog Retin Eye Res,2005,24(5):587-611.
[9]SCHMIDT-ERFURTH U, NIEMEYER M, GEITZENAUER W, et al. Time course and morphology of vascular effects associated with photodynamic therapy [J]. Ophthalmology, 2005,112(12):2061-9.
[10]MASON J O,3RD, COLAGROSS C T, HALEMAN T, et al. Visual outcome and risk factors for light perception and no light perception vision after vitrectomy for diabetic retinopathy [J]. Am J Ophthalmol,2005,140(2):231-5.
[11]LA FONTAINE J, HARKLESS L B, DAVIS C E, et al. Current concepts in diabetic microvascular dysfunction [J]. J Am Podiatr Med Assoc,2006,96(3):245-52.
[12]AGUIAR L G, VILLELA N R, BOUSKELA E. Microcirculation in diabetes: implications for chronic complications and treatment of the disease [J]. Arq Bras Endocrinol Metabol,2007,51(2):204-11.
[13]彭晓燕.眼底病诊断思辨[M].北京：人民卫生出版社,2009.
[14]BROWN G C, MAGARGAL L E, FEDERMAN J L. Ischaemia and neovascularization [J]. Trans Ophthalmol Soc U K,1980,100(3):377-80.
[15]TSUI I, KAINES A, HAVUNJIAN M A, et al. Ischemic index and neovascularization in central retinal vein occlusion [J]. Retina,2011,31(1):105-10.
[16]VESTEINSDOTTIR E, BJORNSDOTTIR S, HREIDARSSON A B, et al. Risk of retinal neovascularization in the second eye in patients with proliferative diabetic retinopathy [J]. Acta Ophthalmol,2010,88(4):449-52.
[17]JACOBS N A, TREW D R. Occlusion of the central retinal artery and ocular neovascularisation:an indirect association? [J]. Eye (Lond),1992,6 (Pt 6)(599-602.
[18]宋鄂.糖尿病视网膜病变中VEGF及bFGF作用的分子病理机制研究[D].长春：吉林大学,2000.
[19]陈有信.视网膜血管性疾病[M].北京：科学出版社,2011.
[20]FRANK R N, DUTTA S, MANCINI M A. Pericyte coverage is greater in the retinal than in the cerebral capillaries of the rat [J]. Invest Ophthalmol Vis Sci,1987,28(7):1086-91.
[21]CHAKRAVARTHY U, GARDINER T A. Endothelium-derived agents in pericyte function/dysfunction [J]. Prog Retin Eye Res,1999,18(4):511-27.
[22]HAMANAKA T, AKABANE N, YAJIMA T, et al. Retinal ischemia and angle neovascularization in proliferative diabetic retinopathy [J]. Am J Ophthalmol,2001,132(5): 648-58.
[23]BOERI D, MAIELLO M, LORENZI M. Increased prevalence of microthromboses in retinal capillaries of diabetic individuals [J]. Diabetes,2001,50(6):1432-9.
[24]REHAK J, REHAK M. Branch retinal vein occlusion:pathogenesis, visual prognosis, and treatment modalities [J]. Curr Eye Res,2008,33(2):111-31.
[25]FRAENKL S A, MOZAFFARIEH M, FLAMMER J. Retinal vein occlusions:The potential impact of a dysregulation of the retinal veins [J]. EPMA J,2010,1(2):253-61.
[26]CUNHA-VAZ J, LOBO C, SOUSA J C, et al. Progression of retinopathy and alteration of the blood-retinal barrier in patients with type 2 diabetes:a 7-year prospective follow-up study [J]. Graefes Arch Clin Exp Ophthalmol,1998,236(4):264-8.
[27]KAUR C, FOULDS W S, LING E A. Blood-retinal barrier in hypoxic ischaemic conditions:basic concepts, clinical features and management [J]. Prog Retin Eye Res,2008, 27(6):622-47.
[28]BUSIK J V, OLSON L K, GRANT M B, et al. Glucose-induced activation of glucose uptake in cells from the inner and outer blood-retinal barrier [J]. Invest Ophthalmol Vis Sci, 2002,43(7):2356-63.
[29]RIZZOLO L J. Development and role of tight junctions in the retinal pigment epithelium [J]. Int Rev Cytol,2007,258(195-234.
[30]崔浩.试论中药成份对眼部屏障的通透性[J].黑龙江医学,2005,29(1)：1-3.
[31]REICHENBACH A, WURM A, PANNICKE T, et al. Muller cells as players in retinal degeneration and edema [J]. Graefes Arch Clin Exp Ophthalmol,2007,245(5):627-36.
[32]张惠蓉.眼底病图谱[M].北京：人民卫生出版社,2007.
[33]ABBOTT N J. Astrocyte-endothelial interactions and blood-brain barrier permeability [J]. J Anat,2002,200(6):629-38.
[34]HOSOYA K, TACHIKAWA M. Inner blood-retinal barrier transporters:role of retinal drug delivery [J]. Pharm Res,2009,26(9):2055-65.
[35]DELAEY C, VAN DE VOORDE J. Regulatory mechanisms in the retinal and choroidal circulation [J]. Ophthalmic Res,2000,32(6):249-56.
[36]SCHMETTERER L, WOLZT M. Ocular blood flow and associated functional deviations in diabetic retinopathy [J]. Diabetologia,1999,42(4):387-405.
[37]GARHOFER G, ZAWINKA C, RESCH H, et al. Reduced response of retinal vessel diameters to flicker stimulation in patients with diabetes [J]. Br J Ophthalmol,2004,88(7): 887-91.
