CTLA4-Ig基因转移抑制角膜移植术后免疫排斥反应的实验研究
摘要
目的
免疫排斥反应是角膜移植手术失败的主要原因，特别是高危病例，排斥率高达90％以上。研究如何预防和控制高危角膜移植术后的免疫排斥反应具有重要的意义。
T淋巴细胞介导的迟发性免疫排斥反应是角膜移植排斥反应的主要方式。最近大量的研究发现，基因重组合成的细胞毒T淋巴细胞抗原4(cytotoxic T lymphocyte antigen 4, CTLA4)可以高亲和力、特异地与APC细胞表面的B7结合，从而阻断CD28／B7这一共激活途径，使T淋巴细胞不能活化，进而阻止T淋巴细胞介导的免疫反应。目前在肾脏、心脏、皮肤等其它大器官移植中，人们已经发现利用CTLA4-Ig可以有效地延长移植器官的存活时间，甚至可以造成免疫耐受。
但是CTLA4-Ig应用于抑制器官移植免疫排斥反应仍存在许多问题。诸如CTLA4-Ig的半衰期短，只有50-70小时，临床应用时需反复用药；全身用药不方便，可能造成其它副作用等。因此，对CTLA4-Ig的局部应用，特别是通过转基因方法的应用是研究的重点。
目前CTLA4-Ig在角膜移植中的应用和研究很少，都没有完全阻止免疫排斥反应的发生。而且在转基因预防角膜移植免疫排斥反应的研究中，大部分研究是针对角膜内皮细胞的，理由是角膜内皮细胞是免疫排斥反应攻击的主要靶细胞。但同时又有大量研究表明，角膜基质和上皮细胞是主要的致敏
原，因此，在用药途径上，不应该只针对角膜内皮细胞，而应该重视供受体
的角膜基质和上皮。
本研究利用转基因技术，将cTLA4一19基因分别转染高危角膜移植动物
模型兔供体眼角膜基质或内皮细胞中，或兔受体眼前房中，然后观察何种途
径是抑制穿透性角膜移植术后免疫排斥反应的主要途径，并对CTLA4一Ig局
部转基因在抑制高危角膜移植免疫排斥反应中的作用作出评价。
材料和方法
一、携带目的基因的病毒载体
所有用于本实验的重组腺病毒载体都是来自El区缺失的5型人腺病毒
(一)携带报告基因的病毒载体
1.Ad一LacZ:携带LacZ基因的腺病毒载体。由美国宾西法尼亚大学医学
院基因治疗实验室陈有海教授惠赠。
2.Ad一GFP:携带编码绿荧光蛋白(green fluoreseenee protein，GFp)基因
的腺病毒载体。由美国宾西法尼亚大学医学院基因治疗实验室梁凤歧教授惠
赠。
(二)携带治疗基因的病毒载体
1.Ad一CTLA4一Ig(human):携带编码人细胞毒T淋巴细胞抗原4(cytotoxic
TLymphocyteantigen，CTLA4)细胞外部分和人IgGFc的复制缺陷腺病毒，
目的基因CTLA4一19由CMV启动子控制，分泌的CTLA4一Ig是可溶性的。由
台湾长庚大学医学院，长庚纪念医院内分泌科Brend Ray一Sea Hsu教授惠增。
2.Ad一CTLA4一Ig(mice):携带编码鼠CTLA4和人IgGFc的腺病毒载体。
由日本Yamaguchi University Sehool of Medieine，First Department of Surgery
的李桃生讲师惠赠。
(三)病毒扩增和纯化
根据以往报道的方法，将携带各种基因的重组腺病毒转染293细胞，大
量扩增后，反复冻融3次，经过两次CsCI梯度离心纯化。用快速空斑实验
测定病毒滴度。
二、病毒转染效率的检测
(一)病毒转染293细胞的检测
1.Ad一GFP检测:转染293细胞出现典型CPE后，荧光显微镜下观察照
才目。
2.Ad一cTLA4一lg检测:
1)Ad一CTLA4一Ig接种培养于24孔板的293细胞，内设1排空白孔。
2)按常规方法进行免疫组化检测，不同一抗分别用于不同培养孔。一抗
包括羊抗人CTLA4(DAKO，SC一1628)，羊抗人xgG(SxGMA，I一 3352)，羊抗
鼠CTLA4(Santa Cruz Bioteehnology，Ine.se一630，日本李桃生讲师惠赠)，
Hamster Anti一Mouse CTLA4一FITC(Fluoreseein eonjungate)(Southern
Bioteehnol。gy Assoeiates，Ine.1 790一02，李桃生讲师赠送)。
3)荧光标记二抗为(兔抗羊，FITC一eonjugated Rabbit Anti一Goat 19，DAKO，
FO25O)。
4)碳酸甘油封片，荧光显微镜下(Olympus BX6o，日本)观察，照相。
三.、病毒转染兔角膜组织
新西兰大白兔(New Zealand White Rabbit，NZW)，体重2.skg一3.okg，雌
雄不限。所有动物都是在山东省眼科学重点实验室动物饲养中心饲养，并符
合ARvO有关用于研究目的动物的相关规定(ARVOResolution on the Use of
Animals in Research)。
(二)角膜上皮转染
1.Ad一LacZ:取6只NZW兔，右眼角膜上皮划痕法接种100扒1
l.4x一o8PFU/mlAd一Laez病毒液。取2只眼作为空白对照，接种Hank’S液。
在接种后24h，48h，72h各处死2只兔，取角膜片，x一Gal检测法检测基因表达。
2，Ad一GFP:不划痕结膜囊滴入1 00 p 1 1 .4x10sPFu/m1Ad一GFP，关闭眼
睑，按摩30秒。共接种6只兔的右眼，另外设2只空白对照眼，接种Hank’s
液。在接种后24h，48h，72h各处死2只兔，取角膜片，荧光显微镜下观察有无
荧光着色。空白对照眼进行同样观察。
(砚)结膜下转染
1.Ad一Lacz:取6只Nzw兔，分别在右眼结膜下注射loopl 1.4xl护
PFU/ml Ad一LacZ和Ad一GFP。检测方法同上。
(四)角膜内皮转染:?
Experimental study on suppress of high-risk corneal immune rejection by local CTLA4-Ig expression Shaowei Li, Directed by Professor Lixin Xie.
Purpose
