组织工程化角膜上皮与内皮的构建和移植
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摘要
角膜作为眼球光学系统的重要组成部分，其上皮的完整性和透明度是良好视力的基
本要求，临床上常见各种原因所引起的角膜缘干细胞及角膜上皮的损伤和缺失，使患者
视力下降或致盲。上世纪九十年代以来，随着细胞培养技术和组织工程技术的兴起，组
织工程化人工角膜、角膜上皮、内皮：基质细胞植片的构建与移植研究也正在兴起。为
探索从根本上解决角膜疾患的新方法与途径，本研究用人流产儿、家兔和山羊角膜体外
分离角膜缘干细胞、角膜基质细胞和内皮细胞；采用无饲养层细胞培养体系和冻存的角
膜缘干细胞构建家兔和山羊角膜上皮组织，并自体移植到角膜缘干细胞完全缺失的动物
模型角膜表面，恢复病损角膜上皮；同时，探索来源于自体的表皮干细胞替代角膜缘干
细胞用于角膜上皮重建的可行性；利用体外快速扩增获得角膜内皮细胞，以羊膜和角膜
基质为载体构建组织工程化角膜内皮组织，为组织工程角膜内皮层移植实验奠定基础。
实验研究主要取得以下六个方面成果和进展：
1．流产儿角膜细胞经体外分离培养可甩于组织工程化角膜组织构建，并且认为移
植时同种异体免疫反应更弱。但是，就人胎儿角膜细胞体外分离培养的条件和各类细胞
的生长特性未见系统报道，资料相对缺乏。为了充分利用好胎儿角膜组织这一资源，本
研究对30例人流产胎儿角膜上皮细胞，基质细胞和内皮细胞进行体外分离培养。发现
胎儿角膜上皮细胞采用dispase消化法和完整的全层角膜组织贴壁法可获得纯化的角膜
上皮细胞，传至10代冷冻保存；胎儿角膜基质细胞无论采用消化法还是组织块法都可
得到纯化培养的目的，传至20代冷冻保存；而角膜内皮细胞体外纯化培养比较困难。
2．山羊或家兔角膜缘上皮组织用1．2 IU／ml dispase酶和0．25％胰蛋白酶4℃冷消化，
认为1．2 IU／ml dispase酶是分离角膜缘干细胞较理想的消化酶，其作用时间以冷消化14h～16h为宜。比较DMEM／F12，RPMll640和EpilifeTM三种培养液培养原代山羊和家兔角膜缘干细胞用DMEM／F12培养液可提高细胞的传代次数。山羊角膜缘干细胞传至20代，家兔角膜缘干细胞己传至14代，于一196℃液态氮冷冻保存，获得稳定增殖的角膜缘干细胞。将角膜缘干细胞分别保存在4℃、一20℃、一80℃冰箱和一196℃液态氮中，分别在12～48 h，l～7 d、1～6周和1～6月解冻，经体外培养扩增，细胞在形态上和增殖能力上与冻存前基本相似。可以满足不同的实验条件下，对角膜缘干细胞的运输、保存和增殖的要求，为组织工程技术构建角膜上皮提供丰富的细胞来源。
3．为了制作角膜缘干细胞完全缺失，用于移植角膜缘干细胞的动物模型，将角膜
缘上皮板层切除，中央角膜用1 N NaOH擦除上皮层的方法，成功制作实验性角膜缘干
细胞移植的病理模型。
4．本研究首次采用冻存的角膜缘干细胞和无饲养层细胞培养体系构建家兔角膜上
皮组织，自体移植后观察对角膜缘干细胞缺失的治疗效果。结果7例移植羊膜角膜上皮
的家兔，有2例有效，移植后角膜的透明度增加，新生血管和结膜组织明显减少，4例部分
有效，1例无效；仅移植羊膜的4只家兔有3例无效，l例部分有效；未移植的3只家兔全部
无效。认为构建的角膜上皮组织自体移植后可以修复角膜缘干细胞缺失的角膜上皮，为
临床上角膜缘干细胞缺失所致的角膜疾患治疗提供新的措施。(该方法己申请了发明专
利，申请专利号：200410026154．4)。
5．为了探索利用自体来源的表皮干细胞替代角膜缘干细胞用于角膜表面的重建的
可行性，本文设计一种羊膜负载山羊表皮干细胞构建角膜上皮的新方法，进行移植治疗
角膜缘干细胞完全缺损。通过与角膜缘上皮细胞构建的角膜上皮比较发现，表皮干细胞
与角膜缘上皮细胞一样都可以在羊膜上培养形成复层上皮结构，细胞间形成致密连接，
细胞在羊膜上形成半桥粒，重建了上皮基底膜。构建羊膜植片移植到角膜缘完全缺失的
病理模型，在观察期内，表皮干细胞的异体移植组，观察期6～10个月，4只中有3只
部分有效1只无效；表皮干细胞的自体移植组，观察期3～6个月，5只中有3只部分有
效2只有待于进一步观察；角膜缘干细胞的自体移植组，观察期3～5个月，4只中有2
只部分有效2只有待于进一步观察。3组移植效果未见明显不同，与仅移植羊膜组和对
照组比较效果明显，恢复或部分恢复角膜上皮功能。但是，表皮干细胞替代角膜缘上皮
细胞用于角膜表面的重建的机制和长期作用有待于进一步深入研究。(该方法已申请了
发明专利，申请专利号：200410026031．0)。
6．体外扩增角膜内皮细胞时，在原代培养，以浓度为l×10-5×105个细胞／孔(即
5×104个细胞／cm2)的细胞悬液并添加消化后的．Descemt膜，96小时后细胞形成与在
体细胞相似的密集单层，快速获取纯化的角膜内皮细胞。传代培养时，以1×10-5×10
个细膨孔的浓度传代，72小时生长成密集单层并可继续传代，现已传至第10代。用2～
4代的角膜内皮细胞，以1×105个细胞／mI 的浓度接种分别到去上皮的人羊膜和去除