[38]HUDSON C, FLANAGAN J G, TURNER G S, et al. Exaggerated relative nasal-temporal asymmetry of macular capillary blood flow in patients with clinically significant diabetic macular oedema [J]. Br J Ophthalmol,2005,89(2):142-6.
[39]PORTA M, LA SELVA M, MOLINATTI P, et al. Endothelial cell function in diabetic microangiopathy [J]. Diabetologia,1987,30(8):601-9.
[40]HADI H A, SUWAIDI J A. Endothelial dysfunction in diabetes mellitus [J]. Vasc Health Risk Manag,2007,3(6):853-76.
[41]HAEFLIGER I O, FLAMMER J, LUSCHER T F. Nitric oxide and endothelin-1 are important regulators of human ophthalmic artery [J]. Invest Ophthalmol Vis Sci,1992,33(7): 2340-3.
[42]GOLDIN E, CASADEVALL M, MOURELLE M, et al. Role of prostaglandins and nitric oxide in gastrointestinal hyperemia of diabetic rats [J]. Am J Physiol,1996,270(4 Pt 1): G684-90.
[43]KIM I, MOON S O, KIM S H, et al. Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells [J]. J Biol Chem, 2001,276(10):7614-20.
[44]RABELINK T J, BAKRIS G L. The renin-angiotensin system in diabetic nephropathy: the endothelial connection [J]. Miner Electrolyte Metab,1998,24(6):381-8.
[45]COWAN D B, LANGILLE B L. Cellular and molecular biology of vascular remodeling [J]. Curr Opin Lipidol,1996,7(2):94-100.
[46]WILLIAMS B. A potential role for angiotensin Ⅱ-induced vascular endothelial growth factor expression in the pathogenesis of diabetic nephropathy? [J]. Miner Electrolyte Metab, 1998,24(6):400-5.
[47]BIEGELSEN E S, LOSCALZO J. Endothelial function and atherosclerosis [J]. Coron Artery Dis,1999,10(4):241-56.
[48]BUSSE R, FLEMING I, HECKER M. Signal transduction in endothelium-dependent vasodilatation [J]. Eur Heart J,1993,14 Suppl 1(2-9.
[49]MINSHALL R D, SESSA W C, STAN R V, et al. Caveolin regulation of endothelial function [J]. Am J Physiol Lung Cell Mol Physiol,2003,285(6):L1179-83.
[50]PORTA M, MOLINATTI P, LA SELVA M, et al. Endothelium and its morphofunctional changes in the pathogenesis of diabetic microangiopathy [J]. Minerva Med, 1988,79(11):915-29.
[51]DE VRIESE A S, VERBEUREN T J, VAN DE VOORDE J, et al. Endothelial dysfunction in diabetes [J]. Br J Pharmacol,2000,130(5):963-74.
[52]GIANNINI C, DYCK P J. Ultrastructural morphometric abnormalities of sural nerve endoneurial microvessels in diabetes mellitus [J]. Ann Neural,1994,36(3):408-15.
[53]LAMMIE G A. Hypertensive cerebral small vessel disease and stroke [J]. Brain Pathol, 2002,12(3):358-70.
[54]ALLT G, LAWRENSON J G. Pericytes:cell biology and pathology [J]. Cells Tissues Organs,2001,169(1):1-11.
[55]AUGUSTIN H G, BRAUN K, TELEMENAKIS I, et al. Ovarian angiogenesis. Phenotypic characterization of endothelial cells in a physiological model of blood vessel growth and regression [J]. Am J Pathol,1995,147(2):339-51.
[56]STONE J, ITIN A, ALON T, et al. Development of retinal vasculature is mediated by hypoxia-induced vascular endothelial growth factor (VEGF) expression by neuroglia [J]. J Neurosci,1995,15(7 Pt 1):4738-47.
[57]张惠蓉.眼微循环及其相关疾病[M].北京：北京医科大学中国协和医科大学联合出版社,1993.
[58]NISHIKAWA S I, FRASER S, FUJIMOTO T, et al. All B cells are progeny of endothelial cells:a new perspective [J]. Immunol Rev,2000,175(112-9.
[59]RISAU W, FLAMME I. Vasculogenesis [J]. Annu Rev Cell Dev Biol,1995,11(73-91.
[60]OTANI A, KINDER K, EWALT K, et al. Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogenesis [J]. Nat Med,2002,8(9): 1004-10.
[61]GRANT M B, MAY W S, CABALLERO S, et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization [J]. Nat Med,2002, 8(6):607-12.
[62]SUZUKI T, NISHIDA M, FUTAMI S, et al. Neoendothelialization after peripheral blood stem cell transplantation in humans:a case report of a Tokaimura nuclear accident victim [J]. Cardiovasc Res,2003,58(2):487-92.
[63]RISAU W. Mechanisms of angiogenesis [J]. Nature,1997,386(6626):671-4.
[64]SIMO R, CARRASCO E, GARCIA-RAMIREZ M, et al. Angiogenic and antiangiogenic factors in proliferative diabetic retinopathy [J]. Curr Diabetes Rev,2006,2(1): 71-98.
[65]AMES A,3RD, LI Y Y, HEHER E C, et al. Energy metabolism of rabbit retina as related to function:high cost of Na+ transport [J]. J Neurosci,1992,12(3):840-53.
[66]ROSEN D A, MARSHALL J, KOHNER E M, et al. Experimental retinal vein occlusion in the rhesus monkey. Radioactive microsphere and radioautographic studies [proceedings] [J]. Trans Ophthalmol Soc U K,1976,96(2):198.
[67]ROSEN D A, MARSHALL J, KOHNER E M, et al. Experimental retinal branch vein occlusion in rhesus monkeys. Ⅱ. Retinal blood flow studies [J]. Br J Ophthalmol,1979,63(6): 388-92.
