组织工程人角膜内皮的体外重建及其在兔角膜内皮移植中的作用研究
发布时间：2018-09-09 09:01
【摘要】： 完整的人角膜内皮层(human corneal endothelium, HCE)是维持角膜正常厚度和透明度的关键,而角膜透明性是维持眼正常视功能的重要条件。成年后的人角膜内皮细胞(human corneal endothelial cell,HCEC)几乎没有增殖能力,局部细胞损伤后只能依靠邻近细胞扩张和移行来填补缺损区,一旦细胞密度低于维持角膜内皮生理功能之临界密度后就会引起不可逆病变,即角膜内皮盲。我国现有角膜内皮盲患者80余万人,全世界1100余万人,而且每年还在增加。角膜内皮盲患者绝大部分可以通过角膜移植而复明,但由于捐献角膜的数量非常有限,致使绝大部分患者因得不到移植角膜而无法复明。近年来,角膜组织工程的兴起为组织工程人角膜内皮(tissue-engineered corneal endothelium, TE-HCE)的体外重建和患者通过临床角膜移植而重建光明带来了新的希望。为此,本文在进一步对HCEC细胞系分子鉴定的基础上,从该细胞系中筛选出核型正常的单克隆细胞株,建立去上皮层修饰羊膜(mdAM)载体支架的制备与修饰技术,进而以HCEC单克隆细胞株为种子细胞、以mdAM为载体支架,对TE-HCE的体外重建及其动物移植作用进行了研究,旨在建立TE-HCE体外规模化重建的技术工艺条件,获得可长期维持兔角膜透明的TE-HCE,为角膜内皮盲的临床治疗和患者重见光明创造条件。 为了进一步鉴定本研究室所建立的国际上唯一1个非转染、无致瘤性HCEC细胞系的属性与功能,本文利用Western blot对HCEC的标志蛋白、利用免疫荧光对HCEC的细胞连接蛋白、利用实时荧光定量PCR对HCEC的功能蛋白进行了鉴定。Western blot的鉴定结果显示,该细胞系对人Ⅳ型胶原(角膜中内皮细胞的特异性分泌物)和血管内皮生长因子受体-2(FLK-1)有阳性表达,而对人血管性假血友病因子(human von Willebrand Factor,vWF)和角蛋白(keratin)的表达为阴性,表明该细胞系的确具有HCEC细胞系的固有属性；对细胞连接蛋白的免疫荧光检测结果显示,该细胞系具有紧密连接蛋白1(ZO-1)、N-钙粘蛋白(N-cadherin)、间隙连接蛋白-43(connecxin-43)和整联蛋白αv/β5(integrinαv/β5)的阳性表达,表明该细胞系仍具有形成细胞间以及细胞与细胞外基质间细胞连接的潜能；对膜运输蛋白的实时荧光定量PCR检测结果显示,该细胞系具有水孔蛋白(AQP-1)、Na+/K+泵α1肽链、电位依赖性阴离子通道蛋白(hVDAC2和hVDAC3)、氯离子通道蛋白(hCLCN2和hCLCN3)、Na+/HCO3-协同运输蛋白(hSLC4A4)和囊性纤维化跨膜转运调控蛋白(CFTR)的阳性表达,表明该细胞系仍具有发挥HCEC正常水孔蛋白通道、离子通道和泵的跨膜运输潜能。 为了获得可用于TE-HCE体外重建的核型正常的HCEC(?)中子细胞,本文利用20%胎牛血清(FBS)-DMEM/F12培养液,在37℃、5%CO2培养条件下,采用有限稀释法对所建立的HCEC细胞系进行了克隆化实验。对所获得的13个细胞单克隆进行染色体分析的结果显示,有7个单克隆细胞株的染色体数目为46条,并具有正常的二倍体核型。取其中一个单克隆细胞株C3B进行扩增培养,以用作TE-HCE体外重建的种子细胞。 为了获得可用于TE-HCE体外重建的理想载体支架,本文又利用胰蛋白酶倒置消化和信号分子包被法对新鲜羊膜进行了去上皮层处理与修饰。对所得mdAM的检测结果显示,所得mdAM表面平整,没有上皮细胞残留。对mdAM生物相容性的光镜观察、冰冻切片和茜素红染色的检测结果显示,HCEC在mdAM上培养116h便可长成完整的细胞单层,细胞间连接紧密并形成了细胞连接,表明所制备的mdAM可被用作TE-HCE体外重建的理想载体支架。 为了建立TE-HCE体外规模化重建的技术工艺条件,本文以核型正常的C3B单克隆细胞株为种子细胞、以mdAM为载体支架,使用20% FBS-DMEM/F12培养液在37℃、5%CO2的培养条件下进行了TE-HCE的体外重建,并利用光镜观察、冰冻切片、茜素红染色、免疫荧光、扫描电镜和透射电镜技术对重建TE-HCE进行了形态结构鉴定。光镜观察结果显示,TE-HCE种子细胞在载体支架上生长状态良好,启动重建116h后便可长成紧密的细胞单层,其细胞密度高达3611个/mm2；冰冻切片染色结果显示,种子细胞在载体支架上形成了连续的细胞单层；茜素红染色和免疫荧光检测结果显示,种子细胞在载体支架上形成了连接紧密的细胞单层,在细胞间形成了广泛的细胞连接,并具有细胞连接蛋白ZO-1、N-钙粘蛋白、间隙连接蛋白-43和整联蛋白αv/β5的阳性表达；扫描电镜观察结果显示,种子细胞在载体支架上形成了连续的细胞单层,形态为多角形内皮样,细胞间连接紧密；透射电镜观察结果显示,种子细胞在载体支架上形成了连续的细胞单层,其超微结构与正常HCEC相似,且在细胞之间以及细胞与载体支架之间形成了大量的细胞连接。表明所重建的TE-HCE不仅具有正常HCE的形态结构,而且其细胞密度高达3611个/mm2,相当于10-11岁儿童HCE的细胞密度。 