【摘要】： 完整的人角膜內皮層(human corneal endothelium, HCE)是維持角膜正常厚度和透明度的關鍵,而角膜透明性是維持眼正常視功能的重要條件。成年后的人角膜內皮細胞(human corneal endothelial cell,HCEC)幾乎沒有增殖能力,局部細胞損傷后只能依靠鄰近細胞擴張和移行來填補缺損區(qū),一旦細胞密度低于維持角膜內皮生理功能之臨界密度后就會引起不可逆病變,即角膜內皮盲。我國現(xiàn)有角膜內皮盲患者80余萬人,全世界1100余萬人,而且每年還在增加。角膜內皮盲患者絕大部分可以通過角膜移植而復明,但由于捐獻角膜的數(shù)量非常有限,致使絕大部分患者因得不到移植角膜而無法復明。近年來,角膜組織工程的興起為組織工程人角膜內皮(tissue-engineered corneal endothelium, TE-HCE)的體外重建和患者通過臨床角膜移植而重建光明帶來了新的希望。為此,本文在進一步對HCEC細胞系分子鑒定的基礎上,從該細胞系中篩選出核型正常的單克隆細胞株,建立去上皮層修飾羊膜(mdAM)載體支架的制備與修飾技術,進而以HCEC單克隆細胞株為種子細胞、以mdAM為載體支架,對TE-HCE的體外重建及其動物移植作用進行了研究,旨在建立TE-HCE體外規(guī)�；亟ǖ募夹g工藝條件,獲得可長期維持兔角膜透明的TE-HCE,為角膜內皮盲的臨床治療和患者重見光明創(chuàng)造條件。 為了進一步鑒定本研究室所建立的國際上唯一1個非轉染、無致瘤性HCEC細胞系的屬性與功能,本文利用Western blot對HCEC的標志蛋白、利用免疫熒光對HCEC的細胞連接蛋白、利用實時熒光定量PCR對HCEC的功能蛋白進行了鑒定。Western blot的鑒定結果顯示,該細胞系對人Ⅳ型膠原(角膜中內皮細胞的特異性分泌物)和血管內皮生長因子受體-2(FLK-1)有陽性表達,而對人血管性假血友病因子(human von Willebrand Factor,vWF)和角蛋白(keratin)的表達為陰性,表明該細胞系的確具有HCEC細胞系的固有屬性；對細胞連接蛋白的免疫熒光檢測結果顯示,該細胞系具有緊密連接蛋白1(ZO-1)、N-鈣粘蛋白(N-cadherin)、間隙連接蛋白-43(connecxin-43)和整聯(lián)蛋白αv/β5(integrinαv/β5)的陽性表達,表明該細胞系仍具有形成細胞間以及細胞與細胞外基質間細胞連接的潛能；對膜運輸?shù)鞍椎膶崟r熒光定量PCR檢測結果顯示,該細胞系具有水孔蛋白(AQP-1)、Na+/K+泵α1肽鏈、電位依賴性陰離子通道蛋白(hVDAC2和hVDAC3)、氯離子通道蛋白(hCLCN2和hCLCN3)、Na+/HCO3-協(xié)同運輸?shù)鞍?hSLC4A4)和囊性纖維化跨膜轉運調控蛋白(CFTR)的陽性表達,表明該細胞系仍具有發(fā)揮HCEC正常水孔蛋白通道、離子通道和泵的跨膜運輸潛能。 為了獲得可用于TE-HCE體外重建的核型正常的HCEC(?)中子細胞,本文利用20%胎牛血清(FBS)-DMEM/F12培養(yǎng)液,在37℃、5%CO2培養(yǎng)條件下,采用有限稀釋法對所建立的HCEC細胞系進行了克隆化實驗。對所獲得的13個細胞單克隆進行染色體分析的結果顯示,有7個單克隆細胞株的染色體數(shù)目為46條,并具有正常的二倍體核型。取其中一個單克隆細胞株C3B進行擴增培養(yǎng),以用作TE-HCE體外重建的種子細胞。 為了獲得可用于TE-HCE體外重建的理想載體支架,本文又利用胰蛋白酶倒置消化和信號分子包被法對新鮮羊膜進行了去上皮層處理與修飾。對所得mdAM的檢測結果顯示,所得mdAM表面平整,沒有上皮細胞殘留。對mdAM生物相容性的光鏡觀察、冰凍切片和茜素紅染色的檢測結果顯示,HCEC在mdAM上培養(yǎng)116h便可長成完整的細胞單層,細胞間連接緊密并形成了細胞連接,表明所制備的mdAM可被用作TE-HCE體外重建的理想載體支架。 為了建立TE-HCE體外規(guī)�；亟ǖ募夹g工藝條件,本文以核型正常的C3B單克隆細胞株為種子細胞、以mdAM為載體支架,使用20% FBS-DMEM/F12培養(yǎng)液在37℃、5%CO2的培養(yǎng)條件下進行了TE-HCE的體外重建,并利用光鏡觀察、冰凍切片、茜素紅染色、免疫熒光、掃描電鏡和透射電鏡技術對重建TE-HCE進行了形態(tài)結構鑒定。