[68]MONTEZUMA S R, VAVVAS D, MILLER J W. Review of the ocular angiogenesis animal models [J]. Semin Ophthalmol,2009,24(2):52-61.
[69]ARJAMAA O, NIKINMAA M. Oxygen-dependent diseases in the retina:role of hypoxia-inducible factors [J]. Exp Eye Res,2006,83(3):473-83.
[70]POURNARAS C J, RUNGGER-BRANDLE E, RIVA C E, et al. Regulation of retinal blood flow in health and disease [J]. Prog Retin Eye Res,2008,27(3):284-330.
[71]SHREENIWAS R, OGAWA S, COZZOLINO F, et al. Macrovascular and microvascular endothelium during long-term hypoxia:alterations in cell growth, monolayer permeability, and cell surface coagulant properties [J]. J Cell Physiol,1991,146(1):8-17.
[72]JOSKO J, GWOZDZ B. The influence of hypoxia on blood vessels endothelial cell function in brain [J]. Postepy Hig Med Dosw,2003,57(4):445-54.
[73]SCHALKWIJK C G, STEHOUWER C D. Vascular complications in diabetes mellitus: the role of endothelial dysfunction [J]. Clin Sci (Lond),2005,109(2):143-59.
[74]SCHEURER S B, RYBAK J N, ROSLI C, et al. Modulation of gene expression by hypoxia in human umbilical cord vein endothelial cells:A transcriptomic and proteomic study [J]. Proteomics,2004,4(6):1737-60.
[75]PUGH C W, RATCLIFFE P J. Regulation of angiogenesis by hypoxia:role of the HIF system [J]. Nat Med,2003,9(6):677-84.
[76]WISE G N. Retinal neovascularization [J]. Trans Am Ophthalmol Soc,1956, 54(729-826.
[77]CAMPOCHIARO P A. Molecular targets for retinal vascular diseases [J]. J Cell Physiol,2007,210(3):575-81.
[78]ITO Y, HASUDA H, TERAI H, et al. Culture of human umbilical vein endothelial cells on immobilized vascular endothelial growth factor [J]. J Biomed Mater Res A,2005, 74(4):659-65.
[79]BHISITKUL R B. Vascular endothelial growth factor biology:clinical implications for ocular treatments [J]. Br J Ophthalmol,2006,90(12):1542-7.
[80]SHAMS N, IANCHULEV T. Role of vascular endothelial growth factor in ocular angiogenesis [J]. Ophthalmol Clin North Am,2006,19(3):335-44.
[81]HELLSTROM A, PERRUZZI C, JU M, et al. Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells:direct correlation with clinical retinopathy of prematurity [J]. Proc Natl Acad Sci U S A,2001,98(10):5804-8.
[82]RUBERTE J, AYUSO E, NAVARRO M, et al. Increased ocular levels of IGF-1 in transgenic mice lead to diabetes-like eye disease [J]. J Clin Invest,2004,113(8):1149-57.
[83]POULAKI V, JOUSSEN A M, MITSIADES N, et al. Insulin-like growth factor-I plays a pathogenetic role in diabetic retinopathy [J]. Am J Pathol,2004,165(2):457-69.
[84]YAN Q, LI Y, HENDRICKSON A, et al. Regulation of retinal capillary cells by basic fibroblast growth factor, vascular endothelial growth factor, and hypoxia [J]. In Vitro Cell Dev Biol Anim,2001,37(1):45-9.
[85]BERANEK M, KOLAR P, TSCHOPLOVA S, et al. Genetic variation and plasma level of the basic fibroblast growth factor in proliferative diabetic retinopathy [J]. Diabetes Res Clin Pract,2008,79(2):362-7.
[86]CASTELLON R, HAMDI H K, SACERIO I, et al. Effects of angiogenic growth factor combinations on retinal endothelial cells [J]. Exp Eye Res,2002,74(4):523-35.
[87]HOLLBORN M, KRAUSSE C, IANDIEV I, et al. Glial cell expression of hepatocyte growth factor in vitreoretinal proliferative disease [J]. Lab Invest,2004,84(8):963-72.
[88]ANITUA E, ANDIA I, SANCHEZ M, et al. Autologous preparations rich in growth factors promote proliferation and induce VEGF and HGF production by human tendon cells in culture [J]. J Orthop Res,2005,23(2):281-6.
[89]RAHMAN S, PATEL Y, MURRAY J, et al. Novel hepatocyte growth factor (HGF) binding domains on fibronectin and vitronectin coordinate a distinct and amplified Met-integrin induced signalling pathway in endothelial cells [J]. BMC Cell Biol,2005,6(1):8.
[90]BAROUCH F C, MILLER J W. Potential future targets for treating ocular neovascularization [J]. Ophthalmol Clin North Am,2006,19(3):401-9.
[91]GRANT M B, AFZAL A, SPOERRI P, et al. The role of growth factors in the pathogenesis of diabetic retinopathy [J]. Expert Opin Investig Drugs,2004,13(10):1275-93.
[92]ZHAO S, OVERBEEK P A. Elevated TGFbeta signaling inhibits ocular vascular development [J]. Dev Biol,2001,237(1):45-53.
[93]LI X, PONTEN A, AASE K, et al. PDGF-C is a new protease-activated ligand for the PDGF alpha-receptor [J]. Nat Cell Biol,2000,2(5):302-9.
[94]WILKINSON-BERKA J L, BABIC S, DE GOOYER T, et al. Inhibition of platelet-derived growth factor promotes pericyte loss and angiogenesis in ischemic retinopathy [J]. Am J Pathol,2004,164(4):1263-73.
