【作者】 张凯；

【导师】 吴欣怡；

【作者基本信息】 山东大学 ， 眼科学， 2014， 博士

【摘要】 角膜病患病率高、致盲性强,位居全球致盲原因第二位,目前,全球约有超过1000万的角膜盲患者；同时,角膜病也是我国第二大致盲性眼病,调查显示,我国现有角膜盲患者约100多万。迄今为止,同种异体角膜移植是治疗不可逆性角膜盲最有效的方法。然而,角膜供体的极度匮乏使其应用受到了限制,远远满足不了角膜盲患者的需求；我国每年约有30万患者需行角膜移植术,但因供体角膜有限,每年施行移植手术的患者尚不足4000例(1.3%)。因此,寻找有效的角膜替代物用于角膜移植一直是眼科界研究的热点。目前人工角膜(Keratoprosthesis)是唯一被批准用于临床的角膜替代物,但其生物相容性差、术后并发症多,限制了其在临床的广泛应用。组织工程角膜是近年来研究的热点。其材料来源丰富、生物相容性好,可最大程度的模拟天然角膜结构和功能,有望替代天然角膜用于角膜移植,缓解角膜供体极其匮乏的压力。因此,组织工程角膜的研究具有重大社会意义及显著经济效益。组织工程角膜是通过将种子细胞(角膜上皮细胞、基质细胞和内皮细胞)接种于支架材料,经过体外三维培养构建而成。种子细胞和支架材料是组织工程角膜构建的两大关键要素。我们课题组前期研制的脱细胞猪角膜基质(acellular porcine corneal matrix, APCM),其中的异种细胞及遗传物质被完全脱除,同时胶原及其天然排列结构得到了保留,从而具有良好的透光性、生物相容性以及机械力学性能；组织工程角膜前、后板层的成功构建充分证实APCM是组织工程角膜构建的良好支架材料。种子细胞必须具备较强的增殖能力,能在体外大量扩增。人角膜基质细胞在体外有较强的增殖能力,而上皮细胞和内皮细胞增殖能力有限,尤其是内皮细胞,几乎没有增殖能力,难以在体外培养、扩增,因此寻找新的内皮细胞来源成为亟待解决的问题。目前由于尚未找到理想的内皮细胞来源,大部分有关组织工程角膜构建的研究都是利用增殖能力相对较强的低等动物的角膜内皮细胞或永生化人角膜内皮细胞系等作为内皮细胞的种子细胞,此类内皮细胞构建的组织工程角膜可用于体外实验研究,但是难以满足临床应用的要求。胚胎干细胞(embryonic stem cells, ESCs)因其所具有的强大增殖能力和分化全能性可作为一种良好的种子细胞来源,为组织工程器官构建提供无限的细胞供给。目前已有人胚胎干细胞(human embryonic stem cells, hESCs)诱导分化为心肌细胞、平滑肌细胞、多巴胺神经元及运动神经元等体细胞的报道。然而,迄今为止,国内外尚无hESCs诱导分化成角膜内皮细胞的研究报道。因此如能实现hESCs向角膜内皮细胞的诱导分化,这将为组织工程角膜的构建提供理想的种子细胞来源。因此,本研究探讨了将hESCs定向诱导分化为角膜内皮样细胞(corneal endothelial cell-like cells, CEC-like cells)的可行性,获得了在形态和功能上接近正常角膜内皮细胞的CEC-like cells;将CEC-like cells接种于后板层脱细胞猪角膜支架(posterior acellular porcine corneal matrix, PAPCM)后弹力层表面构建组织工程角膜内皮植片,体内、体外实验证实构建物具有天然角膜后板层的形态和功能。[目的]角膜脱水状态和透明性的维持很大程度上依赖于角膜内皮细胞的屏障及泵功能。人角膜内皮细胞在体内没有增殖能力,体外培养条件下增殖能力也极其有限。因此,获取大量具有正常角膜内皮细胞功能的细胞有利于角膜内皮细胞基础研究和角膜内皮功能失代偿的治疗。本研究探讨体外诱导人胚胎干细胞经眼周间充质干细胞分化为角膜内皮样细胞的可行性。[方法]通过Transwell共培养体系,将人胚胎干细胞与正在向成纤维细胞转化的人角膜基质细胞共培养5天,分化为眼周间充质干细胞；改为人晶状体上皮细胞源性条件培养基继续培养已向眼周间充质干细胞分化的人胚胎干细胞14天,使其进一步分化为角膜内皮样细胞。利用免疫荧光法检测诱导细胞眼周间充质干细胞标记物CD73和FOXC1以及角膜内皮细胞分化标记物N型钙粘素、FOXC1、PITX2、Na+/K+ATP酶和闭锁小带蛋白-1(zonula occluden-1, ZO-1)的表达。流式细胞仪分选CD73表达阳性的眼周间充质干细胞；N型钙粘素/Vimentin双阳性的角膜内皮样细胞。为进一步证明人胚胎干细胞向角膜内皮细胞分化,对分选出的眼周间充质干细胞和角膜内皮样细胞行免疫印迹(Western blotting)和实时定量聚合酶链式反应(real time-polymerase chain reaction, RT-PCR)检测,评估Na+/K+ATP酶在这两种细胞内的表达情况。分选出的角膜内皮样细胞在体外传代培养,扩增后,行免疫荧光检测Na+/K+ATP酶、ZO-1、N型钙粘素及Vimentin表达情况；扩增的角膜内皮细胞经羧基荧光素琥珀酰亚胺酯(Carboxyfluorescein Diacetate Succinimidyl Ester, CFDA SE)标记后,接种于脱细胞猪角膜基质后板层构建角膜内皮植片。角膜内皮植片泵功能通过体外尤氏灌流系统及体内动物移植实验进行评估。实验动物术后观察2月,定期行裂隙灯显微镜检查照相、角膜内皮镜、激光共聚焦显微镜及取材病理切片HE染色等检查。[结果]人胚胎干细胞与分化的人角膜基质细胞共培养5天,可见大量梭形细胞从贴壁的拟胚体爬出,表达眼周间充质干细胞标记物CD73和FOXC1;更换为人晶状体上皮细胞源性条件培养基继续培养7天,可见眼周间充质干细胞由梭形逐渐变为多边形,表达人角膜内皮细胞标记物N型钙粘素、FOXC1、PITX2、 Na+/K+ATP酶和ZO-1。流式细胞仪成功分选出CD73表达阳性的眼周间充质干细胞及N型钙粘素/Vimentin双阳性的角膜内皮样细胞。Western blotting和实时定量聚合酶链式反应(real time-polymerase chain reaction, RT-PCR)检测表明角膜内皮样细胞较眼周间充质干细胞Na+/K+ATP酶表达明显上调。角膜内皮样细胞在体外传代培养2代,扩增后的细胞Na+/K+ATP酶、ZO-1、N型钙粘素及Vimentin表达阳性。扩增的角膜内皮样细胞经CFDA SE标记后,荧光显微镜下见绿色荧光,接种于脱细胞角膜基质后板层成功构建角膜内皮植片。构建物具有正常人角膜后板层类似的形态结构；尤氏灌流系统检测内皮泵功能参数,角膜内皮植片与正常人角膜泵功能参数接近；将构建的角膜内皮植片移植到角膜内皮功能失代偿兔动物模型眼内(实验组),角膜逐渐恢复透明,而单纯移植脱细胞角膜基质后板层的兔眼角膜水肿混浊持续加重(对照组)。角膜内皮镜检查见实验组角膜后表面多边形细胞结构,荧光显微镜下发绿色荧光,HE染色见单层细胞贴附于脱细胞猪角膜后弹力层,而对照组中央角膜后表面未见细胞结构。[结论]人胚胎干细胞诱导分化、流式细胞仪分选获取的角膜内皮样细胞具有正常人角膜内皮细胞的形态结构和内皮泵功能。这为人角膜内皮细胞生物学特性的进一步研究及细胞替代治疗提供了大量细胞来源。更多还原

【Abstract】 Corneal disease is the second most common cause for blindness worldwide for its high prevalence and severe blinding. At present more than10million individuals suffer from corneal blindness in the world. At the same time it is also the second most common cause for blindness in China. Date shows there are over1million individuals suffering from corneal blindness in China. Up to now corneal transplantation is the most effective treatment for irreversible corneal blindness. However the suitable cornea donors are severely shortage, far from meeting the increasing need of patients. There are300thousands patients waiting for