为了鉴定体外重建TE-HCE在动物角膜移植中的作用,本文利用带有DiI荧光标记的TE-HCE对撕除内皮层和后弹力层的新西兰兔进行了后板层角膜内皮移植试验,并利用裂隙灯显微镜、荧光显微镜、冰冻切片、茜素红染色、扫描电镜和透射电镜技术对移植角膜的透明度、角膜内皮及角膜的形态结构进行了检测与鉴定。跟踪观察与裂隙灯显微镜检测结果显示,移植后新西兰兔的角膜未出现水肿和排斥等不良反应，其角膜保持透明的时间-已长达280天；而撕除内皮层和后弹力层后仅移植mdAM载体支架以及不行任何移植直接缝合的新西兰兔眼角膜均出现了明显的水肿,其角膜混浊、不透明。角膜内皮面的荧光观察结果显示,移植兔眼角膜的内皮移植区细胞均带有DiI荧光标记,表明其内皮细胞均来源于TE-HCE;茜素红染色结果显示,种子细胞形成了连接紧密的细胞单层,细胞形态几乎全为六角形,并在细胞间形成了广泛的细胞连接,利用网格目微尺进行细胞计数的结果显示,移植新西兰兔右眼角膜内皮移植区的细胞密度约为2307个/mm2；冰冻切片染色结果显示,TE-HCE种子细胞形成了连续的细胞单层,且移植兔眼角膜的厚度与对照兔眼的角膜厚度相近；扫描电镜和透射电镜观察结果显示,角膜移植区的内皮层完整、细胞间嵌合紧密,种子细胞形态绝大多数为六角形,细胞超微结构正常,含有大量的糙面内质网和线粒体,并分泌产生了后弹力层。新西兰兔角膜内皮移植实验的结果表明,所移植TE-HCE形成了形态结构正常的角膜内皮层,并具有长期使移植兔角膜保持透明的功能。 此外,为了模拟角膜内皮盲临床治疗的角膜移植方式,本文还利用角膜内皮细胞刮除法建立了新西兰兔角膜内皮盲模型,并使用此模型进行了TE-HCE的后板层角膜内皮移植试验,利用裂隙灯显微镜对移植角膜的透明度进行了检测。跟踪观察与裂隙灯显微镜检测结果显示,移植TE-HCE后新西兰兔角膜的水肿情况逐渐消失,角膜逐渐恢复透明,术后第30天移植角膜与正常兔角膜的透明度几乎相同,目前移植兔角膜保持透明的时间长达198天。新西兰兔角膜内皮盲模型的移植结果表明,所移植TE-HCE能达到治愈角膜内皮盲的目的,可使移植兔角膜长期保持透明。 综上所述,本文以从HCEC细胞系中筛选出的核型正常HCEC单克隆细胞株为种子细胞、以去上皮层处理和修饰的羊膜为载体支架,体外重建出了形态结构与在体HCE相似的TE-HCE,其移植后在兔角膜内皮面形成了形态结构正常的角膜内皮层,具有使移植兔角膜长期保持透明的功能,并可用于兔角膜内皮盲的治疗。本文成功体外重建的TE-HCE,具有在体行使角膜内皮层的功能,可望作为临床角膜移植的HCE替代物从根本上解决角膜供体材料匮乏的问题,为角膜内皮盲通过TE-HCE移植进行临床治疗和患者重见光明带来了希望,不仅具有重要的理论意义,也将能产生出巨大的经济效益和重大的社会效益。
[Abstract]:Intact human corneal endothelium (HCE) is the key to maintain normal corneal thickness and transparency, and corneal transparency is an important condition to maintain normal visual function. Corneal endothelial blindness (CEB) is an irreversible lesion once the cell density is below the critical density for maintaining the physiological function of corneal endothelium. In recent years, the rise of corneal tissue engineering (TE-HCE) has become the focus of in vitro reconstruction of tissue-engineered corneal endothelium (TE-HCE) and clinical keratoplasty. Therefore, on the basis of further molecular identification of HCEC cell lines, we screened out normal karyotype monoclonal cell lines, established the preparation and modification technology of epithelial-free modified amniotic membrane (mdAM) carrier scaffold, and then used HCEC monoclonal cell lines as seed cells and mdAM as carrier. Body scaffolds were used to study the in vitro reconstruction of TE-HCE and its effect on animal transplantation. The purpose of this study was to establish the technological conditions for large-scale reconstruction of TE-HCE in vitro and to obtain TE-HCE which could maintain the transparency of rabbit cornea for a long time.
