羟基喜树碱诱导大鼠器官移植免疫耐受的实验研究
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摘要
器官移植是某些终末期疾病的唯一治疗手段，虽然免疫抑制剂的应用使急性排斥得到了比较满意的解决，但它有难以避免的副作用，长期大量使用将致肝肾功能损害、免疫功能受损，而且也不可能完全避免排斥，这已成为进一步提高病人远期存活的巨大障碍，寻找新型的免疫抑制药物和诱导宿主对移植器官的特异免疫耐受是目前移植免疫学上急待解决的两个问题。实际上任何一种对抗细胞增殖的药物都有一定的免疫抑制作用，但均有非特异性和停、减药后免疫抑制作用快速逆转的缺点，相比而言克隆清除同种移植反应T细胞有更长时间的效果并至少对外周无反应起部分作用，克隆清除在外周T细胞耐受中的作用已被很多实验模型所正实。HCPT是从珙桐科植物喜树中提取出的一种生物碱，研究证实为I型DNA拓扑异构酶的抑制剂，可通过抑制DNA的复制而抑制细胞的增殖，临床上用于抗肿瘤治疗。已有研究报导HCPT有诱导淋巴细胞凋亡的作用，我们发现HCPT有免疫抑制作用，单独应用或与CsA、移植后输入供体白细胞结合可诱导受者对移植抗原的特异性免疫耐受，对HCPT在移植免疫中的作用机理进行了探讨，摘要报告如下。
第一部分 羟基喜树碱加环孢素A诱导异基因大鼠肾脏移植免疫耐受的实验研究
以近交系DA大鼠为供者、近交系Lewis大鼠为受者行异基因，肾脏移植，移植后受者大鼠接受不同剂量HCPT、CsA治疗或二者联合应用。A组：移植后不接受任何治疗；B组：HCPT1.0mg kg~(-1)，腹腔注射，1次d~(-1)；C组：HCPT2.0mg kg~(-1)，腹腔注射，1次d~(-1)；D组：CsA 10mg kg~(-1)，导管灌胃，1次d~(-1)；E组：HCPT1.0mg kg~(-1)，腹腔注射，1次d~(-1)，加CsA 10mg kg~(-1)，导管灌胃，1次d~(-1)。肾脏移植物术后存活30d停用免疫抑制剂。体外MLR及再次移植DA、SHR大鼠皮肤证实免疫耐受
第四军医人学唐土研究生学位论文
的特异性。并通过观察外源几 对耐受大鼠外周血淋巴细胞对供体特异性MLR反
应的影响，及应用RT－PCR方法检测耐受和排斥大鼠脾细胞和移植物中细胞因子
InRNA的表达情况探讨耐受的形成机制。结果发现，各组移植后存活时间显著
长于对照组。C组（HCPTZmg－‘·kg d－’中 4门0和 E组（HCPT．omgkt’d－’＋CsA
10tug kg－’d－’）中5门 受者大鼠形成特异性免疫耐受。耐受组＊4。IL10tnRNA
表达明显高于排斥组，而几、IFN Y明显低于排斥组。受者脾内细胞因子表达量
近似于移植物内。耐受大鼠外周血淋巳细胞对供体特异。叮MLR低反应不能被外源
ILZ所逆转。说明大剂量HCPT或小剂量HCPT与CSA合用可诱导异基因大鼠肾
脏移植免疫耐受。检测受者脾内细胞因子mRNA表达可代表移植物内表达细胞因
于表达结果，免疫耐受形成机制可能不是T细胞无能，耐受形成机制之一是细胞
因子偏离向ThZ亚类。
第二部分羟基喜树碱加供者白细胞诱导大鼠肾脏移植免疫耐受的实验研究
在第一部分基础上应用 HCPTZ．omg／kg加供者白细胞外周血管输入，诱导
DA－Lewis肾脏移植免疫耐受，通过皮肤移植、DTH及MLR确定耐受形成。为了
进一步探讨耐受的形成机制，进行序列的肾脏移植，定期处死受者，动态观察耐
受诱导及维持过程中嵌合体、CD25。淋巴细胞凋亡的变化。并通过过继转移实验
观察有无抑制细胞的存在。结果发现，羟基喜树碱加供者白细胞可 100％的诱导
DA工ewiS肾脏移植免疫耐受形成。将耐受大鼠脾细胞 2 X旷个经尾静脉注入正常
同系Lewis大鼠体内，7天后移植DA大鼠皮肤，可使皮肤存活时间增至26天。耐
受大鼠脾脏及胸腺内早期（移植后id）及晚期（移植后150d）均可检测到供者来
源细胞，耐受大鼠移植后第一天即有CD25的高表达，第h天即达高峰，随即快速
降低，而排斥组CD25早期低表达，而后呈进行性表达增加。耐受组早期即可检测
到大量的凋亡淋巴细胞，而排斥组淋巴细胞凋亡出现较晚。说明羟基喜树碱加供
者白细胞可诱导DA－Lewis肾脏移植免疫耐受形成。免疫耐受形成与供者细胞快速
移入受者淋巴组织并长期存在有关。免疫耐受形成与受者淋巴细胞被移植抗原早
5
第四军医大学博土研究生学位论文
期激活并迅速凋亡有关。免疫耐受与调节机制有关，本模型中至少涉及两种调节
机制，一为抑制细胞的作用，二为细胞因于偏离。
第三部分HCPT诱导超抗原激活的淋巴细胞凋亡
基于前两部分的研究结果，利用超抗原的特性进一步探讨HCPT诱导大鼠肾
脏移植免疫耐受的机理。Lewis大鼠给予 SEB ling／kg静脉注射，注射R记为 day 0。
HCPT处理组SEB注射前Zh给予HCPTZmg／kg 腹腔注射，以后给予HCPTZmg／kg／d。
空白对照组以PBS代替SE
Organ transplantation is now a viable therapeutic option for patients with certain end-stage organ failure. However, episodes of rejection, opportunistic infection, and life-theratening side effects of generalized immunosuperessive remain very real problems for these patients. To ques a new tape immunosuperessive drug and to induced transplantation tolerance have become two question anxious for solving. In fact, any sort of cytostatic drugs also have the function of immnosuppressive action, but they have the defect of non-specific, immnosuppressive activity to be rapidly reversible upon dosage reduction or treatment withdrawal, whereas clonal deletion of alloreactive T cells could have more prolonged effects and contribute to partial peripheral unresponsiveness. The contribution of clonal deletion to peripheral T cell tolerance has been demonstrated in several experimental models. HCPT, an ingredient from Chinese herb drugs have been demonstrated as an inhibitor of DNA topoisomerase I, can inhibt cell prolif
