供体凋亡细胞输注诱导大鼠胰岛移植免疫耐受的研究
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摘要
研究背景
胰岛移植重建胰岛素分泌系统,是一种有望彻底根治糖尿病的治疗方法。2000年,Edmonton方案的成功,标志着胰岛移植取得了突破性的进展,从而带动了胰岛移植临床试验性治疗在世界范围内广泛展开。但是免疫排斥以及免疫抑制剂所带来的副作用和潜在危险性仍是难以克服的障碍。目前普遍认为,解决异体移植排斥反应的关键在于诱导受体对供体的细胞、组织或器官产生免疫耐受。由于移植免疫耐受的机制十分复杂,尽管诱导免疫耐受的方法众多,但均未能达到完全可靠持久的特异性耐受。细胞凋亡是生物体内普遍存在的一种生理和病理现象,是正常器官和组织发育、清除有害的、过量的和无功能细胞、维持自身稳态的必要环节。现有研究表明:凋亡是机体免疫状态保持平衡和稳定的重要作用机制,凋亡细胞对免疫系统存在着主动的调节作用。凋亡细胞能分泌脂类趋化因子,改变自身胞膜结构表达“eat-me”信号,诱导吞噬细胞对其进行清除;同时凋亡细胞被抗原提呈细胞吞噬后,通过吞噬细胞分泌抑制性免疫因子如TGF-β、PGE_2、IL-10等,造成了特殊的抗原识别微环境,可以促使相关淋巴细胞针对凋亡抗原产生免疫耐受,而不会引起炎性免疫应答。据此,孙尔维等提出利用供体凋亡细胞输注来诱导供体特异性免疫耐受的理论设想。我们课题组以前的工作证实,凋亡细胞在体外对T淋巴细胞活化增殖有直接抑制作用;凋亡细胞预输注可以显著延长大鼠心脏移植模型、肝移植模型存活时间。因此,我们拟应用供体凋亡脾淋巴细胞静脉输注的方法,来诱导糖尿病大鼠同种异体胰岛移植的免疫耐受,以探求延长胰岛移植物存活的方法。
目的
初步探讨凋亡细胞静脉输注诱导胰岛移植免疫耐受,延长胰岛移植物存活时间的方法,为寻找诱导胰岛移植免疫耐受的方法开辟新的途径,提供新的依据。
方法
1、直线加速器照射对体外培养大鼠脾细胞凋亡的影响:采用研磨法获得Wistar大鼠脾淋巴细胞悬液。将脾细胞悬液加入细胞培养瓶,分四组,A组为对照组,B、C、D组应用Varian医用直线加速器照射,各组吸收剂量分别为:1.5Gy、2.0Gy、3.0Gy。各组细胞处理后置于37℃,5%CO_2恒温培养箱培养。分别在培养后4h、8h、12h应用流式细胞仪检测细胞凋亡率;
2、大鼠胰岛细胞的分离纯化:采用胶原酶P胰管灌注消化,短期低温培养后Ficoll-400不连续梯度纯化Wistar大鼠胰岛;
3、建立糖尿病动物模型:以STZ(链脲佐菌素)(56mg/kg·体重)经腹腔一次性注射,制备SD大鼠实验性糖尿病模型(连续2次血糖＞16.7mmol/L);
4、供体凋亡细胞输注对糖尿病大鼠胰岛移植物的影响:以雄性Wistar大鼠为供体,雄性SD大鼠为受体。将糖尿病SD大鼠随机分为Hank's液注射(A组)、正常供体脾细胞预输注(B组)、供体凋亡脾细胞预输注(C组)及供体坏死脾细胞预输注(D组)四组。各组在预处理7天后行胰岛移植,将1000IEQ的胰岛细胞移植入各组糖尿病大鼠的肾包囊内,比较各组移植物生存时间上的差异。同时各实验组分别在预处理后第7天(即胰岛移植术前)、胰岛移植术后7天、14天、排斥后应用放射免疫法测定大鼠血清胰岛素水平。供体凋亡脾细胞预输注糖尿病SD大鼠则在不同的移植时期(包含血糖正常期、排斥期两个时期)手术将移植物载体肾取出;其余各实验组在糖尿病SD大鼠移植物排斥后2天,取出其移植物载体肾,做胰岛素的免疫组化检测。
5、供体凋亡细胞输注对胰岛移植糖尿病大鼠淋巴细胞增殖反应的影响:将糖尿病SD大鼠按上述方法分组,各组分别于预处理后第7天(即胰岛移植术前)、胰岛移植术后7天、14天、排斥后取大鼠脾脏,分离淋巴细胞,经刀豆蛋白A(ConcanavalinA ConA)刺激培养,利用CFSE(2羧基荧光素二醋酸盐琥珀酰亚胺酯)细胞染色法观察受体淋巴细胞增殖反应程度。
结果
1、直线加速器高能X线照射Wistar大鼠脾细胞,吸收剂量2.0Gy组各个时间点的凋亡率有差异(F=5.930,P=0.026),且与对照组、1.5Gy组比较凋亡率有差异(P值均＜0.050),以2.0Gy组、8小时的凋亡率较高。
2、每条Wistar大鼠胰腺经分离、纯化后平均可获取的胰岛细胞团数量为(1063.91±84.74)IEQ,DTZ(双硫腙)染色显示纯度为(70.51±6.20)%。高糖刺激时胰岛β细胞胰岛素的释放量为低糖刺激时的2.72倍。
3、以STZ(56mg/kg·体重)一次性经腹腔注射的方法,可成功诱发SD大鼠的试验性糖尿病,糖尿病模型建模成功率为94%。
4、输注凋亡细胞组与输注Hank's液组、输注正常细胞组、输注坏死细胞组之间的移植物存活时间有差异(P值均＜0.050)。平均存活天数为32天,最长达42天,以输注凋亡细胞组胰岛移植物存活时间最长。正常脾细胞输注组较输注Hank's液组及坏死细胞组也有所延长。预输注Hank's液及坏死细胞组则无此效应。
5、预输注凋亡细胞组移植前胰岛素水平与移植后7天、移植后14天有差异(P值均＜0.050),以移植后7天、14天较高。移植后14天输注凋亡细胞组与输注正常细胞组的胰岛素水平有差异(T=3.439,P=0.009),以凋亡细胞组胰岛素水平最高,而预输注Hank's液及坏死细胞组各时间点的胰岛素水平无差异(P值均＞0.050)。
6、供体凋亡细胞预输注组胰岛移植物在受体血糖正常时取出做组织切片,经Insulin免疫组织化学染色证实有表达胰岛素阳性的胰岛细胞团存在,呈棕褐色。而在被排斥后取出的移植物进行免疫组化检测,则未发现胰岛素阳性的胰岛细胞团存在。输注Hank's液组、输注供体正常细胞或坏死细胞组的移植物在被排斥后取出行Insulin免疫组化染色检测,亦无胰岛素阳性细胞存在。