Descemt膜的角膜基质上，体外培养构建角膜内皮。结果以羊膜和角膜基质为载体培养
角膜内皮细胞，可体外形成?
The research focuses on the reconstruction and transplantation of tissue engineering cornea epithelium and endothelium. Our experiment studies develop six contexts as below:
1. TO Isolate and culture epithelial cells, endothelial cells and stromal cells from human fetus corneas in vitro. 30 human fetus (that is 60 corneas) used in this study were received in the asepsis condition, in which among them ,44 corneas were from 4 to 6-month-old fetus, the other 16 were from 7 to 9-month-old fetus. The results shows when the corneal limbal epithelium were digested by 0.25% trypsin and 0.02%EDTA, the more dispersed cells and living cells from 7 to 9-month-old embryonic corneas were obtained than those from 4 to 6-month old embryonic corneas (P<0.05). It could shorten the period of forming a monolayer cells and increase the cells proliferation ability after being passages that grown cells in a culture plate together with a limbal explants. There are not significantly different in the dispersed cells and living cells' digested by Dispase and trypsin (P>0.05), but the cells dispersed by Dispase could proliferate to more than 10 generations. In the explants culture, there are no different between the corneal epithelium sides up and down, it could only successfully culture when the cornea was not separated into small pieces. A fewer corneal endothelial cells from 7 to 9-month-old corneas grown together with stromal cells in the primary cells culture could not be pure after subcultured in vitro. At the same time, the corneal endothelium explants cultures after digested by trypsin could not also obtain pure endothelial cells. When the corneal stromal explants were cultured, the stromal cells could outgrow and form a monolayer after culture 12-15days. Moreover, the corneal stromal cells digested by trypsin would grow together with epithelial cells or endothelial cells, but the pure uncontaminated stromal cells could obtain after 4-5 passages. In addition, in the corneal explants culture, the corneal epithelial cells or endothelial cells outgrow more early 5-6 days than stromal cells.
