【作者】 苗莹；

【导师】 樊廷俊；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2013， 博士

【摘要】 角膜位于眼球的最表面，因直接与外界接触故易受到损伤或感染，一旦受到损伤和发生病变，就会引起角膜混浊，造成视力的减退甚至丧失。角膜病是我国主要的致盲病之一，仅次于白内障，我国现有角膜病盲患者近500万人，而全世界现有角膜病盲患者近6000万人，其中大部分是由于角膜基质异常而引起的，而且还在逐年递增。角膜病尤其是角膜盲不仅给患者个人带来巨大的痛苦，在生活上还需要家人等的照顾，给家庭和社会带来了巨大的经济负担。目前，治疗角膜盲的唯一有效方法就是进行角膜移植，但由于角膜供体的严重短缺，无法满足现有众多角膜病盲患者治疗的需要。近年来，角膜组织工程技术的兴起使体外重建出组织工程人角膜成为可能，而体外重建的组织工程人角膜基质，作为捐献角膜的替代物，已成为众多角膜病盲患者通过角膜移植而复明的唯一希望。组织工程人角膜基质的体外重建急需解决足量种子细胞的来源和理想生物相容性载体支架的制备问题。本实验室通过多年研究，已建立了多个非转染人角膜基质细胞系，初步成功解决了种子细胞的来源问题，并初步建立了脱细胞猪角膜基质制备的技术工艺，本文拟在已有技术工艺的基础上，通过改进和优化利用猪角膜基质制备出脱细胞猪角膜基质，并利用本实验室业已建立的非转染人角膜基质细胞系细胞对其进行生物相容性研究，进而以脱细胞猪角膜基质为载体支架、以非转染人角膜基质细胞为种子细胞，开展组织工程人角膜基质的体外重建研究，并对体外重建所得组织工程人角膜基质的形态结构和功能蛋白表达进行鉴定，最后利用新西兰兔和比格犬的板层角膜基质移植进行生物学功能验证，评估组织工程人角膜基质作为捐献角膜替代物用于临床角膜移植的可行性。本文首先开展了脱细胞猪角膜基质的优化制备研究，采用脱氧胆酸钠-原钒酸钠混合液处理、反复冻融和DNA-RNA酶处理等技术工艺对猪角膜基质进行了脱细胞处理，制备出脱细胞猪角膜基质，在利用HE染色、冰冻切片的荧光染色以及透射电子显微镜技术对其形态结构进行鉴定的基础上，又对其透光性能及其孔隙率等理化性质进行了检测。鉴定结果显示，所得脱细胞猪角膜基质中胶原纤维排列规则、结构均匀而完整，没有明显细胞和核物质的残留，且透光性能好、机械性能好，与正常猪角膜无显著差异，表明本文制备所得脱细胞猪角膜基质符合作为组织工程人角膜基质载体支架的条件和要求，可用于组织工程人角膜基质的体外重建研究。在利用人角膜基质细胞检测脱细胞猪角膜基质的生物相容性之前，本文首先利用生长曲线、染色体标本制做和免疫细胞化学技术对第15代人角膜基质细胞的生长特性、核型、标志蛋白和功能蛋白的表达进行了检测和鉴定。检测和鉴定结果发现，第15代人角膜基质细胞的生长分裂状态良好，群体倍增时间为43.57h，其特征性染色体数目为46条，保持有波形蛋白（人角膜基质细胞标志性蛋白）的阳性表达，并具有细胞连接蛋白间隙连接蛋白-43和整联蛋白β1的阳性表达，以及膜运输载体蛋白——钠钾泵和钙泵及乙醛脱氢酶的阳性表达，证明第15代人角膜基质细胞具有正常人角膜基质细胞的属性和发挥正常细胞功能的潜能，符合作为组织工程人角膜基质种子细胞的条件和要求，可用于组织工程人角膜基质的体外重建研究。在获得脱细胞猪角膜基质和人角膜基质细胞的基础上，本文采用微量注射技术将第15代人角膜基质细胞接种到脱细胞猪角膜基质中，利用MTT法和免疫细胞化学技术对脱细胞猪角膜基质与人角膜基质细胞的生物相容性进行了鉴定研究。结果发现，脱细胞猪角膜基质载体支架的浸提液对第15代人角膜基质细胞没有细胞毒性，而接种至载体支架内的人角膜基质细胞在接种后1~5d内一直保持有波形蛋白以及间隙连接蛋白-43和整联蛋白β1的阳性表达，且在接种后第3d细胞的形态、分布和波形蛋白以及间隙连接蛋白-43和整联蛋白β1的表达已经十分理想，表明脱细胞猪角膜基质与人角膜基质细胞具有良好的生物相容性，可以用于组织工程人角膜基质的体外重建研究。在进行生物相容性研究的基础上，本文以脱细胞猪角膜基质为载体支架、以非转染第15代人角膜基质细胞为种子细胞，开展了组织工程人角膜基质的体外重建研究，并对所得组织工程人角膜基质的形态结构和功能蛋白表达进行鉴定。对于制备所得生物相容性良好的脱细胞猪角膜基质载体支架，利用微量注射技术接种第15代人角膜基质细胞，放置于含有10%胎牛血清-DMEM/F12培养液的24孔培养板孔中在37℃二氧化碳培养箱中体外培养3d，获得体外重建的组织工程人角膜基质。石蜡切片HE染色、免疫细胞化学、透射电镜等的鉴定结果显示，体外重建的组织工程人角膜基质的形态结构与活体人角膜相似，其种子细胞仍保持有波形蛋白、整联蛋白β1、间隙连接蛋白-43、乙醛脱氢酶、钠钾泵和钙泵的1阳性表达，表明体外重建的组织工程人角膜基质与活体人角膜基质不仅形态结构相似，还保持有执行人角膜基质功能的潜能。为了验证体外重建的组织工程人角膜基质是否具有正常的生物学功能，本文又选用新西兰兔和比格犬对组织工程人角膜基质进行了板层角膜基质移植实验，并利用裂隙灯显微镜、角膜测厚仪和眼压计对移植动物角膜的的透明度、角膜厚度和眼压进行了跟踪监测。结果表明，术后20d，新西兰兔术眼角膜保持透明，角膜无感染，无新生血管。利用石蜡切片HE染色、免疫细胞化学、透射电镜等技术对移植兔眼进行了离体鉴定，HE染色结果显示组织工程人角膜基质植片与植床愈合情况良好；荧光显微镜观察结果显示植片上有DiI标记的人角膜基质细胞定位，透射电镜结果显示重建的组织工程人角膜基质中胶原纤维排列紧密规则，人角膜基质细胞伸展其间，并且与支架形成了细胞连接。免疫荧光染色结果显示，标志蛋白-波形蛋白，功能蛋白-间隙连接蛋白-43、整联蛋白β1、钠钾泵、钙泵和乙醛脱氢酶的表达均呈阳性，说明人角膜基质细胞移植入体内后有发挥生物学功能的潜能。比格犬角膜移植实验结果表明，移植早期比格犬术眼角膜角膜轻微水肿，到一个月时，角膜逐渐透明，术后120d，比格犬术眼角膜保持透明，角膜无感染，无新生血管，至今仍在跟踪观察。移植眼角膜厚度和眼压与对照眼相比，无显著差异。上述动物移植结果表明，本文体外重建所得组织工程人角膜基质在植入动物眼上以后能发挥生物学功能，有望作为捐献角膜的替代物用于角膜基质病盲患者的临床角膜移植和治疗，动物移植实验的成功为临床实验研究奠定了基础。综上所述，本文首次利用改进的技术方法制备出脱细胞猪角膜基质，并利用属性和功能蛋白表达正常的非转染人角膜基质细胞对其进行了生物相容性研究，进而以脱细胞猪角膜基质为载体支架、以非转染人角膜基质细胞为种子细胞成功体外重建出了形态结构正常且生物学功能正常的组织工程人角膜基质，有望用作捐献角膜的等效替代物用于角膜基质病盲患者的临床角膜移植和治疗，为众多角膜基质病盲患者重见光明创造了条件。更多还原