corneal transplantation per year, but the number of patients accepted corneal transplantation is less than4thousands (1.3%), due to the shortage of cornea donors. Therefore many researchers are attempting to develop cornea substitutes for corneal transplantation. Currently Keratoprosthesis are the sole alternatives for donor corneas which have been approved for clinical application, but its poor biocompatibility and severe complications handicapped extensive adoption.Tissue engineering corneas have experienced major progress during the past few years. The accessibility, perfect biocompatibility and accurately similarity anatomically and functionally with native cornea make it a potential alternative of donor cornea for keratoplasty, which can effectively relieve the extreme shortage of donor corneas. Thus, the construction of tissue engineering cornea has major social significance and economic benefits. The cornea was reconstructed through seeding corneal cells (i.e. corneal epithelial, stromal and endothelial cells) on or in the scaffold, subsequently proceeding to three-dimentional culture using tissue engineering technology. Seed cells and scaffolds are two key elements for construction of tissue engineering cornea. Acellular porcine corneal matrix (APCM) has been prepared in our previous studies, in which the cell components and hereditary materials were completely removed while the organization of collagen fibers preserved. The characteristics of high optical clarity, good biocompatibility, toughness biomechanics and non-immunogenicity and successful fabrication of anterior/posterior corneal lamellas demonstrated APCM is a kind of favorable scaffold for cornea tissue engineering. The seed cells need to posses high proliferative ability and could be amplified in large scale. Human corneal stromal cells can be cultured for many passages in vitro, while the proliferative ability of epithelial and endothelial cells was limited, especially the human corneal endothelial cells which have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human corneal endothelial cells is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Now most tissue engineering corneas are not suitable for clinical use, because they are constructed with animal cells or immortalized human cells, which have stronger immune rejection and safety consideration.Embryonic stem cells are pluripotent cells derived from the inner cell mass of the blastocyst that can be maintained in culture for an extended period of time without losing differentiation potential. The successful isolation of human embryonic stem cells (hESCs) has raised the hope that may provide a potentially unlimited supply of cells that may be directed to differentiate into all cell types within the body and used in regenerative medicine for tissue and cell replacement therapies. At present documents report hESCs are capable of differentiation into multiple cell lineages under specific culture conditions, such as cardiomyocytes, smooth muscle cells, midbrain dopamine neurons, and motoneurons et al. However whether human corneal endothelial cells can be acquired from differentiated hESCs is still unknown. Therefore we assume hESCs would be an ideal source of seed cells for tissue engineering cornea construction if induced to differentiate successfully in vitro.Here we investigate the feasibility of inducing hESCs to differentiate into corneal endothelial cell (CEC)-like cells with phenotypic, genetic and functional characteristics of normal human CECs from hESCs. CEC-like cells were seeded onto posterior acellular porcine corneal matrix (PAPCM) lamellas to construct the CEC-like cells sheets. Pump