角膜缘上皮细胞培养和异体移植的实验研究
|
摘要
目的：检测以羊膜为载体的改良组织块培养法培养的角膜缘上皮细胞片的细胞生物学特性，观察该细胞片移植到角膜缘干细胞严重损伤的异体兔眼角膜上的效果。
方法：切取含有完整上皮细胞的大小约2mm×2mm、厚约200μm异体兔角膜缘组织，剪切成4个1mm×1mm小组织块，经消化酶处理后放在无上皮细胞的羊膜上培养，用倒置显微镜观察细胞的生长情况，定期进行病理组织学、免疫组化染色和电镜检查。环形切除26只成年白兔右眼角膜缘组织后刮除全部角膜上皮细胞，建立角膜缘干细胞损伤模型。随机取24只白兔，将其分为实验组和对照组，每组12只。细胞在羊膜上培养16天左右，实验组兔眼接受含有培养的异体角膜缘上皮细胞的羊膜片移植，对照组单独进行羊膜移植，术后观察角膜混浊度、角膜新生血管、角膜上皮修复等情况，定期进行病理组织学检查。
结果：角膜缘组织块在羊膜上培养第8天，培养的角膜缘上皮细胞出现汇合，并很好地附着在下面的羊膜上；培养第16天，羊膜上培养的角膜缘上皮细胞形成3-4层，羊膜上培养的细胞经广谱角蛋白(Pan-Keratin，PK)抗体和增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)单克隆抗体染色呈阳性反应，角膜缘干细胞损伤模型制作后第6周，全角膜可见新生血管，角膜中央上皮细胞剥脱伴结膜上皮入侵。移植术后第5天，实验组损伤角膜表面全部上皮化，对照组则全部缺损；移植术后第16天开始，实验组逐渐出现排斥反应，对照组则没有出现排斥反应。
结论：以羊膜为底物改良组织块培养法培养的角膜缘上皮细胞片短期内可形成多层，既保持角膜上皮细胞的特性，又具有高的增殖能力；该细胞片可以用来异体移植治疗角膜缘功能缺陷的角膜病；但在异体移植术后早期角膜全部上皮化，晚期
安嫩医科大学硕士学位论文
则出现排斥反应。
Objective To detect the effect of method which limbal epithelial cells were cultivated on amniotic membrane by tissue inoculation. To observe the effect of amniotic membrane containing cultivated limbal epithelial cells were transplanted to allogeneic limbal stem cell damaged rabbit cornea.
Methods Small specimens (size, about 2.0mmx2.0mm; thickness, about 200um)of limbal epithelial cells were excised from the limbal regions in rabbits; each of them was cut into four tissues(lmm x 1mm). Making tissues loosen by enzyme digestion and cultivating them on acellular human amniotic membrane. The cultivated limbal epithelial cells on amniotic membrane were observed by inverted microscope every two or three days, and examined by histology, immunohistochemical and electron microscop. 26 rabbits underwent corneal epithelium curettement and limbal ablation to induce limbal stem cell deficiency and were randomly divided into two groups, experimental group and control group(n=12 for each group). Limbal epithelial cells were cultivated by tissue inoculation on amniotic membrane for about sixteen days, experimental group were transplanted with amniotic membrane that contained cultivated allogeneic limbal epithelial cells. Control group were transplanted with amniotic membrane transplantation only. Outcome(cor
neal opacity, vascularization,the process of corneal reepithelialization) was assessed by daily slit-lamp examination, and histopathologic examination was carried out regularly.
Results A confluent primary culture of limbal epithelial cells was established on acellular human amniotic membrane on the 8th day.The cultivated limbal epithelial cells
on the amniotic membrane formed multilayer after 16 days in culture, the cultivated limbal epithelial cells formed 3~4 layers on acellular human amniotic membrane and attached well to the underlying amniotic membrane. The cultured cells on amniotic membrane were stained positive in Pan-keratin antibody and anti-proliferating cell nuclear antigen(anti-PCNA). 6 weeks later, 26 rabbit eyes of corneal epithelial curettement and limbal ablation developed features of limbal stem cell deficiency including conjunctival epithelial ingrowth, vascularization and chronic inflammation. Experimental group entire corneal were all successfully epithelialied up to 5 days after surgery while control group rabbits eyes of corneal epithelia remained full defected. From the 16th day, experimental group rabbits eyes appeared to undergo rejection while control group not.