【Abstract】 As the frontier and first barrier of the eyeball, cornea is vulnerable and frequentlysubjected to injury. Such injury, when occurred, can result in the maculae cornea andeventually corneal blind. There are approx. five million blind patients in China whilemore than six billion all over the world caused by corneal trauma. Surprisingly, thepopulation is still increasing fast. The only drastic method to solve this problem iscornea transplantation. However, amount of the donated cornea cannot be settled forthe requirement in cornea transplantation for the blind. Tissue-engineered humancorneal stroma, which was invented and rapidly developed in the last decade as asubstitute of human corneal stroma, holds great promise not only for the release ofdonor deficiency but also the sight restoration via graft operation.Plenty of seeders cells and scaffolds possessing ideal bio-compatibility are twoprerequisites to the reconstruction of tissue-engineered human corneal stroma. We haveestablished several non-transfected and non-tumorigenic human corneal stromal celllines that can be utilized as seeder cells in cornea reconstruction. In this study, weplanned to use the acelluar porcine corneal stroma as the scaffold to reconstruct thetissue-engineered corneal stroma after inoculating seeder cells into the acelluar porcinecorneal stroma. A series of animal transplantation and observation were carried outsubsequently. This study provides a new method for large scale scaffold preparationand also offers data for preclinical experiments.The optimisation of acelluar porcinecorneal stroma preparation method is the first milestone in this study. Firstly, weadopted deoxycholic acidsodium salt and sodium orthovanadate mixture to disposefresh porcine corneal stroma. The subsequent repeated freeze-thaw and combinedDNase-RNase digestion removed porcine cells in porcine corneal stroma efficiently.No visible intact cells were detected in porcine corneal stroma after decellularisedprocess via HE and fluoroscent staining. A series of phycial-chemical properties wereperformed. The results demonstrated that after decellularization the collagen fibrils of the acelluar porcine corneal stroma ranged regularly and no sign of disruption ordegradation of collagen fibril were observed, and the physical properties showed thatacelluar porcine corneal stroma was similar with the normal cornea, which could serveas a scaffold for tissue-engineered human corneal stroma.Before the biocompatibility test, human corneal stromal cells at passage15wasexamined by using growth curve, karyotye observation, and immunocytochemistryanalysis. Accordling, the population doubling time of human corneal stromal cells atpassage15is43.57h indicated it keep strong ability to cleavage. Karyotype analysisshowed that human corneal stromal cells have their predominant chromosome numberin46. The immunofluorescence showed that human corneal stromal cells expressed thetheir marker protein vimentin, connection protein i.e. integrin β1and connexin-43andvariousfunctional proteinincluding aldehyde dehydrogenase, Na+-K+-ATPase andCa2+-ATPase positively, which suggested that human corneal stromal cells maitainednormal