基因转移huCTLA4-Ig诱导大鼠移植肝免疫耐受的实验研究
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摘要
免疫抑制剂的应用使器官移植获得了很大的发展，然而，终身免疫抑制所带来的毒性、机会感染及肿瘤的高发病率成为临床器官移植所面临的重要问题。供体特异性器官移植免疫耐受，即在接受组织配型不相容的器官移植或加短疗程治疗后达到不用药(常规免疫抑制剂)、不排斥、不感染的“三不状态”是器官移植发展的方向。免疫干预新策略的应用，有可能给上述问题带来解决之道。与全身性给予免疫抑制性生物制剂相比，基因治疗技术在器官移植领域具有巨大的潜在应用价值。针对移植物局部产生的免疫抑制分子可能会提高它们的治疗效果，并减少它们对全身的影响。
可溶性重组融合蛋白huCTLA4-Ig是一种新颖的免疫抑制剂，分子量约为92KD，由人CTLA-4分子的胞膜外区与人IgG1的Fc段(铰链区与恒定区)融合形成，能牢固地与B7分子结合，阻断CD28介导的共刺激信号，导致免疫反应的抑制。给予huCTLA4-Ig，短期阻断CD28介导的共识别可导致MHC不匹配的啮齿类模型中移植物的长期存活与耐受。腺病毒介导的针对移植物的免疫调节基因的转移，是调控免疫反应的新策略。目前，关于基因转移huCTLA4-Ig诱导同种异体移植物耐受的研究，国外仅有少数实验室开展，国内未见报道。
我们采用AdEasy腺病毒载体系统，自行构建携带huCTLA4-Ig的重组腺病毒，通过先体外后体内(ex vivo)基因转移技术，于供肝冷保存时，将该治疗基因导入大鼠移植肝，使其于移植肝局部表达，观察其抑制排斥反应，诱导大鼠移植肝免疫耐受的作用。通过本研究，旨在探讨：在无常规免疫抑制剂应用的情况下，先体外后体内基因转移免疫抑制分子获得同种异体移植物耐受的可能性。
主要实验结果如下：
1．成功构建了携带融合基因huCTLA4-Ig的重组腺病毒，滴度为6.0×10~(13)PFU／L。该重组病毒在体外能有效感染正常肝细胞株L-O2，受感染细胞能表达、分泌可溶性的重组融合蛋白huCTLA4-Ig；
2．建立了大鼠BN(RT1~n)→LEW(RT1~l)原位肝移植同种异体排斥模型，该模
博士学位论义 基因转移hU＊＊LA个ig诱导大鼠移植肝免疫耐受的实验研究 中文摘要
型排斥反应时间较为恒定，在不应用免疫抑制剂的情况下，生存期＜3周（平均生存时
问人］6 6士 2．5 d，11一引；
3．供肝冷保存时，采用血管夹技术＊lamp technique）经门静脉灌注携带融合基
因hllCTLA4dg的重组腺病毒，于术后3天、7天能定性检测到hllCTLA4Ig在受体外
划血卜1。的外分泌性表达及供肝J］－I‘细胞内的表达，以后迅速下降，至术后2 8天几乎不能
检测到。
个讹注亚组腺炳边组（A组，n—5）或灌注杉引IZ‘报告基囚GFP的亚组腺病毒组（B
纵，；。一4），受仰鼠均在3川内死亡，平均生存时问A组为16石土2．5天，B组为15．5
土3．1大，两组问无显著性差异V－0．35，尸＞0刀5八 而灌注携带融合基因＊＊＊＊A个ig
的重组腺病毒组 ＊组，fi—5）受体鼠均能获得长期生存（＞150天）；A组与C组间、
B组与C组间，生存期均有显著性差异’（P＜0刀1人
A组与B组，在术后8天行移植肝活检，病理学检查证实：移植肝均发生严重的
细胞性排斥。C组，移植肝病理学检查发现：术后8天，可见移植肝轻、中度的汇管
区炎症，但组织损伤程度显著减轻：术后150天，单核细胞浸润亦能观察到，但胆管
保存完好，无血管损伤证据。
术前大鼠血 ＊1卜2浓度为362们土45．84 n＊几；肝移械 术后，＊ 宁1卜2的浓度在
A、B组中即升高到较高水平（约为术前的1．5－2．5倍），相反地，在C组中其始终维
持在一个较低水平（按近或低于术前水平），术后3、7天，血清IL－二的浓度在A、C
组；句及 B、C组 l”。J差异均非常显著（P＜0．of）。
结论：本研究在国内率先建立了简便有效的针对血管化移植物的基因转移系统，
该系统中重组腺病毒在冷保存时能有效感染供肝，并获得融合基因的分泌性表达。移
植肝局部产生的可溶性融合蛋白hllCTLA4Ig，能抑制同种异体免疫反应，在不使用常－
规兔疫抑制剂情况下，获得大鼠同种异体移植肝的长期存活。初步证实本研究建立的
器官移植基因治疗方法具有简便、有效的特点。本研究对于临床肝移植具有一定的潜
在应用价值，为免疫调节基因转移技术用于肝移植临床，以防治移植肝术后免疫排斥
反应的发十，仆狄衍移伙川’的见疫耐受捉仪了初步的实验依抓。
【关键词】肝移植;基因转移;免疫耐受;CTLA4-Ig
With the uSe of immunosuppressive drugs that efficiently control acute rejection, Organ
transplantation has achieved impressive development. Nevertheless, clinical transplantation still
thces some important problems: lilblong immunosuppression is associated with toxicity,
oppottunistic infections and a high incidence of cancer. Donor specific organ transplantation
immune tolerance, which is defined as no medication (routine immunosuppressive agents), no
