深低温保存角膜缘组织活性的实验研究
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摘要
目的 评价深低温保存兔眼及人眼角膜缘组织活性，以探讨深低温保存角膜缘组织应用于临床的可行性。
方法 3只健康新西兰大耳白兔(6只眼)，制备角膜缘组织片(约带2mm宽的周边角膜和2mm宽巩膜)，采用简化二步深低温保存法保存90天；取2只人尸眼角膜缘组织片(约带有2-3mm宽周边角膜及2-3mm宽巩膜)，来源于简化二步深低温保存法保存92天(1只眼)及726天(1只眼)的带巩膜环的角膜组织片。分别行光镜(HE染色、PAS染色)、透射电镜观察其组织结构及超微结构，酶组织化学染色[乳酸脱氢酶(LDH)、琥珀酸脱氢酶(SDH)]测定酶活性，并与新鲜眼角膜缘组织对比。
结果 ①光镜下，简化二步深低温保存兔及人角膜缘组织上皮层形态完好，细胞排列紧密，深层柱状上皮细胞胞浆少、核小、染色深。部分上皮从上皮下乳头间向下突出，每隔1-2mm横过角膜缘，呈放射状排列，形成“Vogt栅栏”。有色素颗粒沉着。未见细胞膜破裂，细胞浆均匀，未见空泡形成，细胞核未见脱失，基底膜完整，呈波浪状。基质层为疏松的纤维组织，胶原纤维排列不规则，直径粗细不均。可见血管。兔及人角膜缘上皮层PAS染色阴性；与其毗邻的角膜侧上皮层PAS染色阴性；巩膜侧上皮层偶见PAS染色阳性。②电镜下，简化二步深低温保存兔及人角膜缘组织上皮层细胞胞膜完整，上皮层表面有微绒毛和微皱褶，上皮细胞之间可见多数桥粒及半桥粒连接，细胞连接完好，底部柱状细胞为单层细胞，排列不规则，其基底连同上皮基底膜呈波浪状。胞质致密，细胞内器丰富，含有高浓度的线粒体、高尔基复合体、内质网及细丝状结构。部分线粒体肿胀，少数嵴断裂，内质网池轻度扩张。胞质内可见色素颗粒。核完整，形状不规则，核膜清晰，染色质分布均匀，
中文摘要
变化不明显。③简化二步深低温保存兔及人角膜缘组织上皮层LDH、
SDH的酶组织化学测定可见上皮细胞内LDH为蓝色反应颗粒，分布于
细胞质中;SDH为紫蓝色反应颗粒，分布于细胞质中。与新鲜角膜缘组
织相比其活性无显著性差异(P>0.05)。
结论深低温保存法能保存角膜缘组织活性;能随时按需要为临床
提供活性角膜缘组织材料;为深低温保存角膜缘干细胞移植治疗临床眼
表疾病提供了实验依据。
Objective To evaluate the vitality of limbal tissues with Cryopreservation.
Methods Limbal grafts include 2mm of peripheralcornea and 2mm of sclera of 3 rabbits were preserved by simplified two-step Cryopreservation method for 90 days. The human corneal tissues were cryopreserved by the same method for 92 days and 726 days, and were made into limbal graft include 2-3mm of peripheralcornea and 2-3mm of sclera. Light and transmission electron microscopies were used to study the structure of limbal tissue cells. Enzyme histochemistry methods with image analysis system were used to evaluate the metabolic enzyme activity, lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH).
Results (1) From light microscopic study, the cryopreserved limbal epithelial cells remained intact and lined up tightly. Partial epithelium arrayed radiately and shaped into Vogt bars, which projected downward over the limbus every 1 -2mm from the subepithelial papilla. And a lot of pigment granule was deposited. There were blood vessel tissue in Hypothallus. (2) From transmission electron microscopic study, the cryopreserved limbal epithelial cytomembrane remained intact and zonula occludens was as excellent as before. Partial mitochondria were swollen with no nuclei change in limbal tissue cells preserved by simplified two-step Cryopreservation method. (3) The activity of LDH and SDH in the epithelial cells of limbal tissue remained the same as in the fresh control group, which preserved by simplified two-step Cryopreservation method (P>0.05).
Conclusions Simplified two-step Cryopreservation method can maintain the vitality of limbal tissue cells and provide viable limbal grafts at any time. And can provide an experimental evidence for cryopreserved limbal stem cells to treat ocular surface disorders.