In order to further identify the attributes and functions of the only non-transfected, non-tumorigenic HCEC cell line established by our laboratory in the world, the marker proteins of HCEC were identified by Western blot, the connexins of HCEC were identified by immunofluorescence, and the functional proteins of HCEC were identified by real-time quantitative PCR. The results showed that the cell line had positive expression of human collagen type IV (a specific secretion of corneal endothelial cells) and vascular endothelial growth factor receptor-2 (FLK-1), but negative expression of human von Willebrand Factor (vWF) and keratin, suggesting that the cell line did have a HCEC cell line. Immunofluorescence assay of connexin-1, N-cadherin, connexin-43 and integrin alpha v/beta 5 showed that the cell line still had positive expression of tight junction protein-1 (ZO-1), N-cadherin, connexin-43 and integrin alpha v/beta 5, indicating that the cell line still had intercellular formation and cell-to-cell relationship. The results of real-time fluorescence quantitative PCR showed that the cell line had aquaporin (AQP-1), Na +/K + pump alpha 1 peptide chain, potential-dependent anion channel protein (hVDAC2 and hVDAC3), chloride channel protein (hCLCN2 and hCLCN3), Na +/HCO3-co-transport protein (hSLC4A4) and cystic fibers. The positive expression of chemical transmembrane transport regulator (CFTR) indicates that the cell line still has the potential of transmembrane transport of HCEC through normal aquaporin channels, ion channels and pumps.
In order to obtain HCEC (?) neutron cells with normal karyotype which can be used for TE-HCE reconstruction in vitro, the cloning experiments of HCEC cell lines were carried out by using 20% fetal bovine serum (FBS) -DMEM/F12 culture medium at 37 C and 5% CO2 culture conditions. The results showed that there were 46 chromosomes in 7 monoclonal cell lines with normal diploid karyotype. One of them, C3B, was amplified and cultured to be used as seed cells for TE-HCE reconstruction in vitro.