rejection and no infection after MHC mismatched organ transplantatlon, is the trend of organ
transplantation. Solutions to these problems will come from the application of new strategies of
immunointervefltion. The application of gene therapy techniques to transplantation has great
potential theraPeutic advantages over systemic administration of the immunosuppressive
bioreagents. Local production of immunosuppressive molecules may increase their therapeutic
efficiency and reduce their systemic side-effects.
HuCTLA4-lg,' one of the most potent in1n1unosuppressive molecules, is a soluble
recombinant fusion protein (molecu1ar weight of approximately 92 kD) consisting of the
extracellular domain of human CTLA-4 and a fragment (hinge and constant region) of the Fc
portion of human IgGl. It strongly adheres to the B7 molecule to block the CD28-mediated
costimulatory signal, resulting in inhibition of in vitro and in vivo immune response. With
administration of CTLA4-Ig, short-term blockade of CD28-mediated corecognition resulted in
prolonged graft survival and tolerance in MHC mismatched rodent models.
Adenoviral-mediated transfer to graft of immunoregulatory genes is a novel strategy to
mod[1latil1g in1l11une 'responses.
In this study, with AdEasy vector system, the recombinant adenovirus containing
huCTLA4-Ig gene was constructed. Using ex vivo gene transfer technique, exogenous gene was
introduced to the liver graft during cold preservation and express locally in the graft. The effect
of inhibition of rejection and inducing liver graft tolerance was observed. Through this study,
the possibility of achieving graft tolerance by gene transfer without routine immunosuppressive
drugs was explored.
The results are as followsi
l.The recombinant adenovirus containing huCTLA4-Ig gene was constructed successfully
and its titer was 6 X 10l3 PFU/L. The recombinant virus AdhuCTLA4-Ig prepared in this study
efliciently inltcted L-O2 'cells, and the infected cells expressed a-nd excreted soluble
rccolllbina11t protein huCTLA4-Ig.
2.Rat orthotopic liver trarlsplantation was perfOrmed in the strong rejecter combination of
BN (RTl' ) to LEW (RTl l). This al1ogenic rejection model was stable and the survival time
without immunosuppression agents was less than three weeks (the average survival time was
l 6.6 I 2.5 days, n=5).