Immune rejection is the leading cause of corneal allograft failure,especially for the high-risk cases[l]. The critical role of T cells in allograft rejection is well established. CTLA4-Ig, a recombinant form of CTLA4 has been shown to inhitit T-Cell activation by blocking the costimulatory pathway, and thereby prolong graft survival and even produce donor-specific tolerance in some animal transplant models[2-7].However, there still remain many unresolved issues with respect to how best to use CTLA4-Ig in clinical setting. In of which, the relative shorter serum elimination half-life of the recombind CTLA4-Ig is concerned[3], and led to seeking of the potential of using gene therapy.
Several studies were conducted in preventing corneal allograft by using CTLA4-Ig, but none of them demonstrated long-term graft survival[8-ll].It was noted that most of the studies using gene transfer were focused on endothelial layer. The reaseon is that it is believed that the major target of corneal graft rejection is the corneal endothelium. But numourus studies have shown that it was mainly the corneal stoma and epithelial layer act as the alloantigen[12].
In this study, we explored the potential use of gene therapy in preventing of corneal rejection by transferring Ad-CTLA4-Ig to the different layers or sites of the donor or recipient eye in rabbit model.
Materials and Methods
Recombinant adenoviruses
Ad-LacZ was the kind gift of Dr.Youhai Chen(Department of Molecular and Cellular engineering, University of Pennsylvania, School of Mecicine). Ad-GFP was the kind gift of Dr.Frank Liang(F.M. Kirby Center for Molecualr Ophthalmology, Department of Ophthalmology, University of Pennsylvania). Ad-CTLA4-Ig(human)was the kind of gift of Dr. Brend Ray-Sea Hsu(Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang-Gung Memorial hospital and Chan Gung University Medical College, Taiwan).Ad-CTLA4-Ig(mice) was a kind gift of Dr.Taosheng Li(First Department of Surgery,Yamaguchi University School of Medicine,Japan).
The recombined adenovirus vectors were propagated in 293 cells and purified by centrifugation over caesium chloride gradients and titred following standard protocols[13-15].
Detection of gene expression
Green fluorescence protein expressed by Ad-GFP was observed under fluorescence microscope. LacZ gene expression was detected by X-Gal solution described previously[16]. CTLA4-Ig expression was detected by immunohistochemistry using goat antihuman IgG antibody(Sigma, 1-3382) as primary antibodies, and FITC-conjugated Rabbit Anti-Goat Ig, DAKO, F0250) as secondary antibodies.The results were observed under fluorescence microscope.