Postgraduated: Luo Lin(Ophthalmology) Directed by: Professor Wang Chuanfu
方法 3只健康新西兰大耳白兔(6只眼)，制备角膜缘组织片(约带2mm宽的周边角膜和2mm宽巩膜)，采用简化二步深低温保存法保存90天；取2只人尸眼角膜缘组织片(约带有2-3mm宽周边角膜及2-3mm宽巩膜)，来源于简化二步深低温保存法保存92天(1只眼)及726天(1只眼)的带巩膜环的角膜组织片。分别行光镜(HE染色、PAS染色)、透射电镜观察其组织结构及超微结构，酶组织化学染色[乳酸脱氢酶(LDH)、琥珀酸脱氢酶(SDH)]测定酶活性，并与新鲜眼角膜缘组织对比。
结果 ①光镜下，简化二步深低温保存兔及人角膜缘组织上皮层形态完好，细胞排列紧密，深层柱状上皮细胞胞浆少、核小、染色深。部分上皮从上皮下乳头间向下突出，每隔1-2mm横过角膜缘，呈放射状排列，形成“Vogt栅栏”。有色素颗粒沉着。未见细胞膜破裂，细胞浆均匀，未见空泡形成，细胞核未见脱失，基底膜完整，呈波浪状。基质层为疏松的纤维组织，胶原纤维排列不规则，直径粗细不均。可见血管。兔及人角膜缘上皮层PAS染色阴性；与其毗邻的角膜侧上皮层PAS染色阴性；巩膜侧上皮层偶见PAS染色阳性。②电镜下，简化二步深低温保存兔及人角膜缘组织上皮层细胞胞膜完整，上皮层表面有微绒毛和微皱褶，上皮细胞之间可见多数桥粒及半桥粒连接，细胞连接完好，底部柱状细胞为单层细胞，排列不规则，其基底连同上皮基底膜呈波浪状。胞质致密，细胞内器丰富，含有高浓度的线粒体、高尔基复合体、内质网及细丝状结构。部分线粒体肿胀，少数嵴断裂，内质网池轻度扩张。胞质内可见色素颗粒。核完整，形状不规则，核膜清晰，染色质分布均匀，
中文摘要
变化不明显。③简化二步深低温保存兔及人角膜缘组织上皮层LDH、
SDH的酶组织化学测定可见上皮细胞内LDH为蓝色反应颗粒，分布于
细胞质中;SDH为紫蓝色反应颗粒，分布于细胞质中。与新鲜角膜缘组
织相比其活性无显著性差异(P>0.05)。
结论深低温保存法能保存角膜缘组织活性;能随时按需要为临床
提供活性角膜缘组织材料;为深低温保存角膜缘干细胞移植治疗临床眼
表疾病提供了实验依据。
Objective To evaluate the vitality of limbal tissues with Cryopreservation.
Methods Limbal grafts include 2mm of peripheralcornea and 2mm of sclera of 3 rabbits were preserved by simplified two-step Cryopreservation method for 90 days. The human corneal tissues were cryopreserved by the same method for 92 days and 726 days, and were made into limbal graft include 2-3mm of peripheralcornea and 2-3mm of sclera. Light and transmission electron microscopies were used to study the structure of limbal tissue cells. Enzyme histochemistry methods with image analysis system were used to evaluate the metabolic enzyme activity, lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH).
Results (1) From light microscopic study, the cryopreserved limbal epithelial cells remained intact and lined up tightly. Partial epithelium arrayed radiately and shaped into Vogt bars, which projected downward over the limbus every 1 -2mm from the subepithelial papilla. And a lot of pigment granule was deposited. There were blood vessel tissue in Hypothallus. (2) From transmission electron microscopic study, the cryopreserved limbal epithelial cytomembrane remained intact and zonula occludens was as excellent as before. Partial mitochondria were swollen with no nuclei change in limbal tissue cells preserved by simplified two-step Cryopreservation method. (3) The activity of LDH and SDH in the epithelial cells of limbal tissue remained the same as in the fresh control group, which preserved by simplified two-step Cryopreservation method (P>0.05).
Conclusions Simplified two-step Cryopreservation method can maintain the vitality of limbal tissue cells and provide viable limbal grafts at any time. And can provide an experimental evidence for cryopreserved limbal stem cells to treat ocular surface disorders.
Postgraduated: Luo Lin(Ophthalmology) Directed by: Professor Wang Chuanfu
引文
1 朱志忠．角膜病学．北京，人民卫生出版社，1996：263-265．
2 王传富，刘金刚，鞠明诚，等．深低温长期保存角膜穿透性移植的研究．中华眼科杂志，1990，26：17-20．
3 Wang Chuan-fu, Ju Ming-cheng, Shi Zhen-rong, et al. Evaluation of penetrating keratoplasties with long-term cryopreserved corneas using simplified method. Chinese Medical Journal, 1992, 105: 572-576.
