组织工程人角膜上皮的体外重建、鉴定及其在新西兰兔角膜上皮移植中的作用研究

发布时间：2018-04-28 00:55

本文选题：人角膜上皮种子细胞 + 去上皮层羊膜　；参考：《中国海洋大学》2012年博士论文

【摘要】：人角膜上皮（Human Corneal Epithelium，HCEP）位于角膜最前端，由多层细胞构成，其组织学结构的完整性对于维持角膜的生理功能至关重要。人角膜上皮细胞（Human Corneal Epithelial Cell，HCEPC）是角膜抵御外来致病因子侵害的第一道重要防线，不仅能维持角膜的透明性，还能吸收氧气和营养为角膜供氧/养。HCEP具有自我更新能力，其完整性和更新能力取决于位于角膜缘基底部的角膜缘干细胞（LimbalStem Cell，LSC）的不断增殖与分化。眼球表面的烧伤、灼伤、严重机械创伤、病原体感染、角膜缘部位冷冻治疗、接触镜的长期佩戴以及眼瘢痕性类天疱疮等各种原因均可导致LSC损伤或功能障碍。角膜缘干细胞缺陷症（Limbal Stem Cell Deficiency，LSCD）表现为LSC增殖能力丧失，导致持续性角膜上皮缺损、角膜缘屏障功能下降，致使结膜上皮侵入角膜、新生血管形成，进而影响角膜的光学特性，导致视力受损或致盲。在以前的眼表重建治疗中，一般采用角膜上皮成形术以及自体或异体角膜缘移植的方法，但因供体角膜来源严重不足以及术后排斥反应发生率较高，从而限制了其在临床中的广泛应用。近年来，角膜组织工程的兴起为组织工程人角膜上皮（Tissue-engineered Human Corneal Epithelium，TE-HCEP）的体外重建以及患者通过临床角膜上皮移植而重见光明带来了希望。TE-HCEP体外重建的关键要素包括结构功能正常的HCEPC种子细胞的大量获得和生物相容性理想的载体支架的制备。在种子细胞方面，由于自体LSC来源与数量的限制，而连续性HCEPC细胞系因能提供出大量细胞故被认为是TE-HCEP规模化体外重建的种子细胞的有效来源。在业已建立的HCEPC细胞系中，癌基因转染的永生化HCEPC细胞系因具有潜在的致瘤性无法作为种子细胞用于TE-HCEP的体外重建尤其是临床移植，而非转染HCEPC细胞系因能为理论研究和TE-HCEP提供出足量的正常细胞，故被认为是TE-HCEP种子细胞的理想来源。近年来，国内学者成功建立了非转染的连续性人角膜缘上皮细胞系，但至今仍未见其用于TE-HCEP体外重建的研究报道。在载体支架方面，羊膜（Amniotic Membrane，AM）作为一种天然高分子生物材料，为半透明且抗原性低的薄片组织，其基质中含有大量的生长因子和蛋白酶抑制因子以及抗炎症、抗成纤维化、抗菌、抗血管生成等的相关蛋白，与HCEPC具有理想的生物相容性并能促进HCEPC的细胞分化，已被广泛应用于眼表重建，因此被认为是TE-HCEP的理想载体支架之一。本文在本实验室自主建立的非转染HCEPC细胞系的基础上，对此细胞系的属性和致瘤性进行进一步鉴定，首次以该细胞为种子细胞建、以去上皮层羊膜（denuded AM，dAM）为载体支架，开展TE-HCEP的体外重建研究，并利用LSCD新西兰兔模型对TE-HCEP在角膜上皮移植中的作用进行鉴定，旨在建立TE-HCEP体外重建的技术工艺条件，获得可长期维持兔角膜透明的TE-HCEP，为TE-HCEP替代捐献角膜用于角膜上皮异常疾病的临床治疗创造条件。为了进一步鉴定非转染HCEPC细胞系的属性、功能蛋白的表达及潜在致瘤性，本文对第80代HCEPC进行了复苏、扩增培养和鉴定。属性检测结果显示，复苏的第80代HCEPC透明度高，形态饱满，呈圆形或卵圆形，镶嵌排列呈铺路状，细胞群体倍增时间为40.75h，生长状态良好，分裂旺盛，特征性染色体数目仍为2n=46。功能蛋白的表达检测结果显示，该细胞能够表达HCEP特异性标志蛋白——角蛋白（keratin）3和12，以及紧密连接蛋白ZO-1、E-钙粘蛋白、间隙连接蛋白-43、整联蛋白β1，表明该细胞仍具有角膜上皮的表型，且具有形成完整HCEP结构的潜能。致瘤性检验结果显示，该细胞无致瘤性。因此，该细胞为TE-HCEP体外重建提供了种子细胞。为了获得TE-HCEP体外重建的理想载体支架，本文以新鲜AM为材料，采用胰酶倒置消化法对其进行去上皮层处理，再使用Ⅳ型胶原进行包被，并通过接种HCEPC研究dAM的生物相容性。通过光镜、电镜以及石蜡切片HE染色等方法对处理后的AM以及HCEPC在经包被的dAM上的生长情况进行了观察和鉴定，结果显示利用胰酶于37℃倒置消化20min，再经Ⅳ型胶原包被后获得的dAM表面平整，无上皮细胞残留，且有基底膜结构；dAM对HCEPC具有理想的生物相容性，且光学性能良好。