Adenovirus-mediated Gene Transfer
The Ad-CTLA4-Ig, Ad-LacZ, Ad-GFP were transferred to the corneal endothelium according to the method described before[ll,17]. Briefly, NZW rabbit corneas were incubated in 1ml 2% fetal calf serum(FCS) in DMEM containing 1.4X105 PFU/ml of Ad-CTLA4-Ig or AdD-LacZ or Ad-GFP for 3 hours at 37C in 5%CO2 After incubation, specimens were washed three times with Hanks' balanced salt solution. The corneas were translated to a healthy NZW rabbit eye. The gene expression was examined at different time point after penetrating keratoplasty(PKP).
Genes were transfected into corneal stoma by direct injection of recombinant adenoviruses using a 1ml syringe with a 30 gage needle. The expression of transferred gene were examined at different time point.
Gene in vivo transferring to rabbit anterior chamber was conducted according to the method described previously[18], in which the recombinant adenoviruses were injected into the anterior chamber.
Penetrating keratplasty
New Zealand White (NZW) rabbits of both sexes weighing between 2.5-3.Okg were used as experimental model. Throughout this study, all animals were handled according to the ARVO Resolution on the Use of Animal
免疫排斥反应是角膜移植手术失败的主要原因，特别是高危病例，排斥率高达90％以上。研究如何预防和控制高危角膜移植术后的免疫排斥反应具有重要的意义。
T淋巴细胞介导的迟发性免疫排斥反应是角膜移植排斥反应的主要方式。最近大量的研究发现，基因重组合成的细胞毒T淋巴细胞抗原4(cytotoxic T lymphocyte antigen 4, CTLA4)可以高亲和力、特异地与APC细胞表面的B7结合，从而阻断CD28／B7这一共激活途径，使T淋巴细胞不能活化，进而阻止T淋巴细胞介导的免疫反应。目前在肾脏、心脏、皮肤等其它大器官移植中，人们已经发现利用CTLA4-Ig可以有效地延长移植器官的存活时间，甚至可以造成免疫耐受。
但是CTLA4-Ig应用于抑制器官移植免疫排斥反应仍存在许多问题。诸如CTLA4-Ig的半衰期短，只有50-70小时，临床应用时需反复用药；全身用药不方便，可能造成其它副作用等。因此，对CTLA4-Ig的局部应用，特别是通过转基因方法的应用是研究的重点。
目前CTLA4-Ig在角膜移植中的应用和研究很少，都没有完全阻止免疫排斥反应的发生。而且在转基因预防角膜移植免疫排斥反应的研究中，大部分研究是针对角膜内皮细胞的，理由是角膜内皮细胞是免疫排斥反应攻击的主要靶细胞。但同时又有大量研究表明，角膜基质和上皮细胞是主要的致敏
原，因此，在用药途径上，不应该只针对角膜内皮细胞，而应该重视供受体
的角膜基质和上皮。
本研究利用转基因技术，将cTLA4一19基因分别转染高危角膜移植动物
模型兔供体眼角膜基质或内皮细胞中，或兔受体眼前房中，然后观察何种途
径是抑制穿透性角膜移植术后免疫排斥反应的主要途径，并对CTLA4一Ig局
部转基因在抑制高危角膜移植免疫排斥反应中的作用作出评价。
材料和方法
一、携带目的基因的病毒载体
所有用于本实验的重组腺病毒载体都是来自El区缺失的5型人腺病毒
(一)携带报告基因的病毒载体
1.Ad一LacZ:携带LacZ基因的腺病毒载体。由美国宾西法尼亚大学医学
院基因治疗实验室陈有海教授惠赠。
2.Ad一GFP:携带编码绿荧光蛋白(green fluoreseenee protein，GFp)基因
的腺病毒载体。由美国宾西法尼亚大学医学院基因治疗实验室梁凤歧教授惠
赠。
(二)携带治疗基因的病毒载体
1.Ad一CTLA4一Ig(human):携带编码人细胞毒T淋巴细胞抗原4(cytotoxic
TLymphocyteantigen，CTLA4)细胞外部分和人IgGFc的复制缺陷腺病毒，
目的基因CTLA4一19由CMV启动子控制，分泌的CTLA4一Ig是可溶性的。由
台湾长庚大学医学院，长庚纪念医院内分泌科Brend Ray一Sea Hsu教授惠增。
2.Ad一CTLA4一Ig(mice):携带编码鼠CTLA4和人IgGFc的腺病毒载体。
由日本Yamaguchi University Sehool of Medieine，First Department of Surgery
的李桃生讲师惠赠。
(三)病毒扩增和纯化
根据以往报道的方法，将携带各种基因的重组腺病毒转染293细胞，大
量扩增后，反复冻融3次，经过两次CsCI梯度离心纯化。用快速空斑实验