7、预输注凋亡细胞组移植前的淋巴细胞增殖指数与移植后14天、排斥后有差异(P值均＜0.050),以移植前的增殖指数最低,移植前、移植后7天各组淋巴细胞增殖指数有差异(F=29.943,P=0.000),以输注凋亡细胞组的增殖指数最低,这种抑制效应至移植后14天仍存在,排斥后则消失。
结论
1、直线加速器高能X线照射能在体外简便安全有效诱导大鼠脾细胞凋亡。
2、采用胶原酶P胰管灌注消化,短期低温培养后Ficoll-400不连续梯度纯化能分离、纯化出较大数量且功能良好的大鼠胰岛细胞。
3、预输注供体凋亡细胞能显著延长同种异体大鼠胰岛移植物的存活时间。应用供体凋亡细胞诱导胰岛移植免疫耐受是一条可行的途径。
4、以体外淋巴细胞增殖实验证实,预输注供体凋亡细胞对受体鼠淋巴细胞经丝裂原ConA刺激的增殖反应具有抑制作用。
Background
Islets transplantation to re-establish the secretion system of insulin has been considered as a most promising treatment to cure diabetes mellitus. Especially the success of Edmonton Protocol in 2000, has demonstrated the great progress which has been made in the territory of islets transplantation, and accordingly exploited an extensive application perspective of islets transplantation to cope with type 1 diabetes. However, transplantation rejection and the potential risk coming with the application of immunosuppressive agents are still the obstacles unable to overcome completely. Now it is generally thought that the key to solve the rejection of allografts is to induce the recipient's immune tolerance to the grafts from the donor. Though there are so many means to induce the tolerance, none of them can achieve the complete permanent specific tolerance. Apoptosis is a kind of physiological or pathological phenomenon generally existing in the biosystem, and plays an important role in maintaining the balance and stabilization of the host's immune state. Apoptotic cells can secrete lipidic haemostatic factors , and present "eat-me" signals on the surface to attracted the phagocyte to remove themselves; at the same time, antigent-presenting cells (monocytes and macrophages, etc ) can secrete many inhibitive immune factors such as TGF-β、PGE2、IL-10 etc, after the phagocytosis. All of these creat a microenvironment for antigen recognition and induce the related lymphocytes' tolerance to apoptotic antigen rather than the inflammatory response. On these grounds, Sun et al proposed to make use of the transfusion of apoptotic cells to induce the donor's specific tolerance. Our workgroup has demonstrated that apoptotic cells can make an inhibitive effect on the proliferation of T lymphocytes in vitro, and researchers have reported that transfusion of apoptotic cells can notably prolong the survival time of rat allografts after the heart transplantation or liver transplantation. So we plan to induce the allogenic islets transplantation tolerance in diabetic SD rat by pre-transfusion of apoptotic cells to investigate the method of prolonging the survival time of the islets grafts.