[95]CHEN J K, LAU Y T, CHU J. Coordinated regulation of vascular endothelial cell growth and prostacyclin production by epidermal growth factor:evidence of clonal variations among rat aortic endothelial cells [J]. In Vitro Cell Dev Biol,1992,28A(2):143-5.
[96]SARKIS S A, ABDULLAH B H, ABDUL MAJEED B A, et al. Immunohistochemical expression of epidermal growth factor receptor (EGFR) in oral squamous cell carcinoma in relation to proliferation, apoptosis, angiogenesis and lymphangiogenesis [J]. Head Neck Oncol, 2010,2(13.
[97]LUO T, XIA Z. A small dose of hydrogen peroxide enhances tumor necrosis factor-alpha toxicity in inducing human vascular endothelial cell apoptosis:reversal with propofol [J]. Anesth Analg,2006,103(1):110-6, table of contents.
[98]MAWARIBUCHI S, TAMURA K, OKANO S, et al. Tumor necrosis factor-alpha attenuates thyroid hormone-induced apoptosis in vascular endothelial cell line XLgoo established from Xenopus tadpole tails [J]. Endocrinology,2008,149(7):3379-89.
[99]NAMBU H, NAMBU R, OSHIMA Y, et al. Angiopoietin 1 inhibits ocular neovascularization and breakdown of the blood-retinal barrier [J]. Gene Ther,2004,11(10): 865-73.
[100]JOUSSEN A M, POULAKI V, TSUJIKAWA A, et al. Suppression of diabetic retinopathy with angiopoietin-1 [J]. Am J Pathol,2002,160(5):1683-93.
[101]HACKETT S F, WIEGAND S, YANCOPOULOS G, et al. Angiopoietin-2 plays an important role in retinal angiogenesis [J]. J Cell Physiol,2002,192(2):182-7.
[102]ASHLEY R A, DUBUQUE S H, DVORAK B, et al. Erythropoietin stimulates vasculogenesis in neonatal rat mesenteric micro vascular endothelial cells [J]. Pediatr Res,2002, 51(4):472-8.
[103]GRIMM C, WENZEL A, GROSZER M, et al. HIF-1-induced erythropoietin in the hypoxic retina protects against light-induced retinal degeneration [J]. Nat Med,2002,8(7): 718-24.
[104]WATANABE D, SUZUMA K, MATSUI S, et al. Erythropoietin as a retinal angiogenic factor in proliferative diabetic retinopathy [J]. N Engl J Med,2005,353(8):782-92.
[105]JIN M, KASHIWAGI K, IIZUKA Y, et al. Matrix metalloproteinases in human diabetic and nondiabetic vitreous [J]. Retina,2001,21(1):28-33.
[106]BARNETT J M, MCCOLLUM G W, FOWLER J A, et al. Pharmacologic and genetic manipulation of MMP-2 and-9 affects retinal neovascularization in rodent models of OIR [J]. Invest Ophthalmol Vis Sci,2007,48(2):907-15.
[107]MARCHESI S, PASQUALINI L, LOMBARDINI R, et al. Prostaglandin El improves endothelial function in critical limb ischemia [J]. J Cardiovasc Pharmacol,2003, 41(2):249-53.
[108]SCHROR K, HOHLFELD T. Mechanisms of anti-ischemic action of prostaglandin E1 in peripheral arterial occlusive disease [J]. Vasa,2004,33(3):119-24.
[109]WILKINSON-BERKA J L, JONES D, TAYLOR G, et al. SB-267268, a nonpeptidic antagonist of alpha(v)beta3 and alpha(v)beta5 integrins, reduces angiogenesis and VEGF expression in a mouse model of retinopathy of prematurity [J]. Invest Ophthalmol Vis Sci, 2006,47(4):1600-5.
[110]CASTELLON R, CABALLERO S, HAMDI H K, et al. Effects of tenascin-C on normal and diabetic retinal endothelial cells in culture [J]. Invest Ophthalmol Vis Sci,2002, 43(8):2758-66.
[111]OLSON J A, WHITELAW C M, MCHARDY K C, et al. Soluble leucocyte adhesion molecules in diabetic retinopathy stimulate retinal capillary endothelial cell migration [J]. Diabetologia,1997,40(10):1166-71.
[112]WALKER T, WENDEL H P, TETZLOFF L, et al. Suppression of ICAM-1 in human venous endothelial cells by small interfering RNAs [J]. Eur J Cardiothorac Surg,2005,28(6): 816-20.
[113]PARK H Y, KWON H M, LIM H J, et al. Potential role of leptin in angiogenesis: leptin induces endothelial cell proliferation and expression of matrix metalloproteinases in vivo and in vitro [J]. Exp Mol Med,2001,33(2):95-102.
[114]UCKAYA G, OZATA M, BAYRAKTAR Z, et al. Is leptin associated with diabetic retinopathy? [J]. Diabetes Care,2000,23(3):371-6.
[115]UCKAYA G, OZATA M, SONMEZ A, et al. Is leptin associated with hypertensive retinopathy? [J]. J Clin Endocrinol Metab,2000,85(2):683-7.
[116]GOETZE S, BUNGENSTOCK A, CZUPALLA C, et al. Leptin induces endothelial cell migration through Akt, which is inhibited by PPARgamma-ligands [J]. Hypertension,2002, 40(5):748-54.
[117]YUUKI T, KANDA T, KIMURA Y, et al. Inflammatory cytokines in vitreous fluid and serum of patients with diabetic vitreoretinopathy [J]. J Diabetes Complications,2001, 15(5):257-9.