In order to obtain an ideal scaffold for TE-HCE reconstruction in vitro, the fresh amniotic membrane was Deepithelialized and modified by trypsin inversion digestion and signal molecule coating. The results of frozen sections and alizarin red staining showed that HCEC could grow into intact cell monolayer after 116 hours of culture on mdAM. The tight junction between cells and the formation of cell junction showed that the prepared mdAM could be used as an ideal carrier for TE-HCE reconstruction in vitro.
In order to establish the technological conditions for large-scale reconstruction of TE-HCE in vitro, we used C3B monoclonal cell line with normal karyotype as seed cells, mdAM as carrier scaffold, 20% FBS-DMEM/F12 medium at 37 C and 5% CO2 as culture medium to reconstruct TE-HCE in vitro. Light microscopy, frozen section, alizarin red staining, immunofluorescence were used. Light, scanning electron microscopy and transmission electron microscopy were used to identify the morphology and structure of reconstructed TE-HCE. The results of light microscopy showed that the seed cells of TE-HCE grew well on the carrier scaffold. After 116 hours of initiation and reconstruction, they could grow into compact cell monolayer with a density of 3611 cells per mm2. Alizarin red staining and immunofluorescence assay showed that seed cells formed tight-junction monolayers on the carrier scaffold, and formed extensive cell junctions between cells, with connexin ZO-1, N-cadherin, connexin 43 and integrin alpha v/beta 5. The results of scanning electron microscopy showed that the seed cells formed a continuous monolayer on the carrier scaffold with polygonal endothelium-like morphology and tight intercellular junction; transmission electron microscopy showed that the seed cells formed a continuous monolayer on the carrier scaffold, and its ultrastructure was similar to that of normal HCEC. The reconstructed TE-HCE not only has normal HCE morphology, but also has a high cell density of 3611 cells/mm2, which is equivalent to the cell density of HCE in 10-11 years old children.
In order to identify the role of reconstructed TE-HCE in animal corneal transplantation, the posterior lamellar corneal endothelial graft of New Zealand rabbits with endothelium and posterior elastic layer were performed by using TE-HCE labeled with DiI fluorescence. Slit lamp microscopy, fluorescence microscopy, frozen section, alizarin red staining, scanning electron microscopy and transmission electron microscopy were used. The results of follow-up observation and slit lamp microscopy showed that the cornea of New Zealand rabbits did not appear edema and rejection, and the cornea remained transparent for 280 days, while the endothelium and the elastic layer were removed. After transplantation, the corneas of New Zealand rabbits were evidently edematous, opaque and opaque. The fluorescence observation on the corneal endothelial surface showed that the cells in the corneal endothelial graft area of transplanted rabbits were labeled with DiI fluorescence, indicating that the endothelial cells were derived from TE-HCE. The results of red staining showed that the seeding cells formed a tightly connected monolayer, almost all cells were hexagonal in shape, and formed extensive cell junctions between cells. Cell counting with mesh micrometer showed that the cell density of corneal endothelial graft area in the right eye of New Zealand rabbits was about 2307/mm2. The results of staining showed that the seeding cells of TE-HCE formed a continuous monolayer, and the corneal thickness of the transplanted rabbits was similar to that of the control rabbits. The results of corneal endothelial transplantation in New Zealand rabbits showed that the transplanted TE-HCE formed a normal corneal endothelium with long-term function of keeping the cornea transparent.
In addition, in order to simulate the clinical treatment of corneal endothelial blindness, the corneal endothelial cell curettage method was used to establish a corneal endothelial blindness model in New Zealand rabbits. The corneal edema of New Zealand rabbits disappeared gradually after TE-HCE transplantation, and the cornea became transparent gradually. The transparency of cornea transplantation was almost the same as that of normal rabbits on the 30th day after transplantation. The cornea of New Zealand rabbits remained transparent for 198 days. The results showed that the transplanted TE-HCE could cure corneal endothelial blindness and keep the cornea transparent for a long time.