Objective
To investigate the methods and mechanism of islets transplantation tolerance induced by transfusion of the apoptotic cells. To provide experimental evidences for establishing new protocols which can induce islets transplantation tolerance.
Methods
1. To investigate the effect of X-irradiation from electron linear accelerator on the rats spleen cell in vitro: the Wistar rat spleen cells obtained by the method of grinding were divided into four groups: control (A) and B, C, D group were irradiated by the absorbed dose of 1.5Gy, 2.0Gy, 3.0Gy respectively. And then incubated in 37℃, 5%CO_2. The early apoptotic cells at 4h, 8h, and 12h were measured by flow cytometry (FCM) with Annexin V-FITC/PI.
2. Isolation and purification of rat islets: The pancreatic islets were digested and isolated by perfusing collagenase P via pancreatic duct and purified by Ficoll-400 discontinuous density gradients after l-2d incubation.
3. To establish animal model of diabetes mellitus: SD rats were rendered diabetic via single intraperitoneal injection of streptozotocin at 56mg/kg ? weight (continuous twice plasma glucose >16.7mmol/L).
4. To study the effect of transfusion of donor apoptotic spleen cells on the survival of islet grafts: Wistar and SD rats were taken respectively as donor and recipient. Diabetic SD rats were randomly divided into 4 groups, and recived infusion of Hank's, normal donor cells, apoptotic donor cells and necrotic donor cells (respectively as A, B, C, D group) via dorsal vein of penis, then, 7 days later, all rats received islets (1000IEQ per STZ SD rat) under their left renal capsule respectively and the survival time of islet grafts were compared. The insulin lever in serum were measured by RIA at 0d, 7d, 14d, after transplantation and at the time of rejection. Grafts from recipients were examined for the expression of insulin by immunohistochemical staining at the time of euglycemic or returning to a hyperglycemic state in C group. Grafts of other groups were examined for the expression of insulin by immunohistochemical staining only after the rejection.