4 Lojda Z, Gossrau R, Schiebler TH. Enzyme histochemistry. A laboratory manualed Berlin: Springer-Verlay, 1979: 48, 258-284.
5 周善璧，陈家祺，黄挺，等．深低温保存角膜缘干细胞移植治疗眼表疾病初步研究．重庆医科大学学报，2000，25：62-64．
6 Tseng SCG, Tsubota K. Important concepts for treating ocular surface and tear disorders. AM J Ophthalmol, 1997, 124: 825-835.
7 Tan DTH, Ficker LA, Buckley RJ. Limbal transplantation. Ophthalmology, 1996, 103: 29-36.
8 Keyon KR, Tseng SCG. Limbal autograft transplantation for ocular surface disorders. Ophthalmology, 1989, 96: 709-723.
9 Kaufman HE, Capella JA. Preserved corneal tissue for transplantation. J Cryosurg, 1968, 1:125-131.
10 Taylor MJ. Clinical cryobiology of tissues: preservation of corneas. Cryobiology, 1986, 23: 323-353.
11 王传富，孙为荣，王大博，等．深低温长期保存巩膜的动物实验和临床应用．青岛医学院学报，1996，32：194-196．
12 王传富，孙为荣，崔向东，等．深低温长期保存结膜的动物实验和临床研究．青岛医学院学报，1997，33：290-292．
13 Durlu YK, Tamai M.Transplantation of retinal pigment epithelium using viable cryo-
preserved cells. Cell-Transplant, 1997, 6:149-162.
14 Canals-M,Costa-J,Potau-JM,et al.Long-term cryopreservation of human donar coreas. Ear J Ophthalmol, 1993, 6: 234-241.
15 Haselbacher G, Groscurth P, Otten U, et al. Long-term cultivation of cryopreserved human fetal brain cells in a chemically defined medium. J Neurosci Methods, 1989, 30: 121-131.
16 Redmond D.E., NaftolinF, Collier T.J.,et al.Fetal neural guaft survival. Lancet, 1990, 33: 820-822
17 Robbins JE. A study of endothelium in keratoplasty and corneal preservation. Arch Ophthalmol, 1965; 73: 242-248.
18 周爱儒主编．生物化学．人民卫生出版社，北京，2002，73-84．
19 王传富，刘金刚，鞠明诚，等．深低温长期保存兔角膜穿透性移植的动物实验．青岛医学院学报，1988，24：97-99．
20 王传富，鞠明诚，刘金刚．深低温长期保存角膜穿透性移植的临床研究．青岛医学院学报，1988，24．101-105．
21 石珍荣，周惠民，王传富．深低温冷冻保存角膜的超微结构观察．青岛医学院学报，1989，25：18-20．
22 孙为荣，王传富．深低温保存角膜内皮细胞的酶组织化学观察．眼科研究，1991，9：1-4．
23 鲍连云，王传富，杨湘娟．深低温保存角膜穿透性移植术后角膜神经再生的研究．中华眼科杂志，1996，32：379-381．
24 王传富，刘金刚，葛凤英，等。深低温长期保存角膜技术的研究：冷冻保护剂的筛选。青岛医学院学报，1988，24：92-95．
25 刘金刚，王传富．深低温冻贮角膜复温后处理的实验研究．青岛医学院学报，1988，24：266-268．
26 Meryman HT, Horblower MA. Simplifed procedure deglycerolizing red blood cells frozen in a high glycerol concentration.Transfusion, 1997, 17: 438-443.
27 Kaden R, Klippel FF, Katzorke T, et al. A new instant cryoprotectant for human sperm. Arch Androl, 1985, 14: 133-137.
28 Lovelock JE, Bishop MWH. Prevention of freezing damage to living cells by dimethylsulphoxide. Nature, 1959, 183: 1394-1400.
29 刘金刚，刘作斌．低温医学．人民卫生出版社，北京，1993，3-28．
30 华泽钊，任禾盛．低温生物医学技术．科学出版社，北京，1994，37-63．
31 Tseng SC. Concept and application of limbal stem cells. Eye, 1989, 3: 141-157.
32 Schermer A, Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratinin vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol, 1986, 103: 49-62.
33 朱志忠．角膜缘潜在性功能的开发应用．眼科研究，2000，15：375-378．
34 Gipson IK. The epithelial basement membrane zone of the limbus. Eye, 1989, 3: 132-140.
35 Zieske JD. Perpetuation of stem cells in the eye. Eye, 1994, 8: 163-169.
2 王传富，刘金刚，鞠明诚，等．深低温长期保存角膜穿透性移植的研究．中华眼科杂志，1990，26：17-20．
3 Wang Chuan-fu, Ju Ming-cheng, Shi Zhen-rong, et al. Evaluation of penetrating keratoplasties with long-term cryopreserved corneas using simplified method. Chinese Medical Journal, 1992, 105: 572-576.