Conclusions The cultivated limbal epithelial cells by tissue inoculation can form multi-layered on amniotic membrane, and some of them have ability in proliferation. Cultivated limbal epithelial cells on amniotic membrane by tissue inoculation can be transplantated to rabbit eyes of limbal stem cell deficiency. All experimental group corneas were all epithelialized in the earlier postoperative period and were undergone rejection in the later postoperative period.
方法：切取含有完整上皮细胞的大小约2mm×2mm、厚约200μm异体兔角膜缘组织，剪切成4个1mm×1mm小组织块，经消化酶处理后放在无上皮细胞的羊膜上培养，用倒置显微镜观察细胞的生长情况，定期进行病理组织学、免疫组化染色和电镜检查。环形切除26只成年白兔右眼角膜缘组织后刮除全部角膜上皮细胞，建立角膜缘干细胞损伤模型。随机取24只白兔，将其分为实验组和对照组，每组12只。细胞在羊膜上培养16天左右，实验组兔眼接受含有培养的异体角膜缘上皮细胞的羊膜片移植，对照组单独进行羊膜移植，术后观察角膜混浊度、角膜新生血管、角膜上皮修复等情况，定期进行病理组织学检查。
结果：角膜缘组织块在羊膜上培养第8天，培养的角膜缘上皮细胞出现汇合，并很好地附着在下面的羊膜上；培养第16天，羊膜上培养的角膜缘上皮细胞形成3-4层，羊膜上培养的细胞经广谱角蛋白(Pan-Keratin，PK)抗体和增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)单克隆抗体染色呈阳性反应，角膜缘干细胞损伤模型制作后第6周，全角膜可见新生血管，角膜中央上皮细胞剥脱伴结膜上皮入侵。移植术后第5天，实验组损伤角膜表面全部上皮化，对照组则全部缺损；移植术后第16天开始，实验组逐渐出现排斥反应，对照组则没有出现排斥反应。
结论：以羊膜为底物改良组织块培养法培养的角膜缘上皮细胞片短期内可形成多层，既保持角膜上皮细胞的特性，又具有高的增殖能力；该细胞片可以用来异体移植治疗角膜缘功能缺陷的角膜病；但在异体移植术后早期角膜全部上皮化，晚期
安嫩医科大学硕士学位论文
则出现排斥反应。
Objective To detect the effect of method which limbal epithelial cells were cultivated on amniotic membrane by tissue inoculation. To observe the effect of amniotic membrane containing cultivated limbal epithelial cells were transplanted to allogeneic limbal stem cell damaged rabbit cornea.
Methods Small specimens (size, about 2.0mmx2.0mm; thickness, about 200um)of limbal epithelial cells were excised from the limbal regions in rabbits; each of them was cut into four tissues(lmm x 1mm). Making tissues loosen by enzyme digestion and cultivating them on acellular human amniotic membrane. The cultivated limbal epithelial cells on amniotic membrane were observed by inverted microscope every two or three days, and examined by histology, immunohistochemical and electron microscop. 26 rabbits underwent corneal epithelium curettement and limbal ablation to induce limbal stem cell deficiency and were randomly divided into two groups, experimental group and control group(n=12 for each group). Limbal epithelial cells were cultivated by tissue inoculation on amniotic membrane for about sixteen days, experimental group were transplanted with amniotic membrane that contained cultivated allogeneic limbal epithelial cells. Control group were transplanted with amniotic membrane transplantation only. Outcome(cor
neal opacity, vascularization,the process of corneal reepithelialization) was assessed by daily slit-lamp examination, and histopathologic examination was carried out regularly.
Results A confluent primary culture of limbal epithelial cells was established on acellular human amniotic membrane on the 8th day.The cultivated limbal epithelial cells
on the amniotic membrane formed multilayer after 16 days in culture, the cultivated limbal epithelial cells formed 3~4 layers on acellular human amniotic membrane and attached well to the underlying amniotic membrane. The cultured cells on amniotic membrane were stained positive in Pan-keratin antibody and anti-proliferating cell nuclear antigen(anti-PCNA). 6 weeks later, 26 rabbit eyes of corneal epithelial curettement and limbal ablation developed features of limbal stem cell deficiency including conjunctival epithelial ingrowth, vascularization and chronic inflammation. Experimental group entire corneal were all successfully epithelialied up to 5 days after surgery while control group rabbits eyes of corneal epithelia remained full defected. From the 16th day, experimental group rabbits eyes appeared to undergo rejection while control group not.