5. To observe the effect of transfusion of donor apoptotic cells on the Proliferative response of the lymphocytes from the recipient rats: Diabetic SD rats were randomly rendered infusion of Hank's, normal donor cells, apoptotic donor cells and necrotic donor cells, then, at 0d, 7d, 14d after transplantation and at the time of rejection, all recipients' spleen were resected and the isolated lymphocytes were stimulated with ConA. Then the extent of the Proliferative response of the recipient's lymphocytes were observed by the method of cell staining with CFSE.
Results
1. The Wistar rat splenocytes were induced by X-irradiated with accelerator linear: the ratio of apoptotic cells in 2.0Gy group has a difference in the different time group (F=5.930,P=0.026). And has a difference compared with the control and 1.5Gy group (P<0.050) . The 2.0 Gy group has a highest Apoptosis rate at the time of 8 h after the Irradiation.
2. After the purification by discontinuous gradient centrifugation, about( 1063.49±84.74) IEQ per rat pancreas were acquired, with an average purity of (70.51±6.20)%, and the purified islets were responded to high concentration glucose stimulation with 2.72 times increase of insulin secretion compared with low concentration glucose stimulation.
3. Pharmacological diabetes on SD rats were induced successfully by single intraperitoneal STZ injection (56mg/kg·weight) with an average model-performed ratio of 94%.
4. The survival time of islet grafts of diabetic SD rats who were pretreated with donor apoptotic cells were dramatically prolonged, compared with who are pretreated with Hank's, normal donor cells and necrotic donor cells (P<0.050), with an average survival time of 32.00 days and the longest survival time of 42 days, the normal donor cells group also has a prolonged survival time compared with the A ,D group. The A, D groups had no prolonged effect.
5. The insulin level in serum of the diabetic SD rats in C group had a notable increase at 7d, 14d, compared with the time of pre-transplantation (P<0.050), and the insulin level in C group at 14d after transplantation was higher than the B group (T=3.439 ,P=0.009) . The A, D groups have no significant changes.
6. Grafts from groups who are pretreated with Hank's, normal donor cells and necrotic donor cells, harvested at the time of return to the diabetic state, revealed the complete absence of insulin-positive cells. Compared with control grafts, the grafts from STZ SD rats being pretreated with donor apoptotic cells, harvested from euglycemic animals, showed intact islets immunohistochemically stained for insulin. In the contrast, the grafts harvested from the same group at the time return to hyperglycemia, showed no staining for islets.
7. After the stimulation by ConA , the Proliferative index of lymphocytes before transplantation and at 7d after transplantation in C group was lowest compared with other groups (F=29.943, P=0.000) . and the C group had a lowest proliferative index before transplantation(P<0.050). the suppressing effect existed until at 14 days after transplantation and disappeares after the rejection.
Conclusion
1. Irradiation by X rays from electron linear accelerator is a convenient, safe and effective way to induce apoptosis of Rat spleen cells.
2. A large quantity of islets which still have good functions can be acquired by isolation by intraductal collagenase P and purification by Ficoll-400 discontinuous density gradients after culturing 1 -2d .
3. The transfusion of donor apoptotic cells can prolong the survival time of islet grafts and suppress transplantation rejection; therefore, the utility of donor apoptotic cells will be a feasible pathway to induce islets transplantation tolerance.
4. We have demonstrated that the transfusion of donor apoptotic cells can have an inhibitive effect on the proliferation response of the recipient's lymphocytes to the stimulation of Con A.