2. This research objective is to obtain and proliferate regularly the goats and rabbits corneal limbal stem cells, and to cryo-preserve them in different conservation condition. The goats or rabbits cornea epithelium were digested by 1.2IU/ml dispase or 0.25% tripsin for 12 hours, 14 hours, 16 hours and 18 hours in 4"C, respectively. After comparing the cells total numbers, proliferation ability from dispase digested with those of tripsin digested, we find that Dispase is more perfecter than tripsin to obtain the corneal limbal stem cells, and its
digestion time should be 14-16 hours in 4 °C.Through comparing the action of DMEM/Fn, RPMI1640 and Epilife? Keratinocyte medium in culturing the corneal limbal stem cells, the DMEM/Fi2 medium could increase more limbal stem cell colony and passage ability than the other two medium. So, the DMEM/Fn medium is perfect medium to culture limbal stem cells to more 20 passages of goats, as well as 14 passages of rabbits. Immunostaining for K19, p63, PCNA AND K3 was positive for almost limbal stem cells. Cryo-preserve limbal stem cells in different conservation condition, and then thawing in different time, we find limbal stem cells could preserve 12-48hours, l-7days, 1-6 weeks and 1-6 months in 4°C\ — 20°CU —80°C refrigeratory and — 196 °C liquid nitrogen container, respectively. These methods of Cryo-preserve limbal stem cells could meet the need of transportation, conservation and proliferation of limbal stem cells in research and clinical field.
3. According to the theory that corneal epithelial stem cells located limbus serve as the ultimate source for maintenance and regeneration of the corneal epithelium. 20 experimental rabbits were randomly divided into four groups, 5 rabbits in one group. The whole or upper-half of corneal limbal epithelium lamella were excised by surgery or burned with 1 mol/L NaOH for exploring the best creating approach of the pathological model of corneal total li
本要求，临床上常见各种原因所引起的角膜缘干细胞及角膜上皮的损伤和缺失，使患者
视力下降或致盲。上世纪九十年代以来，随着细胞培养技术和组织工程技术的兴起，组