To sum up, we reconstructed TE-HCE with similar morphology and structure to HCE in vivo by using normal karyotype HCEC monoclonal cell lines screened from HCEC cell lines as seed cells and amniotic membrane treated and modified by epithelium removal as carrier scaffolds. After transplantation, the corneal endothelium with normal morphology and structure was formed on the corneal endothelium of rabbits. TE-HCE, successfully reconstructed in vitro, can perform the function of corneal endothelium in vivo. It is expected that TE-HCE can be used as an alternative to HCE in clinical corneal transplantation to solve the problem of corneal donor material shortage fundamentally and to transplant corneal endothelium blindness through TE-HCE. The clinical treatment and the patient's return to light bring hope, not only has important theoretical significance, but also will produce enormous economic and social benefits.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R779.65
本文编号：2231924
[Abstract]:Intact human corneal endothelium (HCE) is the key to maintain normal corneal thickness and transparency, and corneal transparency is an important condition to maintain normal visual function. Corneal endothelial blindness (CEB) is an irreversible lesion once the cell density is below the critical density for maintaining the physiological function of corneal endothelium. In recent years, the rise of corneal tissue engineering (TE-HCE) has become the focus of in vitro reconstruction of tissue-engineered corneal endothelium (TE-HCE) and clinical keratoplasty. Therefore, on the basis of further molecular identification of HCEC cell lines, we screened out normal karyotype monoclonal cell lines, established the preparation and modification technology of epithelial-free modified amniotic membrane (mdAM) carrier scaffold, and then used HCEC monoclonal cell lines as seed cells and mdAM as carrier. Body scaffolds were used to study the in vitro reconstruction of TE-HCE and its effect on animal transplantation. The purpose of this study was to establish the technological conditions for large-scale reconstruction of TE-HCE in vitro and to obtain TE-HCE which could maintain the transparency of rabbit cornea for a long time.
In order to further identify the attributes and functions of the only non-transfected, non-tumorigenic HCEC cell line established by our laboratory in the world, the marker proteins of HCEC were identified by Western blot, the connexins of HCEC were identified by immunofluorescence, and the functional proteins of HCEC were identified by real-time quantitative PCR. The results showed that the cell line had positive expression of human collagen type IV (a specific secretion of corneal endothelial cells) and vascular endothelial growth factor receptor-2 (FLK-1), but negative expression of human von Willebrand Factor (vWF) and keratin, suggesting that the cell line did have a HCEC cell line. Immunofluorescence assay of connexin-1, N-cadherin, connexin-43 and integrin alpha v/beta 5 showed that the cell line still had positive expression of tight junction protein-1 (ZO-1), N-cadherin, connexin-43 and integrin alpha v/beta 5, indicating that the cell line still had intercellular formation and cell-to-cell relationship. The results of real-time fluorescence quantitative PCR showed that the cell line had aquaporin (AQP-1), Na +/K + pump alpha 1 peptide chain, potential-dependent anion channel protein (hVDAC2 and hVDAC3), chloride channel protein (hCLCN2 and hCLCN3), Na +/HCO3-co-transport protein (hSLC4A4) and cystic fibers. The positive expression of chemical transmembrane transport regulator (CFTR) indicates that the cell line still has the potential of transmembrane transport of HCEC through normal aquaporin channels, ion channels and pumps.
In order to obtain HCEC (?) neutron cells with normal karyotype which can be used for TE-HCE reconstruction in vitro, the cloning experiments of HCEC cell lines were carried out by using 20% fetal bovine serum (FBS) -DMEM/F12 culture medium at 37 C and 5% CO2 culture conditions. The results showed that there were 46 chromosomes in 7 monoclonal cell lines with normal diploid karyotype. One of them, C3B, was amplified and cultured to be used as seed cells for TE-HCE reconstruction in vitro.
In order to obtain an ideal scaffold for TE-HCE reconstruction in vitro, the fresh amniotic membrane was Deepithelialized and modified by trypsin inversion digestion and signal molecule coating. The results of frozen sections and alizarin red staining showed that HCEC could grow into intact cell monolayer after 116 hours of culture on mdAM. The tight junction between cells and the formation of cell junction showed that the prepared mdAM could be used as an ideal carrier for TE-HCE reconstruction in vitro.