测定病毒滴度。
二、病毒转染效率的检测
(一)病毒转染293细胞的检测
1.Ad一GFP检测:转染293细胞出现典型CPE后，荧光显微镜下观察照
才目。
2.Ad一cTLA4一lg检测:
1)Ad一CTLA4一Ig接种培养于24孔板的293细胞，内设1排空白孔。
2)按常规方法进行免疫组化检测，不同一抗分别用于不同培养孔。一抗
包括羊抗人CTLA4(DAKO，SC一1628)，羊抗人xgG(SxGMA，I一 3352)，羊抗
鼠CTLA4(Santa Cruz Bioteehnology，Ine.se一630，日本李桃生讲师惠赠)，
Hamster Anti一Mouse CTLA4一FITC(Fluoreseein eonjungate)(Southern
Bioteehnol。gy Assoeiates，Ine.1 790一02，李桃生讲师赠送)。
3)荧光标记二抗为(兔抗羊，FITC一eonjugated Rabbit Anti一Goat 19，DAKO，
FO25O)。
4)碳酸甘油封片，荧光显微镜下(Olympus BX6o，日本)观察，照相。
三.、病毒转染兔角膜组织
新西兰大白兔(New Zealand White Rabbit，NZW)，体重2.skg一3.okg，雌
雄不限。所有动物都是在山东省眼科学重点实验室动物饲养中心饲养，并符
合ARvO有关用于研究目的动物的相关规定(ARVOResolution on the Use of
Animals in Research)。
(二)角膜上皮转染
1.Ad一LacZ:取6只NZW兔，右眼角膜上皮划痕法接种100扒1
l.4x一o8PFU/mlAd一Laez病毒液。取2只眼作为空白对照，接种Hank’S液。
在接种后24h，48h，72h各处死2只兔，取角膜片，x一Gal检测法检测基因表达。
2，Ad一GFP:不划痕结膜囊滴入1 00 p 1 1 .4x10sPFu/m1Ad一GFP，关闭眼
睑，按摩30秒。共接种6只兔的右眼，另外设2只空白对照眼，接种Hank’s
液。在接种后24h，48h，72h各处死2只兔，取角膜片，荧光显微镜下观察有无
荧光着色。空白对照眼进行同样观察。
(砚)结膜下转染
1.Ad一Lacz:取6只Nzw兔，分别在右眼结膜下注射loopl 1.4xl护
PFU/ml Ad一LacZ和Ad一GFP。检测方法同上。
(四)角膜内皮转染:?
Experimental study on suppress of high-risk corneal immune rejection by local CTLA4-Ig expression Shaowei Li, Directed by Professor Lixin Xie.
Purpose
Immune rejection is the leading cause of corneal allograft failure,especially for the high-risk cases[l]. The critical role of T cells in allograft rejection is well established. CTLA4-Ig, a recombinant form of CTLA4 has been shown to inhitit T-Cell activation by blocking the costimulatory pathway, and thereby prolong graft survival and even produce donor-specific tolerance in some animal transplant models[2-7].However, there still remain many unresolved issues with respect to how best to use CTLA4-Ig in clinical setting. In of which, the relative shorter serum elimination half-life of the recombind CTLA4-Ig is concerned[3], and led to seeking of the potential of using gene therapy.
Several studies were conducted in preventing corneal allograft by using CTLA4-Ig, but none of them demonstrated long-term graft survival[8-ll].It was noted that most of the studies using gene transfer were focused on endothelial layer. The reaseon is that it is believed that the major target of corneal graft rejection is the corneal endothelium. But numourus studies have shown that it was mainly the corneal stoma and epithelial layer act as the alloantigen[12].
In this study, we explored the potential use of gene therapy in preventing of corneal rejection by transferring Ad-CTLA4-Ig to the different layers or sites of the donor or recipient eye in rabbit model.
