内皮祖细胞眼内移植的示踪及对视网膜血管损伤修复的研究

发布时间：2018-09-17 16:50

【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。
[Abstract]:Diabetic retinopathy, including retinopathy of premature infants, central retinal vein occlusion and other retinal ischemic diseases, is characterized by neovascularization. Additionally, it causes edema and exudation of peripheral tissues, and then leads to ischemia and hypoxia of retinal tissues, and compensatory formation of new blood vessels with incomplete organizational structure, leading to bleeding, proliferation, and even retinal detachment, which seriously threatens the visual acuity of patients. Endothelial growth factor inhibitors, photodynamic therapy, steroid hormones and surgical treatment can reduce angiogenesis to a certain extent, but can not fundamentally eliminate angiogenesis factors, and accompanied by a series of side effects and the possibility of recurrence. Retinal ischemic diseases and pathological neovascularization play an important role in the formation and development of retinal ischemic diseases. Therefore, the key to control retinal neovascularization is to repair damaged vascular endothelium and improve retinal hypoxia and hypoxia. EPCs transplantation can improve the ischemic injury of heart, brain and limbs, increase the blood flow and capillary density in ischemic sites, and improve the therapeutic effect on ischemic diseases. If EPCs can be effectively transplanted to the ischemic and hypoxic retina region It is possible to repair damaged vascular endothelium, improve retinal blood supply, and avoid pathological neovascularization. This study requires not only appropriate cell transplantation, but also a stable and efficient tracing method to provide an objective basis for the experiment.
Carboxyfluorescein diacetate succinimide ester (CFSE) labeled EPCs, Dil labeled acetylated low density lipoprotein (DiI-AcLDL) labeled EPCs and lentivirus mediated green fluorescent protein (GFP) transduction of EPCs were used to compare the tracing of EPCs in vitro and in vivo, and to observe the repairing effect of transplanted EPCs on retinal vascular injury. It provides an experimental basis for selecting suitable markers for cell transplantation and better tracking the efficacy of EPCs transplantation.
Method
(1) Culture and identification of EPCs from human umbilical cord blood: Mononuclear cells from human umbilical cord blood were isolated by hydroxyethyl starch sedimentation and Percoll density gradient centrifugation, and differentiated into EPCs in vitro. The cells were identified by morphology, flow cytometry, immunofluorescence staining and electron microscopy. Methods EPCs were labeled in vitro by CFSE, DiI-AcLDL and lentivirus-mediated GFP gene transduction. The morphological changes of the cells before and after labeling were observed by inverted phase contrast microscope. The viability and adhesion of the labeled EPCs were measured by Trypan blue staining and adherent cell counting. The fluorescence intensity was observed under fluorescence microscope. Flow cytometry was used to determine the positive rate of labeling and compare the advantages and disadvantages of the three methods. (3) Intraocular transplantation of labeled EPCs: C57BL/6N mice retinal vascular injury model was established by multi-wavelength krypton laser selective injury. EPCs labeled with LDL, CFSE and lentivirus mediated GFP gene transduction were transplanted into vitreous cavity by microinjector under operating microscope. Fundus photography, retinal paraffin section, frozen section and retinal paving were observed at different time intervals. The distribution, fluorescence intensity and persistence of labeled cells in longitudinal and transverse plane of retina were observed. Time and other conditions, and compare the repair of retinal vascular injury before and after EPCs transplantation.
Result
(1) The primary cultured EPCs were isolated from human umbilical cord blood and showed typical morphological changes during the culture process. The cells expressed CD34, CD133 and''VEGFR-2 in different degrees. They could phagocytose DiI-AcLDL and combine with FITC-UEA-I. The W-P bodies of endothelial cells were observed under electron microscope, which proved that most of the cultured cells were in large part of the population. The positive rate of EPCs labeled with DiI-AcLDL was up to 80%. The fluorescence intensity of CFSE and DiI-AcLDL labeled EPCs lasted for 4 weeks and gradually decreased with the time of culture. Four days after transduction of GFP gene by lentivirus, the positive rate of EPCs labeled with DiI-AcLDL was up to 95%. The green fluorescence was observed under confocal microscope, and then the green fluorescence positive cells increased gradually, the fluorescence intensity increased gradually, and the transfection efficiency exceeded 30% at 4 weeks after transduction. Changes. (3) In vivo, retinal vascular injury model was successfully established by laser photocoagulation in mice. After intravitreal injection of labeled EPCs, laser spot pigmentation and scar formation were observed in fundus photography at 4 weeks after transplantation, which were less severe than those in non-transplanted eyes. After transplantation, the frozen sections of the retina showed that fluorescent cells could be seen on the surface of the retina 2 days after transplantation of DiI-AcLDL and CFSE-labeled EPCs. At 1 week, fluorescent cells could be seen in the injured area. At 4 weeks, fluorescent cells could be seen in all layers of the retina. There were no fluorescent cells in the frozen sections of retina at each time point after lentivirus-mediated GFP gene transduction of EPCs transplantation. Evans blue perfusion angiography could clearly show the structure of retinal capillary network, and fluorescent leakage could be seen in the laser spot of retinal vascular injury model. Two days after transplantation, green fluorescent labeled cells clustered on the retina. One week after transplantation, green fluorescent labeled cells clustered around the laser injury. Four weeks after transplantation, green fluorescent labeled cells formed a similar tubular structure, confirming that EPCs participated in retinal vascular repair.
conclusion
(1) CFSE and Dil-AcLDL are suitable for short-term tracing of EPCs. CFSE has the highest efficiency, the strongest initial fluorescence, the lowest cost, the simplest operation and the advantages of short-term tracing. CFSE labeling EPCs combined with frozen section of retina and Evans blue perfusion retina paving have established a multi-angle observation method for the tracing of EPCs intraocular transplantation. Gene-transduced EPCs have more potential in long-term tracing. (2) Retinal vascular injury model was established by laser photocoagulation in mice, and intraocular transplantation of EPCs was performed by intravitreal injection.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R774.1

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