[118]DOGANAY S, EVEREKLIOGLU C, ER H, et al. Comparison of serum NO, TNF-alpha, IL-lbeta, sIL-2R, IL-6 and IL-8 levels with grades of retinopathy in patients with diabetes mellitus [J]. Eye (Lond),2002,16(2):163-70.
[119]LI A, DUBEY S, VARNEY M L, et al. IL-8 directly enhanced endothelial cell survival, proliferation, and matrix metalloproteinases production and regulated angiogenesis [J]. J Immunol,2003,170(6):3369-76.
[120]APTE R S, BARREIRO R A, DUH E, et al. Stimulation of neovascularization by the anti-angiogenic factor PEDF [J]. Invest Ophthalmol Vis Sci,2004,45(12):4491-7.
[121]REN J G, JIE C, TALBOT C. How PEDF prevents angiogenesis:a hypothesized pathway [J]. Med Hypotheses,2005,64(1):74-8.
[122]APARICIO S, SAWANT S, LARA N, et al. Expression of angiogenesis factors in human umbilical vein endothelial cells and their regulation by PEDF [J]. Biochem Biophys Res Commun,2005,326(2):387-94.
[123]DIXELIUS J, LARSSON H, SASAKI T, et al. Endostatin-induced tyrosine kinase signaling through the Shb adaptor protein regulates endothelial cell apoptosis [J]. Blood,2000, 95(11):3403-11.
[124]HOHENESTER E, SASAKI T, MANN K, et al. Variable zinc coordination in endostatin [J]. J Mol Biol,2000,297(1):1-6.
[125]NOMA H, FUNATSU H, YAMASHITA H, et al. Regulation of angiogenesis in diabetic retinopathy:possible balance between vascular endothelial growth factor and endostatin [J]. Arch Ophthalmol,2002,120(8):1075-80.
[126]JUNG S P, SIEGRIST B, HORNICK C A, et al. Effect of human recombinant Endostatin protein on human angiogenesis [J]. Angiogenesis,2002,5(1-2):111-8.
[127]O'REILLY M S, HOLMGREN L, SHING Y, et al. Angiostatin:a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma [J]. Cell,1994, 79(2):315-28.
[128]CORNELIUS L A, NEHRING L C, HARDING E, et al. Matrix metalloproteinases generate angiostatin:effects on neovascularization [J]. J Immunol,1998,161(12):6845-52.
[129]MORIKAWA W, YAMAMOTO K, ISHIKAWA S, et al. Angiostatin generation by cathepsin D secreted by human prostate carcinoma cells [J]. J Biol Chem,2000,275(49): 38912-20.
[130]OCHOA A, MONTES DE OCA P, RIVERA J C, et al. Expression of prolactin gene and secretion of prolactin by rat retinal capillary endothelial cells [J]. Invest Ophthalmol Vis Sci,2001,42(7):1639-45.
[131]CORBACHO A M, MACOTELA Y, NAVA G, et al. Human umbilical vein endothelial cells express multiple prolactin isoforms [J]. J Endocrinol,2000,166(1):53-62.
[132]FUNAKI H, SAWAGUCHI S, YAOEDA K, et al. Expression and localization of angiogenic inhibitory factor, chondromodulin-I, in adult rat eye [J]. Invest Ophthalmol Vis Sci, 2001,42(6):1193-200.
[133]FUKUSHIMA A, FUNAKI H, YAOEDA K, et al. Localization and expression of chondromodulin-I in the rat cornea [J]. Arch Histol Cytol,2003,66(5):445-52.
[134]GOOD D J, POLVERINI P J, RASTINEJAD F, et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin [J]. Proc Natl Acad Sci U S A,1990,87(17):6624-8.
[135]VOLPERT O V, LAWLER J, BOUCK N P. A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1 [J]. Proc Natl Acad Sci U S A,1998,95(11):6343-8.
[136]MIYAJIMA-UCHIDA H, HAYASHI H, BEPPU R, et al. Production and accumulation of thrombospondin-1 in human retinal pigment epithelial cells [J]. Invest Ophthalmol Vis Sci,2000,41(2):561-7.
[137]ANDO A, YANG A, NAMBU H, et al. Blockade of nitric-oxide synthase reduces choroidal neovascularization [J]. Mol Pharmacol,2002,62(3):539-44.
[138]GONZALEZ C, CORBACHO A M, EISERICH J P, et al.16K-prolactin inhibits activation of endothelial nitric oxide synthase, intracellular calcium mobilization, and endothelium-dependent vasorelaxation [J]. Endocrinology,2004,145(12):5714-22.
[139]MASOS T, DAN J A, MISKIN R. Plasminogen activator inhibitor-1 mRNA is localized in the ciliary epithelium of the rodent eye [J]. Invest Ophthalmol Vis Sci,2000,41(5): 1006-11.
[140]BROOKS P C, SILLETTI S, VON SCHALSCHA T L, et al. Disruption of angiogenesis by PEX, a noncatalytic metalloproteinase fragment with integrin binding activity [J]. Cell,1998,92(3):391-400.
[141]SILLETTI S, KESSLER T, GOLDBERG J, et al. Disruption of matrix metalloproteinase 2 binding to integrin alpha vbeta 3 by an organic molecule inhibits angiogenesis and tumor growth in vivo [J]. Proc Natl Acad Sci U S A,2001,98(1):119-24.
[142]ROSEWICZ S, DETJEN K, SCHOLZ A, et al. Interferon-alpha:regulatory effects on cell cycle and angiogenesis [J]. Neuroendocrinology,2004,80 Suppl 1(85-93.