eration by obstroction DNA duplication, were used treatment neoplasm in clinical. There are paper report that HCPT can induce lymphocyte apoptosis. We discoved that HCPT has the action of immnosuppressive. HCPT , HCPT combined with CsA or HCPT combined with donor specific leukocyte injection after transplantation can induce allogenic kidney transplantation immune tolerance in rats.
Meanwhile, we studied the tolerance mechanisms on cellular and molecular level. The whole work consists of three parts as follows:
Part One: Immune Tolerance Induced by HCPT and Cyclosporin A in Recipient Rats of Allogenic Kidney Transplantation Induced by HCPT
The inbred DA rats were chosen as kidney donors and Lewis rats as recipients. After transplantation, the recipients were treated with HCPT and/or CsA . Group A received placebo; Group B , HCPTl.Omg kg-'d^LP. ; Group C , HCPT2.0mg kg-'d''L.P. ; Groupe D, CsA lOmg kg-1 d"1 by means of gastric intubation; Group E , HCPTl.Omg kg-1 d"]LP. plus CsA lOmg kg-1 d"1 gastrointubation. Immunosuppression (HCPT and CsA) was withdrawn at 30 days after transplantation. Immune tolerance was verified by challenged with DA and SHR rats skin . Mixed lymphocyte reaction (MLR) and IL2 to the system was investigated in vitro. The levels of cytokine expression were assessed using RT-PCR. The results showed that 4 of 10 group C (HCPT 2.0mg) and 5 of 10 group E (HCPT+CsA) kidney grafts survived longer than 150 days even immunosuppression (HCPT and CsA) was withdrawn at 30 days after transplantation. Immune tolerance was verified by challenged with DA and SHR rats skin . The level of IL2 and EFNr mRNA expression is significantly lower in the grafts of tolerant rats than in the grafts of rejected rats ; the level of IL4 and IL10 mRNA is significantly higer in the grafts of tolerant rats than in the grafts of rejected rats.The level of cytokine mRNA
expression in the grafts is similar to that of spleen of recipients. The lower reaction of MLR to DA lymphocyte could not be reversed by adding IL2 to the system. The study demonstrated that high dosage of HCPT or low dosage of HCPT combined with CsA could induce tolerance in allogenic kidney transplantation in the rats of recipients. Cytokine deviation is one of the mechanisms of allogenic kidney transplantation immune tolerance induced by hydrocamptothecin . The level of cytokine mRNA expression in the grafts is similar to that of spleen of recipients.