3.Using clamp technique, ex vivo gene transfer into liver graft was performed during cold
preservation via perfusion of the portal vein with 5ml Ringer's solution containing
replication-defective adenovirus vector AdhuCTLA4-Ig. The expression of huCTLA4-Ig
protein was detected in recipient serum and liver graft within the first seven days after
transplantation, rapidly declifning to undetectable levels by day 28. No recipient of group A
(without any treatment, n=5) or group B (treated with AdGFP, n=4) died within three weeks
after transplantation and severe acute rejection (massive periportal infiltrate, endothelilitis,
damage to biliary epithelium and severe tissue destruction) was confirmed by pathological
examination of the graft. In contrast, all recipients of group C (treated with AdhuCTLA4-Ig,
n=5) achieved lohg-term liver allograft survival (>l50d). Histological examination of
AdhuCTLA4-Ig transduced allografts demonstrated a mild to moderate peripoftal inflammation
and mild injury to liver graft on day 8 posttransplant. A mild mononuclear infiltrate persisted,
however, there is complete preservation of the bile ducts and no evidence of vascular injury on
day l50
可溶性重组融合蛋白huCTLA4-Ig是一种新颖的免疫抑制剂，分子量约为92KD，由人CTLA-4分子的胞膜外区与人IgG1的Fc段(铰链区与恒定区)融合形成，能牢固地与B7分子结合，阻断CD28介导的共刺激信号，导致免疫反应的抑制。给予huCTLA4-Ig，短期阻断CD28介导的共识别可导致MHC不匹配的啮齿类模型中移植物的长期存活与耐受。腺病毒介导的针对移植物的免疫调节基因的转移，是调控免疫反应的新策略。目前，关于基因转移huCTLA4-Ig诱导同种异体移植物耐受的研究，国外仅有少数实验室开展，国内未见报道。
我们采用AdEasy腺病毒载体系统，自行构建携带huCTLA4-Ig的重组腺病毒，通过先体外后体内(ex vivo)基因转移技术，于供肝冷保存时，将该治疗基因导入大鼠移植肝，使其于移植肝局部表达，观察其抑制排斥反应，诱导大鼠移植肝免疫耐受的作用。通过本研究，旨在探讨：在无常规免疫抑制剂应用的情况下，先体外后体内基因转移免疫抑制分子获得同种异体移植物耐受的可能性。
主要实验结果如下：
1．成功构建了携带融合基因huCTLA4-Ig的重组腺病毒，滴度为6.0×10~(13)PFU／L。该重组病毒在体外能有效感染正常肝细胞株L-O2，受感染细胞能表达、分泌可溶性的重组融合蛋白huCTLA4-Ig；
2．建立了大鼠BN(RT1~n)→LEW(RT1~l)原位肝移植同种异体排斥模型，该模
博士学位论义 基因转移hU＊＊LA个ig诱导大鼠移植肝免疫耐受的实验研究 中文摘要
型排斥反应时间较为恒定，在不应用免疫抑制剂的情况下，生存期＜3周（平均生存时
问人］6 6士 2．5 d，11一引；
3．供肝冷保存时，采用血管夹技术＊lamp technique）经门静脉灌注携带融合基
因hllCTLA4dg的重组腺病毒，于术后3天、7天能定性检测到hllCTLA4Ig在受体外
划血卜1。的外分泌性表达及供肝J］－I‘细胞内的表达，以后迅速下降，至术后2 8天几乎不能
检测到。
个讹注亚组腺炳边组（A组，n—5）或灌注杉引IZ‘报告基囚GFP的亚组腺病毒组（B
纵，；。一4），受仰鼠均在3川内死亡，平均生存时问A组为16石土2．5天，B组为15．5
土3．1大，两组问无显著性差异V－0．35，尸＞0刀5八 而灌注携带融合基因＊＊＊＊A个ig
的重组腺病毒组 ＊组，fi—5）受体鼠均能获得长期生存（＞150天）；A组与C组间、
B组与C组间，生存期均有显著性差异’（P＜0刀1人
A组与B组，在术后8天行移植肝活检，病理学检查证实：移植肝均发生严重的
细胞性排斥。C组，移植肝病理学检查发现：术后8天，可见移植肝轻、中度的汇管
区炎症，但组织损伤程度显著减轻：术后150天，单核细胞浸润亦能观察到，但胆管
保存完好，无血管损伤证据。
术前大鼠血 ＊1卜2浓度为362们土45．84 n＊几；肝移械 术后，＊ 宁1卜2的浓度在
A、B组中即升高到较高水平（约为术前的1．5－2．5倍），相反地，在C组中其始终维
持在一个较低水平（按近或低于术前水平），术后3、7天，血清IL－二的浓度在A、C
组；句及 B、C组 l”。J差异均非常显著（P＜0．of）。
结论：本研究在国内率先建立了简便有效的针对血管化移植物的基因转移系统，
该系统中重组腺病毒在冷保存时能有效感染供肝，并获得融合基因的分泌性表达。移
植肝局部产生的可溶性融合蛋白hllCTLA4Ig，能抑制同种异体免疫反应，在不使用常－
规兔疫抑制剂情况下，获得大鼠同种异体移植肝的长期存活。初步证实本研究建立的
器官移植基因治疗方法具有简便、有效的特点。本研究对于临床肝移植具有一定的潜
在应用价值，为免疫调节基因转移技术用于肝移植临床，以防治移植肝术后免疫排斥
反应的发十，仆狄衍移伙川’的见疫耐受捉仪了初步的实验依抓。
【关键词】肝移植;基因转移;免疫耐受;CTLA4-Ig
With the uSe of immunosuppressive drugs that efficiently control acute rejection, Organ
transplantation has achieved impressive development. Nevertheless, clinical transplantation still
thces some important problems: lilblong immunosuppression is associated with toxicity,
oppottunistic infections and a high incidence of cancer. Donor specific organ transplantation
immune tolerance, which is defined as no medication (routine immunosuppressive agents), no
rejection and no infection after MHC mismatched organ transplantatlon, is the trend of organ
transplantation. Solutions to these problems will come from the application of new strategies of