织工程化人工角膜、角膜上皮、内皮：基质细胞植片的构建与移植研究也正在兴起。为
探索从根本上解决角膜疾患的新方法与途径，本研究用人流产儿、家兔和山羊角膜体外
分离角膜缘干细胞、角膜基质细胞和内皮细胞；采用无饲养层细胞培养体系和冻存的角
膜缘干细胞构建家兔和山羊角膜上皮组织，并自体移植到角膜缘干细胞完全缺失的动物
模型角膜表面，恢复病损角膜上皮；同时，探索来源于自体的表皮干细胞替代角膜缘干
细胞用于角膜上皮重建的可行性；利用体外快速扩增获得角膜内皮细胞，以羊膜和角膜
基质为载体构建组织工程化角膜内皮组织，为组织工程角膜内皮层移植实验奠定基础。
实验研究主要取得以下六个方面成果和进展：
1．流产儿角膜细胞经体外分离培养可甩于组织工程化角膜组织构建，并且认为移
植时同种异体免疫反应更弱。但是，就人胎儿角膜细胞体外分离培养的条件和各类细胞
的生长特性未见系统报道，资料相对缺乏。为了充分利用好胎儿角膜组织这一资源，本
研究对30例人流产胎儿角膜上皮细胞，基质细胞和内皮细胞进行体外分离培养。发现
胎儿角膜上皮细胞采用dispase消化法和完整的全层角膜组织贴壁法可获得纯化的角膜
上皮细胞，传至10代冷冻保存；胎儿角膜基质细胞无论采用消化法还是组织块法都可
得到纯化培养的目的，传至20代冷冻保存；而角膜内皮细胞体外纯化培养比较困难。
2．山羊或家兔角膜缘上皮组织用1．2 IU／ml dispase酶和0．25％胰蛋白酶4℃冷消化，
认为1．2 IU／ml dispase酶是分离角膜缘干细胞较理想的消化酶，其作用时间以冷消化14h～16h为宜。比较DMEM／F12，RPMll640和EpilifeTM三种培养液培养原代山羊和家兔角膜缘干细胞用DMEM／F12培养液可提高细胞的传代次数。山羊角膜缘干细胞传至20代，家兔角膜缘干细胞己传至14代，于一196℃液态氮冷冻保存，获得稳定增殖的角膜缘干细胞。将角膜缘干细胞分别保存在4℃、一20℃、一80℃冰箱和一196℃液态氮中，分别在12～48 h，l～7 d、1～6周和1～6月解冻，经体外培养扩增，细胞在形态上和增殖能力上与冻存前基本相似。可以满足不同的实验条件下，对角膜缘干细胞的运输、保存和增殖的要求，为组织工程技术构建角膜上皮提供丰富的细胞来源。
3．为了制作角膜缘干细胞完全缺失，用于移植角膜缘干细胞的动物模型，将角膜
缘上皮板层切除，中央角膜用1 N NaOH擦除上皮层的方法，成功制作实验性角膜缘干
细胞移植的病理模型。
4．本研究首次采用冻存的角膜缘干细胞和无饲养层细胞培养体系构建家兔角膜上
皮组织，自体移植后观察对角膜缘干细胞缺失的治疗效果。结果7例移植羊膜角膜上皮
的家兔，有2例有效，移植后角膜的透明度增加，新生血管和结膜组织明显减少，4例部分
有效，1例无效；仅移植羊膜的4只家兔有3例无效，l例部分有效；未移植的3只家兔全部
无效。认为构建的角膜上皮组织自体移植后可以修复角膜缘干细胞缺失的角膜上皮，为
临床上角膜缘干细胞缺失所致的角膜疾患治疗提供新的措施。(该方法己申请了发明专
利，申请专利号：200410026154．4)。
5．为了探索利用自体来源的表皮干细胞替代角膜缘干细胞用于角膜表面的重建的
可行性，本文设计一种羊膜负载山羊表皮干细胞构建角膜上皮的新方法，进行移植治疗
角膜缘干细胞完全缺损。通过与角膜缘上皮细胞构建的角膜上皮比较发现，表皮干细胞
与角膜缘上皮细胞一样都可以在羊膜上培养形成复层上皮结构，细胞间形成致密连接，
细胞在羊膜上形成半桥粒，重建了上皮基底膜。构建羊膜植片移植到角膜缘完全缺失的
病理模型，在观察期内，表皮干细胞的异体移植组，观察期6～10个月，4只中有3只
部分有效1只无效；表皮干细胞的自体移植组，观察期3～6个月，5只中有3只部分有
效2只有待于进一步观察；角膜缘干细胞的自体移植组，观察期3～5个月，4只中有2
只部分有效2只有待于进一步观察。3组移植效果未见明显不同，与仅移植羊膜组和对
照组比较效果明显，恢复或部分恢复角膜上皮功能。但是，表皮干细胞替代角膜缘上皮
细胞用于角膜表面的重建的机制和长期作用有待于进一步深入研究。(该方法已申请了
发明专利，申请专利号：200410026031．0)。
6．体外扩增角膜内皮细胞时，在原代培养，以浓度为l×10-5×105个细胞／孔(即
5×104个细胞／cm2)的细胞悬液并添加消化后的．Descemt膜，96小时后细胞形成与在
体细胞相似的密集单层，快速获取纯化的角膜内皮细胞。传代培养时，以1×10-5×10
个细膨孔的浓度传代，72小时生长成密集单层并可继续传代，现已传至第10代。用2～
4代的角膜内皮细胞，以1×105个细胞／mI 的浓度接种分别到去上皮的人羊膜和去除
Descemt膜的角膜基质上，体外培养构建角膜内皮。结果以羊膜和角膜基质为载体培养
角膜内皮细胞，可体外形成?