[143]PAULUKAT J, BOSMANN M, NOLD M, et al. Expression and release of IL-18 binding protein in response to IFN-gamma [J]. J Immunol,2001,167(12):7038-43.
[144]CAO R, FARNEBO J, KURIMOTO M, et al. Interleukin-18 acts as an angiogenesis and tumor suppressor [J]. FASEB J,1999,13(15):2195-202.
[145]WIGGINTON J M, LEE J K, WILTROUT T A, et al. Synergistic engagement of an ineffective endogenous anti-tumor immune response and induction of IFN-gamma and Fas-ligand-dependent tumor eradication by combined administration of IL-18 and IL-2 [J]. J Immunol,2002,169(8):4467-74.
[146]CAUDELL E G, MUMM J B, POINDEXTER N, et al. The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24 [J]. J Immunol,2002,168(12):6041-6.
[147]SCHAEFER G, VENKATARAMAN C, SCHINDLER U. Cutting edge:FISP (IL-4-induced secreted protein), a novel cytokine-like molecule secreted by Th2 cells [J]. J Immunol,2001,166(10):5859-63.
[148]PRAIDOU A, ANDROUDI S, BRAZITIKOS P, et al. Angiogenic growth factors and their inhibitors in diabetic retinopathy [J]. Curr Diabetes Rev,2010,6(5):304-12.
[149JASAHARA T, MUROHARA T, SULLIVAN A, et al. Isolation of putative progenitor endothelial cells for angiogenesis [J]. Science,1997,275(5302):964-7.
[150]CERADINI D J, KULKARNI A R, CALLAGHAN M J, et al. Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of SDF-1 [J]. Nat Med, 2004,10(8):858-64.
[151]GARCIA-BARROS M, PARIS F, CORDON-CARDO C, et al. Tumor response to radiotherapy regulated by endothelial cell apoptosis [J]. Science,2003,300(5622):1155-9.
[152]CAMARGO F D, GREEN R, CAPETANAKI Y, et al. Single hematopoietic stem cells generate skeletal muscle through myeloid intermediates [J]. Nat Med,2003,9(12): 1520-7.
[153]URBICH C, HEESCHEN C, AICHER A, et al. Relevance of monocytic features for neovascularization capacity of circulating endothelial progenitor cells [J]. Circulation,2003, 108(20):2511-6.
[154]MUROHARA T, IKEDA H, DUAN J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization [J]. J Clin Invest,2000, 105(11):1527-36.
[155]周秀娟.人外周血、脐血、脂肪组织中内皮祖细胞生物特性比较研究[D].泸州：泸州医学院2008.
[156]DING H, TRIGGLE C R. Endothelial cell dysfunction and the vascular complications associated with type 2 diabetes:assessing the health of the endothelium [J]. Vasc Health Risk Manag,2005,1(1):55-71.
[157]CABALLERO S, SENGUPTA N, AFZAL A, et al. Ischemic vascular damage can be repaired by healthy, but not diabetic, endothelial progenitor cells [J]. Diabetes,2007,56(4): 960-7.
[158]ZUK P A, ZHU M, MIZUNO H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies [J]. Tissue Eng,2001,7(2):211-28.
[159]PLANAT-BENARD V, SILVESTRE J S, COUSIN B, et al. Plasticity of human adipose lineage cells toward endothelial cells:physiological and therapeutic perspectives [J]. Circulation,2004,109(5):656-63.
[160]MIRANVILLE A, HEESCHEN C, SENGENES C, et al. Improvement of postnatal neovascularization by human adipose tissue-derived stem cells [J]. Circulation,2004,110(3): 349-55.
[161]MOON M H, KIM S Y, KIM Y J, et al. Human adipose tissue-derived mesenchymal stem cells improve postnatal neovascularization in a mouse model of hindlimb ischemia [J]. Cell Physiol Biochem,2006,17(5-6):279-90.
[162]SUMI M, SATA M, TOYA N, et al. Transplantation of adipose stromal cells, but not mature adipocytes, augments ischemia-induced angiogenesis [J]. Life Sci,2007,80(6):559-65.
[163]PARDANAUD L, LUTON D, PRIGENT M, et al. Two distinct endothelial lineages in ontogeny, one of them related to hemopoiesis [J]. Development,1996,122(5):1363-71.
[164]CHOI K. The hemangioblast:a common progenitor of hematopoietic and endothelial cells [J]. J Hematother Stem Cell Res,2002,11(1):91-101.
[165]RIBATTI D, VACCA A, NICO B, et al. Cross-talk between hematopoiesis and angiogenesis signaling pathways [J]. Curr Mol Med,2002,2(6):537-43.
[166]REYES M, DUDEK A, JAHAGIRDAR B, et al. Origin of endothelial progenitors in human postnatal bone marrow [J]. J Clin Invest,2002,109(3):337-46.
[167]LIN Y, WEISDORF D J, SOLOVEY A, et al. Origins of circulating endothelial cells and endothelial outgrowth from blood [J]. J Clin Invest,2000,105(1):71-7.
[168]RAFII S, LYDEN D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration [J]. Nat Med,2003,9(6):702-12.
[169]IWAMI Y, MASUDA H, ASAHARA T. Endothelial progenitor cells:past, state of the art, and future [J]. J Cell Mol Med,2004,8(4):488-97.
[170]ISNER J M, ASAHARA T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularization [J]. J Clin Invest,1999,103(9):1231-6.
[171]MENG Q Y, LI X Q, YU X B, et al. Transplantation of VEGF165-gene-transfected endothelial progenitor cells in the treatment of chronic venous thrombosis in rats [J]. Chin Med J(Engl),2010,123(4):471-7.
[172]方立建.人脐带血内皮祖细胞的培养与鉴定[D].长春：吉林大学,2008.