因此，dAM能够作为理想的载体支架进行TE-HCEP的体外重建研究。为了建立TE-HCEP体外规模化重建的技术工艺条件，本文以生长状态良好的HCEPC为种子细胞，以dAM为载体支架，采用气-液界面培养法体外重建TE-HCEP。利用石蜡切片HE染色、免疫组织化学检测及电镜观察等方法对重建的TE-HCEP形态结构、潜在功能等方面进行了鉴定。结果显示TE-HCEP经气-液界面法培养5d后即可形成6~7层的复层结构，结构与正常HCEP结构最为相近，且光学性能良好，表层的HCEPC平整呈卵圆形，且形成了丰富的微绒毛结构。TE-HCEP仍具有HCEP属性以及形成细胞间、细胞与dAM间连接的潜能，超微结构与正常的角膜上皮层结构相近。因此该方法能够获得理想的TE-HCEP，有望在将来进行规模化生产。为了研究体外重建的TE-HCEP在新西兰兔模型角膜上皮移植中的作用，本文利用DiI标记的第80代HCEPC在经包被的dAM上进行体外重建。根据改良的三种方法分别制作了LSCD新西兰兔模型，并利用TE-HCEP分别对其进行角膜上皮移植。利用角膜测厚、裂隙灯显微镜观察、印迹细胞学检查等方法跟踪记录了创伤以及移植后兔模型在体角膜的厚度、透明度、上皮完整性和新生血管情况；利用石蜡切片HE染色、免疫组织化学、电镜观察等方法进行TE-HCEP角膜上皮移植效果的离体检测。结果显示利用碱烧伤、机械创伤和联合法均能获得LSCD的新西兰兔模型；利用TE-HCEP对碱烧伤的LSCD模型进行角膜上皮移植，，术后80d角膜仍为白瓷色，有新生血管；利用TE-HCEP对机械创伤获得的LSCD模型进行角膜上皮移植，术后第12d角膜开始透明，厚度明显下降，新生血管减少，第25d角膜厚度恢复到正常眼水平，且能够长时间保持较透明状态。术后120d的离体检测结果显示角膜上皮细胞来源于体外重建的TE-HCEP，约4~5层，角蛋白3表达呈阳性，上皮细胞之间存在紧密连接结构，与对照组存在显著性差异；利用TE-HCEP对联合法获得的LSCD模型进行角膜上皮移植，术后第12d角膜厚度明显下降，第49d角膜新生血管明显减少，第70d角膜厚度恢复到正常眼水平，角膜长时间保持较透明状态。术后147d的离体检测结果显示角膜上皮细胞来源于TE-HCEP，约3~4层，角蛋白3表达呈阳性，上皮细胞之间存在紧密连接结构并有基底膜结构，与对照组存在显著性差异。新西兰兔角膜上皮移植实验的结果表明，所移植TE-HCEP形成了形态结构较正常的角膜上皮层，新生血管明显减少，并能够使角膜长时间保持较透明状态。综上所述，本文首次以非转染、无致瘤性的第80代HCEPC为种子细胞，以dAM为载体支架，体外重建出了形态结构和潜在功能与正常HCEP相近的TE-HCEP，其移植后能够长时间保持LSCD兔角膜较透明状态。因此，该TE-HCEP有望作为角膜上皮的替代物从根本上解决角膜供体材料严重不足的现状，为角膜上皮异常疾病的患者带来重见光明的希望，这不仅具有重要的理论意义，而且能产生巨大的社会效益和经济效益。
[Abstract]:Human corneal epithelial cells ( HCEP ) have been used in vitro reconstruction of corneal epithelium ( TE - HCEP ) . Amniotic Membrane ( AM ) is a kind of natural high molecular biological material , which is a semi - transparent and low - antigenicity thin - sheet tissue . It contains a large amount of growth factor and protease inhibitor , as well as anti - inflammation , anti - fibrosis , antibacterial , anti - angiogenesis and other related proteins . It has been widely used in vitro reconstruction of TE - HCEP . The aim of this paper is to establish the TE - HCEP in vitro reconstruction of TE - HCEP .