Conclusions The cultivated limbal epithelial cells by tissue inoculation can form multi-layered on amniotic membrane, and some of them have ability in proliferation. Cultivated limbal epithelial cells on amniotic membrane by tissue inoculation can be transplantated to rabbit eyes of limbal stem cell deficiency. All experimental group corneas were all epithelialized in the earlier postoperative period and were undergone rejection in the later postoperative period.
引文
1. Kruse FE.Stem cells and corneal epithelial regeneration. Eye, 1994;8:170～183
2. Williams KA,Roder D,Esterman A,et al. Factors predictive of corneal graft survival:report from the Australian Corneal Graft Registry. Ophthalmology, 1992,99:403～414
3. Williams-KA, Brereton-HM,Aggarwal-R,et al. Use of DNA polymorphisms and the polymerase chain reaction to examine the survival of a human limbal stem cell allograft. Am J Ophthalmol. 1995 Sep; 120:342～350
4. Kim JC,Tseng. Transplantation of opreserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea. 1995; 14:475～484
5. Pellgrini G, Traverso CE,Franzi AT,et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet, 1997; 349:990～993
6. Zieske Jd,Bukusoglu G,Yankauckas MA.Characterization of a potential marker of corneal epithelial stem cells. Invest Ophthalmol Vis Sci. 1992,33:52～143
7. Kenyon KR,Tseng SC.Limbal autograft transplantation for ocular surface disorders.Ophthalmology, 1989;96:709
8. Davanger M, Evensen A, Role of the pericorneal papillary structure in renewal of corneal epithelium[J].Nature 1971 ;229:560～561
9. Kinoshita S, Friend J, Thoyt RA.Sex chromatin of donor corneal epithelium in rabbits[j]. Invest Ophthalmol Vis Sci 1981 ;21:434～441
10. Thoft RA, Friend J.The XYZ hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci, 1983 ;24:1442
11. Schermer A,Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol. 1986, 103:49～64
12. Tseng SC. Concept and application of limbal stem cells. Eye 1989;3:141～157
13. Tsai RJ, Tseng SCG, Effect of stromal inflammation on the outcome of limbal transplantation for corneal surface reconstructiono Cornea 1995; 14(5): 439
14. Thoft RA.Conj untival transplantion[J] .Arch Ophthalmol. 1977:95; 1425～1427
15. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med. 2000 Jul 13;343(2):86～93.
16. Tsai RJ,Tseng SCG.Human allograft limbal transplatation for corneal surface reconstruction. Cornea. 1994,13:389～400
17. Kenyon KR,Rapoza PA.Limbal allograft transplantation for ocular surface disorder[J]. Invest Ophthalmol Vis Sci. 1990;31:1301～1314
18. Lindberg KL ,Brown ME,Chaves HV, et al. In vitro propagation of human ocular surface epithelial cells for transplantation. Invest Ophthalmol Vis Sci, 1993;34:2672～2679
19. Koizumi N, Inatomi T, Quantock AJ, et al. Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits. Cornea. 2000 Jan; 19(1):65～71.
20. Ardjomand N,berghold A,Reich MeE.Loss of corneal langerhans cells during storage in organ culture medium,optisol and McCarey-Kaufman meduum[J].Eye 1998;12:134～138
21．吴静，徐锦堂，赵松滨。体外重建角膜上皮组织的实验研究。眼科研究，1999；17(2)：84～86
22. He YG, McCulley JP. Growing human corneal epithelium on collagen shield and subsequent transfer to denuded cornea in vitro. Curt Eye Res. 1991 Sep; 10(9):851～863.