Materials and Methods
Recombinant adenoviruses
Ad-LacZ was the kind gift of Dr.Youhai Chen(Department of Molecular and Cellular engineering, University of Pennsylvania, School of Mecicine). Ad-GFP was the kind gift of Dr.Frank Liang(F.M. Kirby Center for Molecualr Ophthalmology, Department of Ophthalmology, University of Pennsylvania). Ad-CTLA4-Ig(human)was the kind of gift of Dr. Brend Ray-Sea Hsu(Division of Endocrinology and Metabolism, Department of Internal Medicine, Chang-Gung Memorial hospital and Chan Gung University Medical College, Taiwan).Ad-CTLA4-Ig(mice) was a kind gift of Dr.Taosheng Li(First Department of Surgery,Yamaguchi University School of Medicine,Japan).
The recombined adenovirus vectors were propagated in 293 cells and purified by centrifugation over caesium chloride gradients and titred following standard protocols[13-15].
Detection of gene expression
Green fluorescence protein expressed by Ad-GFP was observed under fluorescence microscope. LacZ gene expression was detected by X-Gal solution described previously[16]. CTLA4-Ig expression was detected by immunohistochemistry using goat antihuman IgG antibody(Sigma, 1-3382) as primary antibodies, and FITC-conjugated Rabbit Anti-Goat Ig, DAKO, F0250) as secondary antibodies.The results were observed under fluorescence microscope.
Adenovirus-mediated Gene Transfer
The Ad-CTLA4-Ig, Ad-LacZ, Ad-GFP were transferred to the corneal endothelium according to the method described before[ll,17]. Briefly, NZW rabbit corneas were incubated in 1ml 2% fetal calf serum(FCS) in DMEM containing 1.4X105 PFU/ml of Ad-CTLA4-Ig or AdD-LacZ or Ad-GFP for 3 hours at 37C in 5%CO2 After incubation, specimens were washed three times with Hanks' balanced salt solution. The corneas were translated to a healthy NZW rabbit eye. The gene expression was examined at different time point after penetrating keratoplasty(PKP).
Genes were transfected into corneal stoma by direct injection of recombinant adenoviruses using a 1ml syringe with a 30 gage needle. The expression of transferred gene were examined at different time point.
Gene in vivo transferring to rabbit anterior chamber was conducted according to the method described previously[18], in which the recombinant adenoviruses were injected into the anterior chamber.
Penetrating keratplasty
New Zealand White (NZW) rabbits of both sexes weighing between 2.5-3.Okg were used as experimental model. Throughout this study, all animals were handled according to the ARVO Resolution on the Use of Animal
引文
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12． Niederkorn JY, Mayhew E. UVB irradiation renders corneal allografts tolerogenic for allospecific delayed hypersensitivity responses. Immunology 1993;79:278-84．
13． Hill JC, Sarvan J, Maske R, et al. Evidence that UV-G irradiation decreases corneal Langerhans cells and improves corneal graft survival in the rabbit. Transplantation 1994;57:1281-4．
14． Sayegh MH, Turka LA. The role of T-cell costimulatory activation pathways in transplant rejection. The New England Journal of Medicine. 1998;338:1813-21．
15． Janeway CA, Bottomly K. Signals and signs for lymphocyte responses. Cell. 1994;76:275-85．
16． Sayegh MH. Finally, CTLA4-Ig graduates to the clinic. The Journal of Clinical Investigation, 1999;103:1223-5．
17． Srinivas NR, Weiner RS, Shyu WC, et al. A pharmacokinetic study of intravenous CTLA4Ig, a novel immunosuppressive agent, in mice. J. Pharm.Sci, 1996;85:296-298．
18． Lenschow DJ, Zeng Y, Thistlethwaite JR, et al. Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4Ig. Science 1992;257:789-792．
19． Tu Y, Rehman A, Flye NW: CTLA4-Ig treatment prolongs rat orthotopic liver graft survival. Transplant Proc.1997;29:1036-1037．
20． Yin DP, Sankary HN, Williams J, et al. Induction of tolerance to small bowel allografts in high-responder rats by combining anti-CD4 with CTLA4Ig. Transplantation, 1996;62:1537-1539．