In order to establish the technological conditions for large-scale reconstruction of TE-HCE in vitro, we used C3B monoclonal cell line with normal karyotype as seed cells, mdAM as carrier scaffold, 20% FBS-DMEM/F12 medium at 37 C and 5% CO2 as culture medium to reconstruct TE-HCE in vitro. Light microscopy, frozen section, alizarin red staining, immunofluorescence were used. Light, scanning electron microscopy and transmission electron microscopy were used to identify the morphology and structure of reconstructed TE-HCE. The results of light microscopy showed that the seed cells of TE-HCE grew well on the carrier scaffold. After 116 hours of initiation and reconstruction, they could grow into compact cell monolayer with a density of 3611 cells per mm2. Alizarin red staining and immunofluorescence assay showed that seed cells formed tight-junction monolayers on the carrier scaffold, and formed extensive cell junctions between cells, with connexin ZO-1, N-cadherin, connexin 43 and integrin alpha v/beta 5. The results of scanning electron microscopy showed that the seed cells formed a continuous monolayer on the carrier scaffold with polygonal endothelium-like morphology and tight intercellular junction; transmission electron microscopy showed that the seed cells formed a continuous monolayer on the carrier scaffold, and its ultrastructure was similar to that of normal HCEC. The reconstructed TE-HCE not only has normal HCE morphology, but also has a high cell density of 3611 cells/mm2, which is equivalent to the cell density of HCE in 10-11 years old children.
In order to identify the role of reconstructed TE-HCE in animal corneal transplantation, the posterior lamellar corneal endothelial graft of New Zealand rabbits with endothelium and posterior elastic layer were performed by using TE-HCE labeled with DiI fluorescence. Slit lamp microscopy, fluorescence microscopy, frozen section, alizarin red staining, scanning electron microscopy and transmission electron microscopy were used. The results of follow-up observation and slit lamp microscopy showed that the cornea of New Zealand rabbits did not appear edema and rejection, and the cornea remained transparent for 280 days, while the endothelium and the elastic layer were removed. After transplantation, the corneas of New Zealand rabbits were evidently edematous, opaque and opaque. The fluorescence observation on the corneal endothelial surface showed that the cells in the corneal endothelial graft area of transplanted rabbits were labeled with DiI fluorescence, indicating that the endothelial cells were derived from TE-HCE. The results of red staining showed that the seeding cells formed a tightly connected monolayer, almost all cells were hexagonal in shape, and formed extensive cell junctions between cells. Cell counting with mesh micrometer showed that the cell density of corneal endothelial graft area in the right eye of New Zealand rabbits was about 2307/mm2. The results of staining showed that the seeding cells of TE-HCE formed a continuous monolayer, and the corneal thickness of the transplanted rabbits was similar to that of the control rabbits. The results of corneal endothelial transplantation in New Zealand rabbits showed that the transplanted TE-HCE formed a normal corneal endothelium with long-term function of keeping the cornea transparent.
In addition, in order to simulate the clinical treatment of corneal endothelial blindness, the corneal endothelial cell curettage method was used to establish a corneal endothelial blindness model in New Zealand rabbits. The corneal edema of New Zealand rabbits disappeared gradually after TE-HCE transplantation, and the cornea became transparent gradually. The transparency of cornea transplantation was almost the same as that of normal rabbits on the 30th day after transplantation. The cornea of New Zealand rabbits remained transparent for 198 days. The results showed that the transplanted TE-HCE could cure corneal endothelial blindness and keep the cornea transparent for a long time.
To sum up, we reconstructed TE-HCE with similar morphology and structure to HCE in vivo by using normal karyotype HCEC monoclonal cell lines screened from HCEC cell lines as seed cells and amniotic membrane treated and modified by epithelium removal as carrier scaffolds. After transplantation, the corneal endothelium with normal morphology and structure was formed on the corneal endothelium of rabbits. TE-HCE, successfully reconstructed in vitro, can perform the function of corneal endothelium in vivo. It is expected that TE-HCE can be used as an alternative to HCE in clinical corneal transplantation to solve the problem of corneal donor material shortage fundamentally and to transplant corneal endothelium blindness through TE-HCE. The clinical treatment and the patient's return to light bring hope, not only has important theoretical significance, but also will produce enormous economic and social benefits.
【学位授予单位】：中国海洋大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R779.65
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