胰岛移植重建胰岛素分泌系统,是一种有望彻底根治糖尿病的治疗方法。2000年,Edmonton方案的成功,标志着胰岛移植取得了突破性的进展,从而带动了胰岛移植临床试验性治疗在世界范围内广泛展开。但是免疫排斥以及免疫抑制剂所带来的副作用和潜在危险性仍是难以克服的障碍。目前普遍认为,解决异体移植排斥反应的关键在于诱导受体对供体的细胞、组织或器官产生免疫耐受。由于移植免疫耐受的机制十分复杂,尽管诱导免疫耐受的方法众多,但均未能达到完全可靠持久的特异性耐受。细胞凋亡是生物体内普遍存在的一种生理和病理现象,是正常器官和组织发育、清除有害的、过量的和无功能细胞、维持自身稳态的必要环节。现有研究表明:凋亡是机体免疫状态保持平衡和稳定的重要作用机制,凋亡细胞对免疫系统存在着主动的调节作用。凋亡细胞能分泌脂类趋化因子,改变自身胞膜结构表达“eat-me”信号,诱导吞噬细胞对其进行清除;同时凋亡细胞被抗原提呈细胞吞噬后,通过吞噬细胞分泌抑制性免疫因子如TGF-β、PGE_2、IL-10等,造成了特殊的抗原识别微环境,可以促使相关淋巴细胞针对凋亡抗原产生免疫耐受,而不会引起炎性免疫应答。据此,孙尔维等提出利用供体凋亡细胞输注来诱导供体特异性免疫耐受的理论设想。我们课题组以前的工作证实,凋亡细胞在体外对T淋巴细胞活化增殖有直接抑制作用;凋亡细胞预输注可以显著延长大鼠心脏移植模型、肝移植模型存活时间。因此,我们拟应用供体凋亡脾淋巴细胞静脉输注的方法,来诱导糖尿病大鼠同种异体胰岛移植的免疫耐受,以探求延长胰岛移植物存活的方法。
目的
初步探讨凋亡细胞静脉输注诱导胰岛移植免疫耐受,延长胰岛移植物存活时间的方法,为寻找诱导胰岛移植免疫耐受的方法开辟新的途径,提供新的依据。
方法
1、直线加速器照射对体外培养大鼠脾细胞凋亡的影响:采用研磨法获得Wistar大鼠脾淋巴细胞悬液。将脾细胞悬液加入细胞培养瓶,分四组,A组为对照组,B、C、D组应用Varian医用直线加速器照射,各组吸收剂量分别为:1.5Gy、2.0Gy、3.0Gy。各组细胞处理后置于37℃,5%CO_2恒温培养箱培养。分别在培养后4h、8h、12h应用流式细胞仪检测细胞凋亡率;
2、大鼠胰岛细胞的分离纯化:采用胶原酶P胰管灌注消化,短期低温培养后Ficoll-400不连续梯度纯化Wistar大鼠胰岛;
3、建立糖尿病动物模型:以STZ(链脲佐菌素)(56mg/kg·体重)经腹腔一次性注射,制备SD大鼠实验性糖尿病模型(连续2次血糖＞16.7mmol/L);
4、供体凋亡细胞输注对糖尿病大鼠胰岛移植物的影响:以雄性Wistar大鼠为供体,雄性SD大鼠为受体。将糖尿病SD大鼠随机分为Hank's液注射(A组)、正常供体脾细胞预输注(B组)、供体凋亡脾细胞预输注(C组)及供体坏死脾细胞预输注(D组)四组。各组在预处理7天后行胰岛移植,将1000IEQ的胰岛细胞移植入各组糖尿病大鼠的肾包囊内,比较各组移植物生存时间上的差异。同时各实验组分别在预处理后第7天(即胰岛移植术前)、胰岛移植术后7天、14天、排斥后应用放射免疫法测定大鼠血清胰岛素水平。供体凋亡脾细胞预输注糖尿病SD大鼠则在不同的移植时期(包含血糖正常期、排斥期两个时期)手术将移植物载体肾取出;其余各实验组在糖尿病SD大鼠移植物排斥后2天,取出其移植物载体肾,做胰岛素的免疫组化检测。
5、供体凋亡细胞输注对胰岛移植糖尿病大鼠淋巴细胞增殖反应的影响:将糖尿病SD大鼠按上述方法分组,各组分别于预处理后第7天(即胰岛移植术前)、胰岛移植术后7天、14天、排斥后取大鼠脾脏,分离淋巴细胞,经刀豆蛋白A(ConcanavalinA ConA)刺激培养,利用CFSE(2羧基荧光素二醋酸盐琥珀酰亚胺酯)细胞染色法观察受体淋巴细胞增殖反应程度。
结果
1、直线加速器高能X线照射Wistar大鼠脾细胞,吸收剂量2.0Gy组各个时间点的凋亡率有差异(F=5.930,P=0.026),且与对照组、1.5Gy组比较凋亡率有差异(P值均＜0.050),以2.0Gy组、8小时的凋亡率较高。
2、每条Wistar大鼠胰腺经分离、纯化后平均可获取的胰岛细胞团数量为(1063.91±84.74)IEQ,DTZ(双硫腙)染色显示纯度为(70.51±6.20)%。高糖刺激时胰岛β细胞胰岛素的释放量为低糖刺激时的2.72倍。
3、以STZ(56mg/kg·体重)一次性经腹腔注射的方法,可成功诱发SD大鼠的试验性糖尿病,糖尿病模型建模成功率为94%。
4、输注凋亡细胞组与输注Hank's液组、输注正常细胞组、输注坏死细胞组之间的移植物存活时间有差异(P值均＜0.050)。平均存活天数为32天,最长达42天,以输注凋亡细胞组胰岛移植物存活时间最长。正常脾细胞输注组较输注Hank's液组及坏死细胞组也有所延长。预输注Hank's液及坏死细胞组则无此效应。
5、预输注凋亡细胞组移植前胰岛素水平与移植后7天、移植后14天有差异(P值均＜0.050),以移植后7天、14天较高。移植后14天输注凋亡细胞组与输注正常细胞组的胰岛素水平有差异(T=3.439,P=0.009),以凋亡细胞组胰岛素水平最高,而预输注Hank's液及坏死细胞组各时间点的胰岛素水平无差异(P值均＞0.050)。
6、供体凋亡细胞预输注组胰岛移植物在受体血糖正常时取出做组织切片,经Insulin免疫组织化学染色证实有表达胰岛素阳性的胰岛细胞团存在,呈棕褐色。而在被排斥后取出的移植物进行免疫组化检测,则未发现胰岛素阳性的胰岛细胞团存在。输注Hank's液组、输注供体正常细胞或坏死细胞组的移植物在被排斥后取出行Insulin免疫组化染色检测,亦无胰岛素阳性细胞存在。
7、预输注凋亡细胞组移植前的淋巴细胞增殖指数与移植后14天、排斥后有差异(P值均＜0.050),以移植前的增殖指数最低,移植前、移植后7天各组淋巴细胞增殖指数有差异(F=29.943,P=0.000),以输注凋亡细胞组的增殖指数最低,这种抑制效应至移植后14天仍存在,排斥后则消失。