functions of CEC-like cells were measured in a Ussing chamber in Vitro and by transplantation into the eyes of rabbit CE dysfunction models in Vivo respectively and its performance was similar to native cornea.PURPOSE. The maintenance of corneal dehydration and transparency depends on barrier and pump functions of corneal endothelial cells (CECs). The human CECs have no proliferation capacity in vivo and the ability to divide in vitro under culture conditions is dramatically limited. Thus, the acquisition of massive cells analogous to normal human CECs is extremely necessary whether from the perspective of cellular basic research or from clinical applications. Here we investigate the feasibility of inducing human embryonic stem cells (hESCs) to differentiate into functional corneal endothelial cell (CEC)-like cells through the periocular mesenchymal precursors (POMPs) phase in vitro.METHODS. Using the transwell co-culture system of hESCs with differentiated human corneal stromal cells (hCSCs), we induced hESCs to differentiate into POMPs. Then, CEC-like cells were derived from POMPs with lens epithelial cell-conditioned medium (LECCM). Immunofluorescence was used to detect expression of the POMPs marker CD73and FOXC1and corneal endothelium differentiation marker N-cadherin, transcription factors FoxCl and Pitx2and CEC-related marker Na+/K+ATPase and zonula occludens-1(ZO-1). Fluorescence-activated cell sorting (FACS)-based isolation of the CD73and N-Cadherin/vimentin dual positive populations enriches for POMPs and CEC-like cells respectivly. In order to further demonstrate the differentiation to CECs, the Western blotting and RT-PCR were performed to evaluate the expression of Na+/K+ATPase in the isolated POMPs and CEC-like cells. The isolated CEC-like cells were expanded by culturing2passages in vitro. Then, the expanded CEC-like cells were performed on Immunofluorescence for expression of Na+/K+ATPase alpha1, ZO-1, N-Cadherin and vimentin, labeled with CFDA SE and seeded onto posterior acellular porcine corneal matrix (PAPCM) lamellas to construct the CEC-like cells sheets. Pump functions of CEC-like cells were measured in a Ussing chamber in Vitro and by transplantation into the eyes of rabbit CE dysfunction models in Vivo respectively. The animals were observed for as long as2months after surgery and underwent clinical and histological examination.RESULTS. Spindle-like POMPs emigrated from the hESCs-derived embryoid bodies (EBs) expressing CD73and FOXC1and turned to polygonal CEC-like cells which expressed N-cadherin, FoxC1, Pitx2, Na+/K+ATPase and ZO-1. The Western blotting and RT-PCR showed the up-regulation of Na+/K+ATPase alphal and betal subunits between POMPs and CEC-like cells. The amplified CEC-like cells expressed Na+/K+ATPase alphal, ZO-1, N-Cadherin and vimentin. Pump function parameters of the CEC-like cells sheetsapproximated those of human donor corneas. The corneas of the CEC-like cells group (experimental group) were much clearer than those of the PAPCM lamellas group (control group) and the mean corneal thickness in the experimentalgroup was significantly less than in the control group5,7,14,21and28days after surgery. Specular microscope and histological analysis confirmed that green fluorescence-positive CEC-like cells formed a monolayer covering the Descemet’s membrane in the experimental group.CONCLUSIONS. CEC-like cells derived from hESCs displayed characteristics of native human CECs. This renewable source of human CECs offers massive cells for further studies of human CECs biological characteristics and potential applications of replacement therapies as substitution for donor CECs in the future.更多还原