光鏡觀察結果顯示,TE-HCE種子細胞在載體支架上生長狀態(tài)良好,啟動重建116h后便可長成緊密的細胞單層,其細胞密度高達3611個/mm2；冰凍切片染色結果顯示,種子細胞在載體支架上形成了連續(xù)的細胞單層；茜素紅染色和免疫熒光檢測結果顯示,種子細胞在載體支架上形成了連接緊密的細胞單層,在細胞間形成了廣泛的細胞連接,并具有細胞連接蛋白ZO-1、N-鈣粘蛋白、間隙連接蛋白-43和整聯(lián)蛋白αv/β5的陽性表達；掃描電鏡觀察結果顯示,種子細胞在載體支架上形成了連續(xù)的細胞單層,形態(tài)為多角形內皮樣,細胞間連接緊密；透射電鏡觀察結果顯示,種子細胞在載體支架上形成了連續(xù)的細胞單層,其超微結構與正常HCEC相似,且在細胞之間以及細胞與載體支架之間形成了大量的細胞連接。表明所重建的TE-HCE不僅具有正常HCE的形態(tài)結構,而且其細胞密度高達3611個/mm2,相當于10-11歲兒童HCE的細胞密度。 為了鑒定體外重建TE-HCE在動物角膜移植中的作用,本文利用帶有DiI熒光標記的TE-HCE對撕除內皮層和后彈力層的新西蘭兔進行了后板層角膜內皮移植試驗,并利用裂隙燈顯微鏡、熒光顯微鏡、冰凍切片、茜素紅染色、掃描電鏡和透射電鏡技術對移植角膜的透明度、角膜內皮及角膜的形態(tài)結構進行了檢測與鑒定。跟蹤觀察與裂隙燈顯微鏡檢測結果顯示,移植后新西蘭兔的角膜未出現(xiàn)水腫和排斥等不良反應，其角膜保持透明的時間-已長達280天；而撕除內皮層和后彈力層后僅移植mdAM載體支架以及不行任何移植直接縫合的新西蘭兔眼角膜均出現(xiàn)了明顯的水腫,其角膜混濁、不透明。角膜內皮面的熒光觀察結果顯示,移植兔眼角膜的內皮移植區(qū)細胞均帶有DiI熒光標記,表明其內皮細胞均來源于TE-HCE;茜素紅染色結果顯示,種子細胞形成了連接緊密的細胞單層,細胞形態(tài)幾乎全為六角形,并在細胞間形成了廣泛的細胞連接,利用網(wǎng)格目微尺進行細胞計數(shù)的結果顯示,移植新西蘭兔右眼角膜內皮移植區(qū)的細胞密度約為2307個/mm2；冰凍切片染色結果顯示,TE-HCE種子細胞形成了連續(xù)的細胞單層,且移植兔眼角膜的厚度與對照兔眼的角膜厚度相近；掃描電鏡和透射電鏡觀察結果顯示,角膜移植區(qū)的內皮層完整、細胞間嵌合緊密,種子細胞形態(tài)絕大多數(shù)為六角形,細胞超微結構正常,含有大量的糙面內質網(wǎng)和線粒體,并分泌產生了后彈力層。新西蘭兔角膜內皮移植實驗的結果表明,所移植TE-HCE形成了形態(tài)結構正常的角膜內皮層,并具有長期使移植兔角膜保持透明的功能。 此外,為了模擬角膜內皮盲臨床治療的角膜移植方式,本文還利用角膜內皮細胞刮除法建立了新西蘭兔角膜內皮盲模型,并使用此模型進行了TE-HCE的后板層角膜內皮移植試驗,利用裂隙燈顯微鏡對移植角膜的透明度進行了檢測。跟蹤觀察與裂隙燈顯微鏡檢測結果顯示,移植TE-HCE后新西蘭兔角膜的水腫情況逐漸消失,角膜逐漸恢復透明,術后第30天移植角膜與正常兔角膜的透明度幾乎相同,目前移植兔角膜保持透明的時間長達198天。新西蘭兔角膜內皮盲模型的移植結果表明,所移植TE-HCE能達到治愈角膜內皮盲的目的,可使移植兔角膜長期保持透明。 綜上所述,本文以從HCEC細胞系中篩選出的核型正常HCEC單克隆細胞株為種子細胞、以去上皮層處理和修飾的羊膜為載體支架,體外重建出了形態(tài)結構與在體HCE相似的TE-HCE,其移植后在兔角膜內皮面形成了形態(tài)結構正常的角膜內皮層,具有使移植兔角膜長期保持透明的功能,并可用于兔角膜內皮盲的治療。本文成功體外重建的TE-HCE,具有在體行使角膜內皮層的功能,可望作為臨床角膜移植的HCE替代物從根本上解決角膜供體材料匱乏的問題,為角膜內皮盲通過TE-HCE移植進行臨床治療和患者重見光明帶來了希望,不僅具有重要的理論意義,也將能產生出巨大的經(jīng)濟效益和重大的社會效益。
[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.
【學位授予單位】：中國海洋大學
【學位級別】：博士
【學位授予年份】：2010
【分類號】：R779.65

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