23．沈玺．人羊膜移植治疗眼表面疾病．中国实用眼科杂志，1999，11：651～653
24. Adinofli M,Akle CA,Mccoll I,et al. Expression of HLA antigens, β 2-microglobulin and enzyme by human aminotic membrane[J].Nature 1982;295:325～372
25. De Rotth A. Plastic repair of conjunctival defects with fetal membrane.Arch ophthalmol. 1940;23:522～525
26. Tseng-SC;Prabhasawat-P;Lee-SH Amniotic membrane transplantation for conjunctival surface reconstruction. Am-J-Ophthalmol. 1997 Dec; 124(6): 765～774
27. Meller D, Tsteng SCG.In vitro Conjuntival epithelial differentiation on opreserved human amniotic membrane. Invest Ophthalmol Vis Sci. 1998,39(Suppl):S428
28. Shimazaki-J; Yang-HY; Tsubota-K. Amniotic membrane transplantation for ocular surface reconstruction in patients with chemical and thermal burns. Ophthalmology. 1997 Dec; 104(12): 2068～2076
29. Tseng-SC; Li-DQ; Ma-X Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type Ⅱ, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. J-Cell-Physiol. 1999 Jun; 179(3): 325-35
30. Kim-JC; Tseng-SC. The effects on inhibition of corneal neovascularization after human amniotic membrane transplantation in severely damaged rabbit corneas. Korean-J-Ophthalmol. 1995 Jun; 9(1): 32-46
31. Talmi-YP; Sigler-L; Inge-E et al. Placenta. Antibacterial properties of human amniotic membranes. 1991 May-Jun; 12(3): 285-8
32. Meler D,Pires RT,Mack RJ,et al. Amniotic membrane transplantation for acute chemical or thermal burns. Ophthalmology.2000;107:980
33. Koizumi N, Fullwood NJ, Bairaktaris G, et al. Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane. Invest Ophthalmol Vis Sci. 2000 Aug;41:2506～2513
34. Schwab-IR. Cultured corneal epithelia for ocular surface disease. Trans-Am-Ophthalmol-Soc. 1999; 97:891～986
35. Pan ZQ ,Zhang WH. Study on proliferation and differentiation of human limbal stem cells in vitro. Chin Ophthal Res, 1999; 17(2): 81～83
36．薛庆善．体外培养的原理与技术．第一版．北京：科学出版社，2001．375～382
37. Kruse-FE, Tseng-SC. Serum differentially modulates the clonal growth and differentiation of cultured limbal and corneal epithelium. Invest Ophthalmol Vis Sci. 1993; 34(10): 2976～2989
38. Schwab IR, Reyes M, Isseroff RR. Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea, 2000;19(4):421～426
39. Zieske JD,Mason VS, Wasson ME,et al. Basemant membrane assembly and differentition of cultured corneal cells:importance of culture enviroment and endothelial cell interaction. Exp Cell Res. 1994;214:631
40. Bravo R,Frand R,Blundell PA,et al. Cyelin/PCNA is the auxiliary protein of DNA polymerase-δ[J].Nature 1987;326:515
41．潘志强，张文华，武宇影．培养角膜缘干细胞羊膜移植治疗碱烧伤动物的实验研究．中华眼科杂志，2000，36(1)：32～35
42．潘志强，张文华．培养角膜干细胞羊膜片移植治疗角膜缘功能障碍．中国实用眼科杂志，2000，18(9)：524～526
43. McBrida Bw McGill JI; Smith JL. MHC class Ⅰ and class Ⅱ antigen expression in normal human corneas and in corneas from cases of herpetic keratitis..Immunology, 1988;65(4):583-587
44. Treseler PA Foulks GN; Sanfilippo FI. Expression of ABO blood group, hematopoietic, and other cell-specific antigens by cells in the human cornea. Cornea, 1985;4(3): 157
45. Whitsett CF,Stulting RD.The distribution of HLA antigens on human corneal tissue. Invest Ophthalmol Vis Sci. 1984;25(5): 519-524
46. Rodrigues MM Rowden G; Hackett J; et al. Langerhans cells in the normal conjunctiva and peripheral cornea of selected species. Invest Ophthalmol Vis Sci. 1981 ;21 (5):759～765