immunointervefltion. The application of gene therapy techniques to transplantation has great
potential theraPeutic advantages over systemic administration of the immunosuppressive
bioreagents. Local production of immunosuppressive molecules may increase their therapeutic
efficiency and reduce their systemic side-effects.
HuCTLA4-lg,' one of the most potent in1n1unosuppressive molecules, is a soluble
recombinant fusion protein (molecu1ar weight of approximately 92 kD) consisting of the
extracellular domain of human CTLA-4 and a fragment (hinge and constant region) of the Fc
portion of human IgGl. It strongly adheres to the B7 molecule to block the CD28-mediated
costimulatory signal, resulting in inhibition of in vitro and in vivo immune response. With
administration of CTLA4-Ig, short-term blockade of CD28-mediated corecognition resulted in
prolonged graft survival and tolerance in MHC mismatched rodent models.
Adenoviral-mediated transfer to graft of immunoregulatory genes is a novel strategy to
mod[1latil1g in1l11une 'responses.
In this study, with AdEasy vector system, the recombinant adenovirus containing
huCTLA4-Ig gene was constructed. Using ex vivo gene transfer technique, exogenous gene was
introduced to the liver graft during cold preservation and express locally in the graft. The effect
of inhibition of rejection and inducing liver graft tolerance was observed. Through this study,
the possibility of achieving graft tolerance by gene transfer without routine immunosuppressive
drugs was explored.
The results are as followsi
l.The recombinant adenovirus containing huCTLA4-Ig gene was constructed successfully
and its titer was 6 X 10l3 PFU/L. The recombinant virus AdhuCTLA4-Ig prepared in this study
efliciently inltcted L-O2 'cells, and the infected cells expressed a-nd excreted soluble
rccolllbina11t protein huCTLA4-Ig.
2.Rat orthotopic liver trarlsplantation was perfOrmed in the strong rejecter combination of
BN (RTl' ) to LEW (RTl l). This al1ogenic rejection model was stable and the survival time
without immunosuppression agents was less than three weeks (the average survival time was
l 6.6 I 2.5 days, n=5).
3.Using clamp technique, ex vivo gene transfer into liver graft was performed during cold
preservation via perfusion of the portal vein with 5ml Ringer's solution containing
replication-defective adenovirus vector AdhuCTLA4-Ig. The expression of huCTLA4-Ig
protein was detected in recipient serum and liver graft within the first seven days after
transplantation, rapidly declifning to undetectable levels by day 28. No recipient of group A
(without any treatment, n=5) or group B (treated with AdGFP, n=4) died within three weeks
after transplantation and severe acute rejection (massive periportal infiltrate, endothelilitis,
damage to biliary epithelium and severe tissue destruction) was confirmed by pathological
examination of the graft. In contrast, all recipients of group C (treated with AdhuCTLA4-Ig,
n=5) achieved lohg-term liver allograft survival (>l50d). Histological examination of
AdhuCTLA4-Ig transduced allografts demonstrated a mild to moderate peripoftal inflammation
and mild injury to liver graft on day 8 posttransplant. A mild mononuclear infiltrate persisted,
however, there is complete preservation of the bile ducts and no evidence of vascular injury on
day l50
引文
1. Sayegh M. H., Turka L. A. The role of T-cell costimulatory activation pathways in transplant rejection. N Engl J Med. 1998,338:1813-21