[173]FORD J W, WELLING T H,3RD, STANLEY J C, et al. PKH26 and 125I-PKH95: characterization and efficacy as labels for in vitro and in vivo endothelial cell localization and tracking [J]. J Surg Res,1996,62(1):23-8.
[174]SZILVASSY S J, MEYERROSE T E, RAGLAND P L, et al. Differential homing and engraftment properties of hematopoietic progenitor cells from murine bone marrow, mobilized peripheral blood, and fetal liver [J]. Blood,2001,98(7):2108-15.
[175]ZHAN Y, WANG Y, WEI L, et al. Differentiation of hematopoietic stem cells into hepatocytes in liver fibrosis in rats [J]. Transplant Proc,2006,38(9):3082-5.
[176]CANOLA K, ANGENIEUX B, TEKAYA M, et al. Retinal stem cells transplanted into models of late stages of retinitis pigmentosa preferentially adopt a glial or a retinal ganglion cell fate [J]. Invest Ophthalmol Vis Sci,2007,48(1):446-54.
[177]KOPEN G C, PROCKOP D J, PHINNEY D G. Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains [J]. Proc Natl Acad Sci U S A,1999,96(19):10711-6.
[178]CHEN J, LI Y, CHOPP M. Intracerebral transplantation of bone marrow with BDNF after MCAo in rat [J]. Neuropharmacology,2000,39(5):711-6.
[179]KIEL M J, HE S, ASHKENAZI R, et al. Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU [J]. Nature,2007,449(7159):238-42.
[180]PARISH C R. Fluorescent dyes for lymphocyte migration and proliferation studies [J]. Immunol Cell Biol,1999,77(6):499-508.
[181]SHIMOMURA O, JOHNSON F H, SAIGA Y. Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea [J]. J Cell Comp Physiol,1962,59(223-39.
[182]TSIEN R Y. The green fluorescent protein [J]. Annu Rev Biochem,1998,67(509-44.
[183]GUBIN A N, REDDY B, NJOROGE J M, et al. Long-term, stable expression of green fluorescent protein in mammalian cells [J]. Biochem Biophys Res Commun,1997, 236(2):347-50.
[184]BEAUDRY P, HIDA Y, UDAGAWA T, et al. Endothelial progenitor cells contribute to accelerated liver regeneration [J]. J Pediatr Surg,2007,42(7):1190-8.
[185]王征秒.脂质体介导绿色荧光蛋白转染人脐血内皮祖细胞[D].长春：吉林大学,2009.
[186]ZHANG S, ZOU Z, JIANG X, et al. The therapeutic effects of tyrosine hydroxylase gene transfected hematopoetic stem cells in a rat model of Parkinson's disease [J]. Cell Mol Neurobiol,2008,28(4):529-43.
[187]CARTER B J. Adeno-associated virus vectors in clinical trials [J]. Hum Gene Ther, 2005,16(5):541-50.
[188]MIZUGUCHI H, HAYAKAWA T. Adenovirus vectors containing chimeric type 5 and type 35 fiber proteins exhibit altered and expanded tropism and increase the size limit of foreign genes [J]. Gene,2002,285(1-2):69-77.
[189]GOMEZ-NAVARRO J, CONTRERAS J L, ARAFAT W, et al. Genetically modified CD34+ cells as cellular vehicles for gene delivery into areas of angiogenesis in a rhesus model [J]. Gene Ther,2000,7(1):43-52.
[190]LUNDSTROM K. Latest development in viral vectors for gene therapy [J]. Trends Biotechnol,2003,21(3):117-22.
[191]MARTIN M, METZGER D J, MICHALEK S M, et al. Distinct cytokine regulation by cholera toxin and type II heat-labile toxins involves differential regulation of CD40 ligand on CD4(+) T cells [J]. Infect Immun,2001,69(7):4486-92.
[192]INDRACCOLO S, HABELER W, TISATO V, et al. Gene transfer in ovarian cancer cells:a comparison between retroviral and lentiviral vectors [J]. Cancer Res,2002,62(21): 6099-107.
[193]SONG E, LU C W, FANG L J, et al. Culture and identification of endothelial progenitor cells from human umbilical cord blood [J]. International Journal Ophthalmology, 2009,9(11):2045-9.
[194]BROXMEYER H E, DOUGLAS G W, HANGOC G, et al. Human umbilical cord blood as a potential source of transplantable hematopoietic stem/progenitor cells [J]. Proc Natl Acad Sci U S A,1989,86(10):3828-32.
[195]BROXMEYER H E, GLUCKMAN E, AUERBACH A, et al. Human umbilical cord blood:a clinically useful source of transplantable hematopoietic stem/progenitor cells [J]. Int J Cell Cloning,1990,8 Suppl 1(76-89; discussion-91.
[196]BROXMEYER H E, KURTZBERG J, GLUCKMAN E, et al. Umbilical cord blood hematopoietic stem and repopulating cells in human clinical transplantation [J]. Blood Cells, 1991,17(2):313-29.
[197]GLUCKMAN E, BROXMEYER H A, AUERBACH A D, et al. Hematopoietic reconstitution in a patient with Fanconi's anemia by means of umbilical-cord blood from an HLA-identical sibling [J]. N Engl J Med,1989,321(17):1174-8.
[198]杨威.高糖对内皮祖细胞功能的影响及其相关机制的研究[D].长春：吉林大学,2010.
[199]YODER M C. Defining human endothelial progenitor cells [J]. J Thromb Haemost, 2009,7(Suppl 1):49-52.