21． Ginan EC, Boussiotis VA, Neuberg D, et al. Transplantation of anergic histoincompatible bone marrow allografts. N.Engl.L.Med. 1999;340:1704-1714．
22． Srinivas NR, Weiner RS, Shyu WC, et al. A pharmacokinetic study of intravenous CTLA4Ig, a novel immunosuppressive agent, in mice. J. Pharm.Sci,
1996;85:296-298．
23． Gebhardt BM,hodkin M, Varnell ED, et al. Protection of corneal allografts by CTLA4-Ig.,Cornea. 1999;18:314-20．
24． Konig MSA, Zhang EP, Wittig B, et al. Ballistic transfer of minimalistic immunologically defined expression constructs for IL4 and CTLA4 into the corneal epithelium in mice after orthotopic corneal allograft transplantation. Graefes Arch Clin Exp Ophthalmol. 2000;238:701-7．
25． Comer RM, King WJ, Ardjomand N, etal. Effect of administration of CTLA4-Ig as protein or cDNA on corneal allograft survival. Invest Ophthalmol Vis Sci. 2002;43:1095-103．
26． Nicholls SM, Williams NA. MHC matching and mechanisms of alloactivation in corneal transplantation. Transplantation 2001 ;72:1491-7．
27． George AJT, Arancibia-Carcamo CV, Avad HM, et al. Gene delivery to the corneal endothelium. Am J Respir Crit Care Med, 2000;162:S194-200．
28． Hori J, Joyce NC, Streilein JW. Immune privilege and immunogenicity reside among different layers of the mouse cornea. Invest Ophthalmol Vis Sci 2000;41:3032-42．
29． Fehervari Z, Rayner SA, Oral HB, et al. Gene transfer to Ex Vivo stored corneas. Cornea 1997,16:459-64．
30． 王旭，谢立信，史伟云。外源基因转入角膜内皮细胞的实验研究。中华眼科杂志，2002，38：117-118。
31． Budenz DL, Bennett J, Alonso L, et al. In vivo gene transfer into murine corneal endothelial and trabecular meshwork cells. IOVS 1995,36:2211-15．
32: Larkin DFP, Oral HB, Ring CJA, et al. Adenovirus-mediated gene delivery to the corneal endothelium. Transplantation, 1996,61:363-370．
33． Ritter T, et al. Adenovirus-mediated gene transfer of interleukin-4 to corneal endothelial cells and organ cultured corneas leads to high IL-4 expression. Curr Eye Res 1999,69:563-8．
34． Oral HB, Larkin DFP, Fehervari Z, et al. Ex Vivo adenovirus-mediated gene ransfer and immunomodulatory protein production in human cornea. Gene therapy, 1997,4:639-47．
35． Feldman ST, Tozer JR,Wang S, et al. Gene transfer to the anterior segment of the eye by infection with recombinant viral vector. Invest Ophthalmol Vis Sci 1994,35:1383．
36． Zhang EP, Muller A, Schulte F, et al. Minimizing side effects of ballistic gene transfer into the murine corneal epithelium. Graefes Arch Clin Exp Ophthalmol 2002 240: 114-9
37． Torres PF, Siegers TPAM, Peek R, et al. Changes in cytokine mRNA levels in experimental corneal allografts after local clodronate-liposome treatment. IOVS 1999;40:3194-201．
38． Hoffmann F, Zhang EP, Pohl T, Kunzendorf U, et al. Inhibition of corneal allograft reaction by CTLA4-Ig. Graefes Arch Clin Exp Ophthalmol 1997;235:535-40．
39． Rayner SA, Larkin DFP, George AJT. TNF receptor secretion after Ex Vivo adenoviral gene transfer to cornea and effect on in vivo graft survival. IOVS, 2001,42:1568-72．
40． 郭萍，谢立信，史伟云，等．肿瘤坏死因子相关凋亡诱导配体转基因治疗鼠角膜移植免疫排斥的研究．中华眼科杂志，2003，39：19-23．
41． Klebe S, Sykes PJ, Coster DJ, et al. Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10． Transplantation 2001,71:1214-20．
42． Dekaris I, Zhu SN, Dana MR. TNF-a regulates corneal Langerhans cell migration. The Journal of Immunology. 1999;162:4235-39．
43． Steurer W, Nickerson PW, Steele AW, et al. Ex Vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance. The Journal of Immunology, 1995,155:1165-74．
44． Yamagami S, Dana MR. The critical role of lymph nodes in corneal alloimmunization and graft rejection. Invest Ophthalmol Vis Sci. 2001;42:1293-98．
45． Lua WT, Leeb WC, Hsu BRS, et al. effects of virus expressing CTLA4-Ig and IL1-RA on islet xenografts. Transplantation Proceedings, 2001;33:713-14．