Part Two: Immune Tolerance Induced by HCPT Combined Donor
Leukocyte Injection after Transplantation in Recipient Rats of
Allogenic Kidney Transplantation
Based on Part One, we induced DA-Lewis allogenic kidney transplantation immune tolerance by HCPT2.0mg kg'1 d"1 combined donor leukocyte injection after transplantation. Immune tolerance was verified by challenged with DA and SHR rats skin, MLR and DTK. To futher explore tolerance mechanisms, we perform serial kidney transplantation. Grafts and spleens were harvested at definated time for observing the transformation of c
第一部分 羟基喜树碱加环孢素A诱导异基因大鼠肾脏移植免疫耐受的实验研究
以近交系DA大鼠为供者、近交系Lewis大鼠为受者行异基因，肾脏移植，移植后受者大鼠接受不同剂量HCPT、CsA治疗或二者联合应用。A组：移植后不接受任何治疗；B组：HCPT1.0mg kg~(-1)，腹腔注射，1次d~(-1)；C组：HCPT2.0mg kg~(-1)，腹腔注射，1次d~(-1)；D组：CsA 10mg kg~(-1)，导管灌胃，1次d~(-1)；E组：HCPT1.0mg kg~(-1)，腹腔注射，1次d~(-1)，加CsA 10mg kg~(-1)，导管灌胃，1次d~(-1)。肾脏移植物术后存活30d停用免疫抑制剂。体外MLR及再次移植DA、SHR大鼠皮肤证实免疫耐受
第四军医人学唐土研究生学位论文
的特异性。并通过观察外源几 对耐受大鼠外周血淋巴细胞对供体特异性MLR反
应的影响，及应用RT－PCR方法检测耐受和排斥大鼠脾细胞和移植物中细胞因子
InRNA的表达情况探讨耐受的形成机制。结果发现，各组移植后存活时间显著
长于对照组。C组（HCPTZmg－‘·kg d－’中 4门0和 E组（HCPT．omgkt’d－’＋CsA
10tug kg－’d－’）中5门 受者大鼠形成特异性免疫耐受。耐受组＊4。IL10tnRNA
表达明显高于排斥组，而几、IFN Y明显低于排斥组。受者脾内细胞因子表达量
近似于移植物内。耐受大鼠外周血淋巳细胞对供体特异。叮MLR低反应不能被外源
ILZ所逆转。说明大剂量HCPT或小剂量HCPT与CSA合用可诱导异基因大鼠肾
脏移植免疫耐受。检测受者脾内细胞因子mRNA表达可代表移植物内表达细胞因
于表达结果，免疫耐受形成机制可能不是T细胞无能，耐受形成机制之一是细胞
因子偏离向ThZ亚类。
第二部分羟基喜树碱加供者白细胞诱导大鼠肾脏移植免疫耐受的实验研究
在第一部分基础上应用 HCPTZ．omg／kg加供者白细胞外周血管输入，诱导
DA－Lewis肾脏移植免疫耐受，通过皮肤移植、DTH及MLR确定耐受形成。为了
进一步探讨耐受的形成机制，进行序列的肾脏移植，定期处死受者，动态观察耐
受诱导及维持过程中嵌合体、CD25。淋巴细胞凋亡的变化。并通过过继转移实验
观察有无抑制细胞的存在。结果发现，羟基喜树碱加供者白细胞可 100％的诱导
DA工ewiS肾脏移植免疫耐受形成。将耐受大鼠脾细胞 2 X旷个经尾静脉注入正常
同系Lewis大鼠体内，7天后移植DA大鼠皮肤，可使皮肤存活时间增至26天。耐
受大鼠脾脏及胸腺内早期（移植后id）及晚期（移植后150d）均可检测到供者来
源细胞，耐受大鼠移植后第一天即有CD25的高表达，第h天即达高峰，随即快速
降低，而排斥组CD25早期低表达，而后呈进行性表达增加。耐受组早期即可检测
到大量的凋亡淋巴细胞，而排斥组淋巴细胞凋亡出现较晚。说明羟基喜树碱加供
者白细胞可诱导DA－Lewis肾脏移植免疫耐受形成。免疫耐受形成与供者细胞快速
移入受者淋巴组织并长期存在有关。免疫耐受形成与受者淋巴细胞被移植抗原早
5
第四军医大学博土研究生学位论文
期激活并迅速凋亡有关。免疫耐受与调节机制有关，本模型中至少涉及两种调节
机制，一为抑制细胞的作用，二为细胞因于偏离。
第三部分HCPT诱导超抗原激活的淋巴细胞凋亡
基于前两部分的研究结果，利用超抗原的特性进一步探讨HCPT诱导大鼠肾
脏移植免疫耐受的机理。Lewis大鼠给予 SEB ling／kg静脉注射，注射R记为 day 0。
HCPT处理组SEB注射前Zh给予HCPTZmg／kg 腹腔注射，以后给予HCPTZmg／kg／d。
空白对照组以PBS代替SE
Organ transplantation is now a viable therapeutic option for patients with certain end-stage organ failure. However, episodes of rejection, opportunistic infection, and life-theratening side effects of generalized immunosuperessive remain very real problems for these patients. To ques a new tape immunosuperessive drug and to induced transplantation tolerance have become two question anxious for solving. In fact, any sort of cytostatic drugs also have the function of immnosuppressive action, but they have the defect of non-specific, immnosuppressive activity to be rapidly reversible upon dosage reduction or treatment withdrawal, whereas clonal deletion of alloreactive T cells could have more prolonged effects and contribute to partial peripheral unresponsiveness. The contribution of clonal deletion to peripheral T cell tolerance has been demonstrated in several experimental models. HCPT, an ingredient from Chinese herb drugs have been demonstrated as an inhibitor of DNA topoisomerase I, can inhibt cell prolif
eration by obstroction DNA duplication, were used treatment neoplasm in clinical. There are paper report that HCPT can induce lymphocyte apoptosis. We discoved that HCPT has the action of immnosuppressive. HCPT , HCPT combined with CsA or HCPT combined with donor specific leukocyte injection after transplantation can induce allogenic kidney transplantation immune tolerance in rats.