结论
1、直线加速器高能X线照射能在体外简便安全有效诱导大鼠脾细胞凋亡。
2、采用胶原酶P胰管灌注消化,短期低温培养后Ficoll-400不连续梯度纯化能分离、纯化出较大数量且功能良好的大鼠胰岛细胞。
3、预输注供体凋亡细胞能显著延长同种异体大鼠胰岛移植物的存活时间。应用供体凋亡细胞诱导胰岛移植免疫耐受是一条可行的途径。
4、以体外淋巴细胞增殖实验证实,预输注供体凋亡细胞对受体鼠淋巴细胞经丝裂原ConA刺激的增殖反应具有抑制作用。
Background
Islets transplantation to re-establish the secretion system of insulin has been considered as a most promising treatment to cure diabetes mellitus. Especially the success of Edmonton Protocol in 2000, has demonstrated the great progress which has been made in the territory of islets transplantation, and accordingly exploited an extensive application perspective of islets transplantation to cope with type 1 diabetes. However, transplantation rejection and the potential risk coming with the application of immunosuppressive agents are still the obstacles unable to overcome completely. Now it is generally thought that the key to solve the rejection of allografts is to induce the recipient's immune tolerance to the grafts from the donor. Though there are so many means to induce the tolerance, none of them can achieve the complete permanent specific tolerance. Apoptosis is a kind of physiological or pathological phenomenon generally existing in the biosystem, and plays an important role in maintaining the balance and stabilization of the host's immune state. Apoptotic cells can secrete lipidic haemostatic factors , and present "eat-me" signals on the surface to attracted the phagocyte to remove themselves; at the same time, antigent-presenting cells (monocytes and macrophages, etc ) can secrete many inhibitive immune factors such as TGF-β、PGE2、IL-10 etc, after the phagocytosis. All of these creat a microenvironment for antigen recognition and induce the related lymphocytes' tolerance to apoptotic antigen rather than the inflammatory response. On these grounds, Sun et al proposed to make use of the transfusion of apoptotic cells to induce the donor's specific tolerance. Our workgroup has demonstrated that apoptotic cells can make an inhibitive effect on the proliferation of T lymphocytes in vitro, and researchers have reported that transfusion of apoptotic cells can notably prolong the survival time of rat allografts after the heart transplantation or liver transplantation. So we plan to induce the allogenic islets transplantation tolerance in diabetic SD rat by pre-transfusion of apoptotic cells to investigate the method of prolonging the survival time of the islets grafts.