phenotypes and possessed the potential to produce normal human cornealstroma. Herein we verified the human corneal stromal lines established by us couldserve as seed cell of tissue engineering human corneal stroma reconstructed in vitro.We employed microsinge technology to inoculate human corneal stromal cells atpassage15into the acelluar porcine corneal stroma and test its biocompability byusing MTT and and immunofluorescent stainningce. The result revealed that it was notcytotoxic to the human corneal stromal cells. We also performed immunofluorescentstainning to examine marker protein and connection proteins expression in humancorneal stromal cells1~5d after inoculation, respectively. The expression ofconnexin-43(an intercellular gap junction-associated proteins) and integrin β1(acell-matrix anchoring junction-associated protein) showed that the human cornealstromal cells had the capability to perform their specific biological functions.To study the effect of reconstructed tissue-engineered human corneal stroma invitro, the corneal transplantation onto lab animal was conducted. After20d oftransplantation on New Zealand rabbit, the implant was transparent according to slitlamp examination. The implant and plant bed healed well by HE staining. Cells intissue-engineered human corneal stroma, according to DiI labeling, were originatedfrom theinoculated human corneal stromal cells. The expression of vimentin,connexin-43, integrin β1, Na+/K+-ATPase, Ca2+-ATPase, and aldehyde dehydrogenasewere also visualized using immunofluorescent staining, indicating that the humancorneal stromal cells had the capability to perform their biological functions in the tissue-engineered human corneal stroma.In the early stage of reconstructed tissue-engineered human corneal stroma graftonto the beagles, the implant swelled slightly. The implanted cornea becametransparent gradually1month after graft.After120d of transplantation, the cornea wastransparent, and we still follow the tracks of the transparency. The observation ofimplant transparence is still ongoing. Results from lab animals transplantationdemonstrates that our reconstructed tissue-engineered human corneal stroma canperform normal functions, which prove its utilisation in the therapy for cornealstromal blind as a substitute to donated human cornea. The success in animaltransplantation experiment provides a solid base for further clinical study.In conclusion, we successfully prepared acelluar porcine corneal stroma viadecellularization as a scaffold, reconstructed tissue-engineered human corneal stromawith this scaffold and previously obtained non-transfected, non-tumorigenic humancorneal stromal cells and finally conducted lab animal transplantation. Results provesthe implanted tissue-engineered human corneal stroma performed similar biologicalfunctions as native cornea in lab animals. This study plays a vital role on large scalereconstruction in vitro and preclinical evaluation, which sets the stage for sightrestoration of the blind patients.更多还原