47. Vantrappen L. Geboes K; Missotten L;et al. Lymphocytes and Langerhans cells in the normal human cornea. Invest Ophthalmol Vis Sci. 1985;26:220～225
48. Otto WR,Nanchahal J,Lu QL,et al.Survival of allogeneic cells in cultured organotypic skin grafts.Plast Reconstr Surg, 1995,96:166～176
49．李群．人角膜Langerhans细胞、T细胞和组织相容性抗原的免疫组化检测．中华眼科杂志，1992，28(3)：147～149
2. Williams KA,Roder D,Esterman A,et al. Factors predictive of corneal graft survival:report from the Australian Corneal Graft Registry. Ophthalmology, 1992,99:403～414
3. Williams-KA, Brereton-HM,Aggarwal-R,et al. Use of DNA polymorphisms and the polymerase chain reaction to examine the survival of a human limbal stem cell allograft. Am J Ophthalmol. 1995 Sep; 120:342～350
4. Kim JC,Tseng. Transplantation of opreserved human amniotic membrane for surface reconstruction in severely damaged rabbit corneas. Cornea. 1995; 14:475～484
5. Pellgrini G, Traverso CE,Franzi AT,et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet, 1997; 349:990～993
6. Zieske Jd,Bukusoglu G,Yankauckas MA.Characterization of a potential marker of corneal epithelial stem cells. Invest Ophthalmol Vis Sci. 1992,33:52～143
7. Kenyon KR,Tseng SC.Limbal autograft transplantation for ocular surface disorders.Ophthalmology, 1989;96:709
8. Davanger M, Evensen A, Role of the pericorneal papillary structure in renewal of corneal epithelium[J].Nature 1971 ;229:560～561
9. Kinoshita S, Friend J, Thoyt RA.Sex chromatin of donor corneal epithelium in rabbits[j]. Invest Ophthalmol Vis Sci 1981 ;21:434～441
10. Thoft RA, Friend J.The XYZ hypothesis of corneal epithelial maintenance. Invest Ophthalmol Vis Sci, 1983 ;24:1442
11. Schermer A,Galvin S, Sun TT. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol. 1986, 103:49～64
12. Tseng SC. Concept and application of limbal stem cells. Eye 1989;3:141～157
13. Tsai RJ, Tseng SCG, Effect of stromal inflammation on the outcome of limbal transplantation for corneal surface reconstructiono Cornea 1995; 14(5): 439
14. Thoft RA.Conj untival transplantion[J] .Arch Ophthalmol. 1977:95; 1425～1427
15. Tsai RJ, Li LM, Chen JK. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. N Engl J Med. 2000 Jul 13;343(2):86～93.
16. Tsai RJ,Tseng SCG.Human allograft limbal transplatation for corneal surface reconstruction. Cornea. 1994,13:389～400
17. Kenyon KR,Rapoza PA.Limbal allograft transplantation for ocular surface disorder[J]. Invest Ophthalmol Vis Sci. 1990;31:1301～1314
18. Lindberg KL ,Brown ME,Chaves HV, et al. In vitro propagation of human ocular surface epithelial cells for transplantation. Invest Ophthalmol Vis Sci, 1993;34:2672～2679
19. Koizumi N, Inatomi T, Quantock AJ, et al. Amniotic membrane as a substrate for cultivating limbal corneal epithelial cells for autologous transplantation in rabbits. Cornea. 2000 Jan; 19(1):65～71.
20. Ardjomand N,berghold A,Reich MeE.Loss of corneal langerhans cells during storage in organ culture medium,optisol and McCarey-Kaufman meduum[J].Eye 1998;12:134～138
21．吴静，徐锦堂，赵松滨。体外重建角膜上皮组织的实验研究。眼科研究，1999；17(2)：84～86
22. He YG, McCulley JP. Growing human corneal epithelium on collagen shield and subsequent transfer to denuded cornea in vitro. Curt Eye Res. 1991 Sep; 10(9):851～863.