46． Li TS, Hamano K, Kajiwara K, et al. Prolonged survival of xenograft fetal cardiomyocytes by adenovirus-mediated CTLA4-Ig expression. Transplantation, 2001,95:217-26．
47． Lin H, Boiling SF, Linsley PS, et al. Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4-Ig plus donor-specific transfusion. J Exp Med 1993;178:1801-6．
48． Walunas TL, Bakker CY, Bluestone JA. CTLA41igation blocks CD28-dependent T cell activation. J Exp Med 1996; 183:2541-50．
2． Zirm E. Eine erfolgreiche totall keratoplastik. Albrecht Von Graefes Arch Ophthalmol 1906;64:580．
3． The collaborative corneal transplantation studies research group. The collaborative corneal transplantation studies(CCTS):effectiveness of histocompatibility matching in high-risk corneal transplantation. Arch Ophthalmol 1992;110:1392．
4． Foulks GN, Niederkorn JY, Sanfilippo F. Corneal allograft rejection. In:Pepose JS, Holland GN, Wilhelmus KR,eds. Ocular infection and immunity. St.LouisrMosby, 1996:435．
5． Niederkorn JY. Immune privilege and immune regulation in the eye. Adv Immunol 1990;48:191-226．
6． Mader TH, Stulting RD. The high-risk penetrating keratoplasty. Ophthalmol Clin North Am 1991 ;4:411-26．
7． Larkin DFP,Calder VL, Lightman SL. Identification and characterization of cells infiltrating the graft and aqueous humor in rat corneal allograft rejection. Clin Exp Immunol 1997;107:381-91．
8． Pleyer U, Dannowski H, Volk HD, et al. Corneal allograft rejectionxurrent understanding. Ophthalmologica 2001;215:254-262．
9． Sonoda Y, Streilein JW. Impaired cell-mediated immunity in mice bearing healthy orthotopic corneal allografts. J Immunol. 1993; 150:1727-34．
10． Joo CK, Pepose JS, Stuart PM. T-cell mediated responses in a murine model of orthotopic corneal transplantation. Invest Ophthalmol Vis Sci. 1995;36:1530-40．
11． Niederkorn JY, Mellon J. Anterior chamber-associated immune deviation promotes corneal allograft survival. Invest Ophthalmol Vis Sci. 1996;37:2700-07．
12． Niederkorn JY, Mayhew E. UVB irradiation renders corneal allografts tolerogenic for allospecific delayed hypersensitivity responses. Immunology 1993;79:278-84．
13． Hill JC, Sarvan J, Maske R, et al. Evidence that UV-G irradiation decreases corneal Langerhans cells and improves corneal graft survival in the rabbit. Transplantation 1994;57:1281-4．
14． Sayegh MH, Turka LA. The role of T-cell costimulatory activation pathways in transplant rejection. The New England Journal of Medicine. 1998;338:1813-21．
15． Janeway CA, Bottomly K. Signals and signs for lymphocyte responses. Cell. 1994;76:275-85．
16． Sayegh MH. Finally, CTLA4-Ig graduates to the clinic. The Journal of Clinical Investigation, 1999;103:1223-5．
17． Srinivas NR, Weiner RS, Shyu WC, et al. A pharmacokinetic study of intravenous CTLA4Ig, a novel immunosuppressive agent, in mice. J. Pharm.Sci, 1996;85:296-298．
18． Lenschow DJ, Zeng Y, Thistlethwaite JR, et al. Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4Ig. Science 1992;257:789-792．
19． Tu Y, Rehman A, Flye NW: CTLA4-Ig treatment prolongs rat orthotopic liver graft survival. Transplant Proc.1997;29:1036-1037．
20． Yin DP, Sankary HN, Williams J, et al. Induction of tolerance to small bowel allografts in high-responder rats by combining anti-CD4 with CTLA4Ig. Transplantation, 1996;62:1537-1539．
21． Ginan EC, Boussiotis VA, Neuberg D, et al. Transplantation of anergic histoincompatible bone marrow allografts. N.Engl.L.Med. 1999;340:1704-1714．
22． Srinivas NR, Weiner RS, Shyu WC, et al. A pharmacokinetic study of intravenous CTLA4Ig, a novel immunosuppressive agent, in mice. J. Pharm.Sci,
1996;85:296-298．
23． Gebhardt BM,hodkin M, Varnell ED, et al. Protection of corneal allografts by CTLA4-Ig.,Cornea. 1999;18:314-20．