Meanwhile, we studied the tolerance mechanisms on cellular and molecular level. The whole work consists of three parts as follows:
Part One: Immune Tolerance Induced by HCPT and Cyclosporin A in Recipient Rats of Allogenic Kidney Transplantation Induced by HCPT
The inbred DA rats were chosen as kidney donors and Lewis rats as recipients. After transplantation, the recipients were treated with HCPT and/or CsA . Group A received placebo; Group B , HCPTl.Omg kg-'d^LP. ; Group C , HCPT2.0mg kg-'d''L.P. ; Groupe D, CsA lOmg kg-1 d"1 by means of gastric intubation; Group E , HCPTl.Omg kg-1 d"]LP. plus CsA lOmg kg-1 d"1 gastrointubation. Immunosuppression (HCPT and CsA) was withdrawn at 30 days after transplantation. Immune tolerance was verified by challenged with DA and SHR rats skin . Mixed lymphocyte reaction (MLR) and IL2 to the system was investigated in vitro. The levels of cytokine expression were assessed using RT-PCR. The results showed that 4 of 10 group C (HCPT 2.0mg) and 5 of 10 group E (HCPT+CsA) kidney grafts survived longer than 150 days even immunosuppression (HCPT and CsA) was withdrawn at 30 days after transplantation. Immune tolerance was verified by challenged with DA and SHR rats skin . The level of IL2 and EFNr mRNA expression is significantly lower in the grafts of tolerant rats than in the grafts of rejected rats ; the level of IL4 and IL10 mRNA is significantly higer in the grafts of tolerant rats than in the grafts of rejected rats.The level of cytokine mRNA
expression in the grafts is similar to that of spleen of recipients. The lower reaction of MLR to DA lymphocyte could not be reversed by adding IL2 to the system. The study demonstrated that high dosage of HCPT or low dosage of HCPT combined with CsA could induce tolerance in allogenic kidney transplantation in the rats of recipients. Cytokine deviation is one of the mechanisms of allogenic kidney transplantation immune tolerance induced by hydrocamptothecin . The level of cytokine mRNA expression in the grafts is similar to that of spleen of recipients.
Part Two: Immune Tolerance Induced by HCPT Combined Donor
Leukocyte Injection after Transplantation in Recipient Rats of
Allogenic Kidney Transplantation
Based on Part One, we induced DA-Lewis allogenic kidney transplantation immune tolerance by HCPT2.0mg kg'1 d"1 combined donor leukocyte injection after transplantation. Immune tolerance was verified by challenged with DA and SHR rats skin, MLR and DTK. To futher explore tolerance mechanisms, we perform serial kidney transplantation. Grafts and spleens were harvested at definated time for observing the transformation of c
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25． White J, Herman A, Pullen AM, Kubo R, Kappler JW, Marrack P. The V beta-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell 1989; 56: 27．
26． Martin SJ, Reutelingsperger CP, McGahon AJ, et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.J Exp Med 1995; 182: 1545．
27． Gavrieli Y, Sherman Y, Ben-Sasson SA. Identification of programmed cell
death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol1992;119:493．
28． Webb S, Morris C, Sprent J. Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity. Cell 1990; 63: 1249．
29． Wells AD, Li XC, Li Y, et al. Requirement for T-cell apoptosis in the induction of peripheral transplantation tolerance. Nat Med 1999; 5: 1303．
30． Vanier LE, Prud'homme GJ. Cyclosporin A markedly enhances superantigen-induced peripheral T cell deletion and inhibits anergy induction. J Exp Med 1992; 176: 37．
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