In order to further identify the properties of non - transfected HCEPC cell lines , the expression of functional protein and the potential oncogenicity of HCEPC cell lines were studied .

In order to obtain the ideal carrier scaffold for the in vitro reconstruction of TE - HCEP , the growth of AM and HCEPC was investigated and identified by trypan inverted digestion with fresh AM as material . The results showed that the growth of AM and HCEPC was studied by light microscope , electron microscope and paraffin section HE staining .
dAM has ideal biocompatibility and good optical performance for HCEPC . Therefore , dAM can be used as an ideal carrier for in vitro reconstruction of TE - HCEP .

In order to establish the technological process conditions for the in vitro large - scale reconstruction of TE - HCEP , the recombinant TE - HCEP was reconstructed in vitro by using HCEPC as seed cell and dAM as carrier . The results showed that the structure of TE - HCEP was similar to normal HCEP structure , and its structure was similar to that of normal HCEP structure . The structure of TE - HCEP was similar to normal HCEP structure .

In order to study the role of TE - HCEP reconstructed in vitro in rabbit model corneal epithelium transplantation in New Zealand , a novel rabbit model of LSCD New Zealand rabbit model was constructed by using DiI - labeled 80 - generation HCEPC in vitro . The corneal epithelium transplantation was performed by using TE - HCEP .
The ex vivo detection of TE - HCEP corneal epithelium transplantation was carried out by means of HE staining , immunohistochemistry and electron microscopy . The results showed that the new Zealand rabbit model with LSCD was obtained by alkali burn , mechanical trauma and combined method .
The corneal epithelium transplantation was performed on the LSCD model of alkali burn by TE - HCEP .
The corneal epithelium transplantation was performed on the LSCD model obtained by mechanical trauma by TE - HCEP . The corneal epithelial cells began to be transparent at the 12th day after the operation , the thickness of the corneal epithelium decreased , the thickness of the corneal epithelium decreased , the thickness of the corneal epithelium was reduced to the normal eye level for a long time , and the corneal epithelial cells were able to stay in a relatively transparent state for a long time .
Corneal epithelium transplantation was performed by using the LSCD model obtained by using TE - HCEP for corneal epithelium transplantation . The corneal thickness of corneal epithelium decreased significantly after operation . The corneal thickness of cornea epithelium was decreased significantly . The corneal epithelial cells were positive in cornea epithelium . There was a significant difference between epithelial cells . The results of corneal epithelium transplantation in New Zealand rabbits showed that the transplanted TE - HCEP formed a normal epithelium layer with a normal morphology , and the neovascularization was significantly reduced and the cornea was maintained in a transparent state for a long time .

Therefore , the TE - HCEP is expected to be used as a substitute for corneal epithelium to solve the serious deficiency of corneal donor material , which not only has important theoretical significance , but also can produce great social benefit and economic benefit .

【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R779.65

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