24． Konig MSA, Zhang EP, Wittig B, et al. Ballistic transfer of minimalistic immunologically defined expression constructs for IL4 and CTLA4 into the corneal epithelium in mice after orthotopic corneal allograft transplantation. Graefes Arch Clin Exp Ophthalmol. 2000;238:701-7．
25． Comer RM, King WJ, Ardjomand N, etal. Effect of administration of CTLA4-Ig as protein or cDNA on corneal allograft survival. Invest Ophthalmol Vis Sci. 2002;43:1095-103．
26． Nicholls SM, Williams NA. MHC matching and mechanisms of alloactivation in corneal transplantation. Transplantation 2001 ;72:1491-7．
27． George AJT, Arancibia-Carcamo CV, Avad HM, et al. Gene delivery to the corneal endothelium. Am J Respir Crit Care Med, 2000;162:S194-200．
28． Hori J, Joyce NC, Streilein JW. Immune privilege and immunogenicity reside among different layers of the mouse cornea. Invest Ophthalmol Vis Sci 2000;41:3032-42．
29． Fehervari Z, Rayner SA, Oral HB, et al. Gene transfer to Ex Vivo stored corneas. Cornea 1997,16:459-64．
30． 王旭，谢立信，史伟云。外源基因转入角膜内皮细胞的实验研究。中华眼科杂志，2002，38：117-118。
31． Budenz DL, Bennett J, Alonso L, et al. In vivo gene transfer into murine corneal endothelial and trabecular meshwork cells. IOVS 1995,36:2211-15．
32: Larkin DFP, Oral HB, Ring CJA, et al. Adenovirus-mediated gene delivery to the corneal endothelium. Transplantation, 1996,61:363-370．
33． Ritter T, et al. Adenovirus-mediated gene transfer of interleukin-4 to corneal endothelial cells and organ cultured corneas leads to high IL-4 expression. Curr Eye Res 1999,69:563-8．
34． Oral HB, Larkin DFP, Fehervari Z, et al. Ex Vivo adenovirus-mediated gene ransfer and immunomodulatory protein production in human cornea. Gene therapy, 1997,4:639-47．
35． Feldman ST, Tozer JR,Wang S, et al. Gene transfer to the anterior segment of the eye by infection with recombinant viral vector. Invest Ophthalmol Vis Sci 1994,35:1383．
36． Zhang EP, Muller A, Schulte F, et al. Minimizing side effects of ballistic gene transfer into the murine corneal epithelium. Graefes Arch Clin Exp Ophthalmol 2002 240: 114-9
37． Torres PF, Siegers TPAM, Peek R, et al. Changes in cytokine mRNA levels in experimental corneal allografts after local clodronate-liposome treatment. IOVS 1999;40:3194-201．
38． Hoffmann F, Zhang EP, Pohl T, Kunzendorf U, et al. Inhibition of corneal allograft reaction by CTLA4-Ig. Graefes Arch Clin Exp Ophthalmol 1997;235:535-40．
39． Rayner SA, Larkin DFP, George AJT. TNF receptor secretion after Ex Vivo adenoviral gene transfer to cornea and effect on in vivo graft survival. IOVS, 2001,42:1568-72．
40． 郭萍，谢立信，史伟云，等．肿瘤坏死因子相关凋亡诱导配体转基因治疗鼠角膜移植免疫排斥的研究．中华眼科杂志，2003，39：19-23．
41． Klebe S, Sykes PJ, Coster DJ, et al. Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10． Transplantation 2001,71:1214-20．
42． Dekaris I, Zhu SN, Dana MR. TNF-a regulates corneal Langerhans cell migration. The Journal of Immunology. 1999;162:4235-39．
43． Steurer W, Nickerson PW, Steele AW, et al. Ex Vivo coating of islet cell allografts with murine CTLA4/Fc promotes graft tolerance. The Journal of Immunology, 1995,155:1165-74．
44． Yamagami S, Dana MR. The critical role of lymph nodes in corneal alloimmunization and graft rejection. Invest Ophthalmol Vis Sci. 2001;42:1293-98．
45． Lua WT, Leeb WC, Hsu BRS, et al. effects of virus expressing CTLA4-Ig and IL1-RA on islet xenografts. Transplantation Proceedings, 2001;33:713-14．
46． Li TS, Hamano K, Kajiwara K, et al. Prolonged survival of xenograft fetal cardiomyocytes by adenovirus-mediated CTLA4-Ig expression. Transplantation, 2001,95:217-26．
47． Lin H, Boiling SF, Linsley PS, et al. Long-term acceptance of major histocompatibility complex mismatched cardiac allografts induced by CTLA4-Ig plus donor-specific transfusion. J Exp Med 1993;178:1801-6．
48． Walunas TL, Bakker CY, Bluestone JA. CTLA41igation blocks CD28-dependent T cell activation. J Exp Med 1996; 183:2541-50．