[200]方立建,宋鄂,栾瑛,等.人脐带血内皮祖细胞体外培养和鉴定[J].中国组织工 程研究与临床康复,2010,14(45)：8450-4.
[201]谢松涛,陈璧,陶克.人脐血中CD133+血管内皮祖细胞的分离培养和生物学特性[J].中国组织工程研究与临床康复,2007,11(7)：1287-9.
[202]LI Y J, DUAN C L, LIU J X, et al. Pro-angiogenic actions of Salvianolic acids on in vitro cultured endothelial progenitor cells and chick embryo chorioallantoic membrane model [J]. J Ethnopharmacol,2010,131(3):562-6.
[203]WEIBEL E R, PALADE G E. NEW CYTOPLASMIC COMPONENTS IN ARTERIAL ENDOTHELIA [J]. J Cell Biol,1964,23(101-12.
[204]VALENTIJN K M, SADLER J E, VALENTIJN J A, et al. Functional architecture of Weibel-Palade bodies [J]. Blood,2011,
[205]SHI H, YANG W, CUI Z-H, et al. Tracking of CFSE-labeled endothelial progenitor cells in laser-injured mouse retina [J]. Chinese Medical Journal,2011,124(5):751-7.
[206]DUMITRIU I E, MOHR W, KOLOWOS W, et al.5,6-carboxyfluorescein diacetate succinimidyl ester-labeled apoptotic and necrotic as well as detergent-treated cells can be traced in composite cell samples [J]. Anal Biochem,2001,299(2):247-52.
[207]LI X, DANCAUSSE H, GRIJALVA I, et al. Labeling Schwann cells with CFSE-an in vitro and in vivo study [J]. J Neurosci Methods,2003,125(1-2):83-91.
[208]LYONS A B. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution [J]. J Immunol Methods,2000,243(1-2):147-54.
[209]PARISH C R, GLIDDEN M H, QUAH B J, et al. Use of the intracellular fluorescent dye CFSE to monitor lymphocyte migration and proliferation [J]. Curr Protoc Immunol,2009, 4(4-9.
[210]NUGENT M A, NUGENT H M, IOZZO R V, et al. Perlecan is required to inhibit thrombosis after deep vascular injury and contributes to endothelial cell-mediated inhibition of intimal hyperplasia [J]. Proc Natl Acad Sci U S A,2000,97(12):6722-7.
[211]左玲.人血管内皮生长因子受体KDR胞外段1-3区基因转染抑制视网膜新生血管的实验研究[D].长春：吉林大学,2008.
[212]BHATWADEKAR A D, GLENN J V, CURTIS T M, et al. Retinal endothelial cell apoptosis stimulates recruitment of endothelial progenitor cells [J]. Invest Ophthalmol Vis Sci, 2009,50(10):4967-73.
[213]BUTLER J M, GUTHRIE S M, KOC M, et al. SDF-1 is both necessary and sufficient to promote proliferative retinopathy [J]. J Clin Invest,2005,115(1):86-93.
[214]SEGAL M S, SHAH R, AFZAL A, et al. Nitric oxide cytoskeletal-induced alterations reverse the endothelial progenitor cell migratory defect associated with diabetes [J]. Diabetes, 2006,55(1):102-9.
[215]ITAYA M, SAKURAI E, NOZAKI M, et al. Upregulation of VEGF in murine retina via monocyte recruitment after retinal scatter laser photocoagulation [J]. Invest Ophthalmol Vis Sci,2007,48(12):5677-83.
[216]DOT C, PARIER V, BEHAR-COHEN F, et al. Influence of age on retinochoroidal healing processes after argon photocoagulation in C57b1/6j mice [J]. Mol Vis,2009,15(670-84.
[217]赵林,王雷,贾三庆,等.人脐血干细胞移植促进心肌梗死大鼠血管新生的机制研究[J].临床心血管病杂志,2008,24(7)：540-4.
[218]吴海英,罗清礼.人视网膜激光光凝后的组织病理学研究概况[J].中华眼底病杂志,1997,13(4)：246-7.
[219]MARTIN K R, KLEIN R L, QUIGLEY H A. Gene delivery to the eye using adeno-associated viral vectors [J]. Methods,2002,28(2):267-75.
[220]SAKAMOTO T, UENO H, GOTO Y, et al. A vitrectomy improves the transfection efficiency of adenoviral vector-mediated gene transfer to Muller cells [J]. Gene Ther,1998, 5(8):1088-97.
[221]YILMAZ T, WEAVER C D, GALLAGHER M J, et al. Intravitreal triamcinolone acetonide injection for treatment of refractory diabetic macular edema:a systematic review [J]. Ophthalmology,2009,116(5):902-11; quiz 12-3.
[222]SOBRADO-CALVO P, VIDAL-SANZ M, VILLEGAS-PEREZ M P. Rat retinal microglial cells under normal conditions, after optic nerve section, and after optic nerve section and intravitreal injection of trophic factors or macrophage inhibitory factor [J]. J Comp Neurol, 2007,501(6):866-78.
[223]CASTANHEIRA P, TORQUETTI L T, MAGALHAS D R, et al. DAPI diffusion after intravitreal injection of mesenchymal stem cells in the injured retina of rats [J]. Cell Transplant,2009,18(4):423-31.
[224JAARTSEN W M, VAN CLEEF K W, PELLISSIER L P, et al. GFAP-driven GFP expression in activated mouse Muller glial cells aligning retinal blood vessels following intravitreal injection of AAV2/6 vectors [J]. PLoS One,2010,5(8):e12387.
[225]HUBSCHMAN J P, COFFEE R E, BOURGES J L, et al. Experimental model of intravitreal injection techniques [J]. Retina,2010,30(1):167-73.