23．沈玺．人羊膜移植治疗眼表面疾病．中国实用眼科杂志，1999，11：651～653
24. Adinofli M,Akle CA,Mccoll I,et al. Expression of HLA antigens, β 2-microglobulin and enzyme by human aminotic membrane[J].Nature 1982;295:325～372
25. De Rotth A. Plastic repair of conjunctival defects with fetal membrane.Arch ophthalmol. 1940;23:522～525
26. Tseng-SC;Prabhasawat-P;Lee-SH Amniotic membrane transplantation for conjunctival surface reconstruction. Am-J-Ophthalmol. 1997 Dec; 124(6): 765～774
27. Meller D, Tsteng SCG.In vitro Conjuntival epithelial differentiation on opreserved human amniotic membrane. Invest Ophthalmol Vis Sci. 1998,39(Suppl):S428
28. Shimazaki-J; Yang-HY; Tsubota-K. Amniotic membrane transplantation for ocular surface reconstruction in patients with chemical and thermal burns. Ophthalmology. 1997 Dec; 104(12): 2068～2076
29. Tseng-SC; Li-DQ; Ma-X Suppression of transforming growth factor-beta isoforms, TGF-beta receptor type Ⅱ, and myofibroblast differentiation in cultured human corneal and limbal fibroblasts by amniotic membrane matrix. J-Cell-Physiol. 1999 Jun; 179(3): 325-35
30. Kim-JC; Tseng-SC. The effects on inhibition of corneal neovascularization after human amniotic membrane transplantation in severely damaged rabbit corneas. Korean-J-Ophthalmol. 1995 Jun; 9(1): 32-46
31. Talmi-YP; Sigler-L; Inge-E et al. Placenta. Antibacterial properties of human amniotic membranes. 1991 May-Jun; 12(3): 285-8
32. Meler D,Pires RT,Mack RJ,et al. Amniotic membrane transplantation for acute chemical or thermal burns. Ophthalmology.2000;107:980
33. Koizumi N, Fullwood NJ, Bairaktaris G, et al. Cultivation of corneal epithelial cells on intact and denuded human amniotic membrane. Invest Ophthalmol Vis Sci. 2000 Aug;41:2506～2513
34. Schwab-IR. Cultured corneal epithelia for ocular surface disease. Trans-Am-Ophthalmol-Soc. 1999; 97:891～986
35. Pan ZQ ,Zhang WH. Study on proliferation and differentiation of human limbal stem cells in vitro. Chin Ophthal Res, 1999; 17(2): 81～83
36．薛庆善．体外培养的原理与技术．第一版．北京：科学出版社，2001．375～382
37. Kruse-FE, Tseng-SC. Serum differentially modulates the clonal growth and differentiation of cultured limbal and corneal epithelium. Invest Ophthalmol Vis Sci. 1993; 34(10): 2976～2989
38. Schwab IR, Reyes M, Isseroff RR. Successful transplantation of bioengineered tissue replacements in patients with ocular surface disease. Cornea, 2000;19(4):421～426
39. Zieske JD,Mason VS, Wasson ME,et al. Basemant membrane assembly and differentition of cultured corneal cells:importance of culture enviroment and endothelial cell interaction. Exp Cell Res. 1994;214:631
40. Bravo R,Frand R,Blundell PA,et al. Cyelin/PCNA is the auxiliary protein of DNA polymerase-δ[J].Nature 1987;326:515
41．潘志强，张文华，武宇影．培养角膜缘干细胞羊膜移植治疗碱烧伤动物的实验研究．中华眼科杂志，2000，36(1)：32～35
42．潘志强，张文华．培养角膜干细胞羊膜片移植治疗角膜缘功能障碍．中国实用眼科杂志，2000，18(9)：524～526
43. McBrida Bw McGill JI; Smith JL. MHC class Ⅰ and class Ⅱ antigen expression in normal human corneas and in corneas from cases of herpetic keratitis..Immunology, 1988;65(4):583-587
44. Treseler PA Foulks GN; Sanfilippo FI. Expression of ABO blood group, hematopoietic, and other cell-specific antigens by cells in the human cornea. Cornea, 1985;4(3): 157
45. Whitsett CF,Stulting RD.The distribution of HLA antigens on human corneal tissue. Invest Ophthalmol Vis Sci. 1984;25(5): 519-524
46. Rodrigues MM Rowden G; Hackett J; et al. Langerhans cells in the normal conjunctiva and peripheral cornea of selected species. Invest Ophthalmol Vis Sci. 1981 ;21 (5):759～765
47. Vantrappen L. Geboes K; Missotten L;et al. Lymphocytes and Langerhans cells in the normal human cornea. Invest Ophthalmol Vis Sci. 1985;26:220～225
48. Otto WR,Nanchahal J,Lu QL,et al.Survival of allogeneic cells in cultured organotypic skin grafts.Plast Reconstr Surg, 1995,96:166～176
49．李群．人角膜Langerhans细胞、T细胞和组织相容性抗原的免疫组化检测．中华眼科杂志，1992，28(3)：147～149