2. He T.C., Zhou S.B., Costa LT.D., et al. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. 1998,95:2509-14.
3. Ilan Y, Saito H, Thummala NR, et al. Adenovirus-mediated gene therapy of liver diseases. Seminars in liver disease. 1999, 19:49-59
4. Sayegh MH, Turka LA. The role of T-cell costimulatory activation pathways in transplant rejection. N Engl J Med. 1998,338:1813-21
5. Harper, K., Balzano,C., Rouvier, E., et al. CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location. J. Immunol. 1991,147:1037-44
6. Linsley PS, Wallace PM, Johnson J et al. Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule. Science. 1992,257:792-5
7. Larsen C.P., Elwood E.T., Alexander D.Z., et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature. 1996,381:434-8
8. Tomasoni S., Azzollini N., Casiraghi F., et al. CTLA4Ig gene transfer prolongs survival and induces donor-specific tolerance in a rat renal allograft. J Am Soc Nephrol. 2000,11:747-52
9. Feng S., Quickel R., Hollister-lock J., et al. Prolonged xenograft survival of islets infected with small doses of adenovirus expressing CTLA4-Ig. Transplant. 1999,67:1607-13
10. Abrams JR, Lebwohl MG, Guzzo CA, et al. CTLA4Ig-mediated blockade of T-cell costimulation in patients with psoriasis vulgaris. J Clin Invest, 1999,103:1243-52
11. Guinan EC, Boussiotis VA, Neuberg D, et al. Transplantation of anergic histoincompatible bone marrow allografts. N Engl J Med, 1999,340:1704-14
12. Guillot C., Mauff B. L., Cuturi MC., et al. Gene therapy in transplantation in the year 2000:moving towards clinical applications? Gene Therapy. 2000,7:14-9
2. He T.C., Zhou S.B., Costa LT.D., et al. A simplified system for generating recombinant adenoviruses. Proc. Natl. Acad. Sci. 1998,95:2509-14.
3. Ilan Y, Saito H, Thummala NR, et al. Adenovirus-mediated gene therapy of liver diseases. Seminars in liver disease. 1999, 19:49-59
4. Sayegh MH, Turka LA. The role of T-cell costimulatory activation pathways in transplant rejection. N Engl J Med. 1998,338:1813-21
5. Harper, K., Balzano,C., Rouvier, E., et al. CTLA-4 and CD28 activated lymphocyte molecules are closely related in both mouse and human as to sequence, message expression, gene structure, and chromosomal location. J. Immunol. 1991,147:1037-44
6. Linsley PS, Wallace PM, Johnson J et al. Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule. Science. 1992,257:792-5
7. Larsen C.P., Elwood E.T., Alexander D.Z., et al. Long-term acceptance of skin and cardiac allografts after blocking CD40 and CD28 pathways. Nature. 1996,381:434-8
8. Tomasoni S., Azzollini N., Casiraghi F., et al. CTLA4Ig gene transfer prolongs survival and induces donor-specific tolerance in a rat renal allograft. J Am Soc Nephrol. 2000,11:747-52
9. Feng S., Quickel R., Hollister-lock J., et al. Prolonged xenograft survival of islets infected with small doses of adenovirus expressing CTLA4-Ig. Transplant. 1999,67:1607-13
10. Abrams JR, Lebwohl MG, Guzzo CA, et al. CTLA4Ig-mediated blockade of T-cell costimulation in patients with psoriasis vulgaris. J Clin Invest, 1999,103:1243-52
11. Guinan EC, Boussiotis VA, Neuberg D, et al. Transplantation of anergic histoincompatible bone marrow allografts. N Engl J Med, 1999,340:1704-14
12. Guillot C., Mauff B. L., Cuturi MC., et al. Gene therapy in transplantation in the year 2000:moving towards clinical applications? Gene Therapy. 2000,7:14-9