4 Lojda Z, Gossrau R, Schiebler TH. Enzyme histochemistry. A laboratory manualed Berlin: Springer-Verlay, 1979: 48, 258-284.
5 周善璧，陈家祺，黄挺，等．深低温保存角膜缘干细胞移植治疗眼表疾病初步研究．重庆医科大学学报，2000，25：62-64．
6 Tseng SCG, Tsubota K. Important concepts for treating ocular surface and tear disorders. AM J Ophthalmol, 1997, 124: 825-835.
7 Tan DTH, Ficker LA, Buckley RJ. Limbal transplantation. Ophthalmology, 1996, 103: 29-36.
8 Keyon KR, Tseng SCG. Limbal autograft transplantation for ocular surface disorders. Ophthalmology, 1989, 96: 709-723.
9 Kaufman HE, Capella JA. Preserved corneal tissue for transplantation. J Cryosurg, 1968, 1:125-131.
10 Taylor MJ. Clinical cryobiology of tissues: preservation of corneas. Cryobiology, 1986, 23: 323-353.
11 王传富，孙为荣，王大博，等．深低温长期保存巩膜的动物实验和临床应用．青岛医学院学报，1996，32：194-196．
12 王传富，孙为荣，崔向东，等．深低温长期保存结膜的动物实验和临床研究．青岛医学院学报，1997，33：290-292．
13 Durlu YK, Tamai M.Transplantation of retinal pigment epithelium using viable cryo-
preserved cells. Cell-Transplant, 1997, 6:149-162.
14 Canals-M,Costa-J,Potau-JM,et al.Long-term cryopreservation of human donar coreas. Ear J Ophthalmol, 1993, 6: 234-241.
15 Haselbacher G, Groscurth P, Otten U, et al. Long-term cultivation of cryopreserved human fetal brain cells in a chemically defined medium. J Neurosci Methods, 1989, 30: 121-131.
16 Redmond D.E., NaftolinF, Collier T.J.,et al.Fetal neural guaft survival. Lancet, 1990, 33: 820-822
17 Robbins JE. A study of endothelium in keratoplasty and corneal preservation. Arch Ophthalmol, 1965; 73: 242-248.
18 周爱儒主编．生物化学．人民卫生出版社，北京，2002，73-84．
19 王传富，刘金刚，鞠明诚，等．深低温长期保存兔角膜穿透性移植的动物实验．青岛医学院学报，1988，24：97-99．
20 王传富，鞠明诚，刘金刚．深低温长期保存角膜穿透性移植的临床研究．青岛医学院学报，1988，24．101-105．
21 石珍荣，周惠民，王传富．深低温冷冻保存角膜的超微结构观察．青岛医学院学报，1989，25：18-20．
22 孙为荣，王传富．深低温保存角膜内皮细胞的酶组织化学观察．眼科研究，1991，9：1-4．
23 鲍连云，王传富，杨湘娟．深低温保存角膜穿透性移植术后角膜神经再生的研究．中华眼科杂志，1996，32：379-381．
24 王传富，刘金刚，葛凤英，等。深低温长期保存角膜技术的研究：冷冻保护剂的筛选。青岛医学院学报，1988，24：92-95．
25 刘金刚，王传富．深低温冻贮角膜复温后处理的实验研究．青岛医学院学报，1988，24：266-268．
26 Meryman HT, Horblower MA. Simplifed procedure deglycerolizing red blood cells frozen in a high glycerol concentration.Transfusion, 1997, 17: 438-443.
27 Kaden R, Klippel FF, Katzorke T, et al. A new instant cryoprotectant for human sperm. Arch Androl, 1985, 14: 133-137.
28 Lovelock JE, Bishop MWH. Prevention of freezing damage to living cells by dimethylsulphoxide. Nature, 1959, 183: 1394-1400.
29 刘金刚，刘作斌．低温医学．人民卫生出版社，北京，1993，3-28．
30 华泽钊，任禾盛．低温生物医学技术．科学出版社，北京，1994，37-63．
31 Tseng SC. Concept and application of limbal stem cells. Eye, 1989, 3: 141-157.
32 Schermer A, Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratinin vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol, 1986, 103: 49-62.
33 朱志忠．角膜缘潜在性功能的开发应用．眼科研究，2000，15：375-378．
34 Gipson IK. The epithelial basement membrane zone of the limbus. Eye, 1989, 3: 132-140.
35 Zieske JD. Perpetuation of stem cells in the eye. Eye, 1994, 8: 163-169.
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