The research focuses on the reconstruction and transplantation of tissue engineering cornea epithelium and endothelium. Our experiment studies develop six contexts as below:
1. TO Isolate and culture epithelial cells, endothelial cells and stromal cells from human fetus corneas in vitro. 30 human fetus (that is 60 corneas) used in this study were received in the asepsis condition, in which among them ,44 corneas were from 4 to 6-month-old fetus, the other 16 were from 7 to 9-month-old fetus. The results shows when the corneal limbal epithelium were digested by 0.25% trypsin and 0.02%EDTA, the more dispersed cells and living cells from 7 to 9-month-old embryonic corneas were obtained than those from 4 to 6-month old embryonic corneas (P<0.05). It could shorten the period of forming a monolayer cells and increase the cells proliferation ability after being passages that grown cells in a culture plate together with a limbal explants. There are not significantly different in the dispersed cells and living cells' digested by Dispase and trypsin (P>0.05), but the cells dispersed by Dispase could proliferate to more than 10 generations. In the explants culture, there are no different between the corneal epithelium sides up and down, it could only successfully culture when the cornea was not separated into small pieces. A fewer corneal endothelial cells from 7 to 9-month-old corneas grown together with stromal cells in the primary cells culture could not be pure after subcultured in vitro. At the same time, the corneal endothelium explants cultures after digested by trypsin could not also obtain pure endothelial cells. When the corneal stromal explants were cultured, the stromal cells could outgrow and form a monolayer after culture 12-15days. Moreover, the corneal stromal cells digested by trypsin would grow together with epithelial cells or endothelial cells, but the pure uncontaminated stromal cells could obtain after 4-5 passages. In addition, in the corneal explants culture, the corneal epithelial cells or endothelial cells outgrow more early 5-6 days than stromal cells.
2. This research objective is to obtain and proliferate regularly the goats and rabbits corneal limbal stem cells, and to cryo-preserve them in different conservation condition. The goats or rabbits cornea epithelium were digested by 1.2IU/ml dispase or 0.25% tripsin for 12 hours, 14 hours, 16 hours and 18 hours in 4"C, respectively. After comparing the cells total numbers, proliferation ability from dispase digested with those of tripsin digested, we find that Dispase is more perfecter than tripsin to obtain the corneal limbal stem cells, and its
digestion time should be 14-16 hours in 4 °C.Through comparing the action of DMEM/Fn, RPMI1640 and Epilife? Keratinocyte medium in culturing the corneal limbal stem cells, the DMEM/Fi2 medium could increase more limbal stem cell colony and passage ability than the other two medium. So, the DMEM/Fn medium is perfect medium to culture limbal stem cells to more 20 passages of goats, as well as 14 passages of rabbits. Immunostaining for K19, p63, PCNA AND K3 was positive for almost limbal stem cells. Cryo-preserve limbal stem cells in different conservation condition, and then thawing in different time, we find limbal stem cells could preserve 12-48hours, l-7days, 1-6 weeks and 1-6 months in 4°C\ — 20°CU —80°C refrigeratory and — 196 °C liquid nitrogen container, respectively. These methods of Cryo-preserve limbal stem cells could meet the need of transportation, conservation and proliferation of limbal stem cells in research and clinical field.
3. According to the theory that corneal epithelial stem cells located limbus serve as the ultimate source for maintenance and regeneration of the corneal epithelium. 20 experimental rabbits were randomly divided into four groups, 5 rabbits in one group. The whole or upper-half of corneal limbal epithelium lamella were excised by surgery or burned with 1 mol/L NaOH for exploring the best creating approach of the pathological model of corneal total li
引文
第一章 参考文献
谢立信,主编.角膜移植学.北京：人民卫生出版社，2000:14 ~ 25
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