Objective
To investigate the methods and mechanism of islets transplantation tolerance induced by transfusion of the apoptotic cells. To provide experimental evidences for establishing new protocols which can induce islets transplantation tolerance.
Methods
1. To investigate the effect of X-irradiation from electron linear accelerator on the rats spleen cell in vitro: the Wistar rat spleen cells obtained by the method of grinding were divided into four groups: control (A) and B, C, D group were irradiated by the absorbed dose of 1.5Gy, 2.0Gy, 3.0Gy respectively. And then incubated in 37℃, 5%CO_2. The early apoptotic cells at 4h, 8h, and 12h were measured by flow cytometry (FCM) with Annexin V-FITC/PI.
2. Isolation and purification of rat islets: The pancreatic islets were digested and isolated by perfusing collagenase P via pancreatic duct and purified by Ficoll-400 discontinuous density gradients after l-2d incubation.
3. To establish animal model of diabetes mellitus: SD rats were rendered diabetic via single intraperitoneal injection of streptozotocin at 56mg/kg ? weight (continuous twice plasma glucose >16.7mmol/L).
4. To study the effect of transfusion of donor apoptotic spleen cells on the survival of islet grafts: Wistar and SD rats were taken respectively as donor and recipient. Diabetic SD rats were randomly divided into 4 groups, and recived infusion of Hank's, normal donor cells, apoptotic donor cells and necrotic donor cells (respectively as A, B, C, D group) via dorsal vein of penis, then, 7 days later, all rats received islets (1000IEQ per STZ SD rat) under their left renal capsule respectively and the survival time of islet grafts were compared. The insulin lever in serum were measured by RIA at 0d, 7d, 14d, after transplantation and at the time of rejection. Grafts from recipients were examined for the expression of insulin by immunohistochemical staining at the time of euglycemic or returning to a hyperglycemic state in C group. Grafts of other groups were examined for the expression of insulin by immunohistochemical staining only after the rejection.
5. To observe the effect of transfusion of donor apoptotic cells on the Proliferative response of the lymphocytes from the recipient rats: Diabetic SD rats were randomly rendered infusion of Hank's, normal donor cells, apoptotic donor cells and necrotic donor cells, then, at 0d, 7d, 14d after transplantation and at the time of rejection, all recipients' spleen were resected and the isolated lymphocytes were stimulated with ConA. Then the extent of the Proliferative response of the recipient's lymphocytes were observed by the method of cell staining with CFSE.
Results
1. The Wistar rat splenocytes were induced by X-irradiated with accelerator linear: the ratio of apoptotic cells in 2.0Gy group has a difference in the different time group (F=5.930,P=0.026). And has a difference compared with the control and 1.5Gy group (P<0.050) . The 2.0 Gy group has a highest Apoptosis rate at the time of 8 h after the Irradiation.
2. After the purification by discontinuous gradient centrifugation, about( 1063.49±84.74) IEQ per rat pancreas were acquired, with an average purity of (70.51±6.20)%, and the purified islets were responded to high concentration glucose stimulation with 2.72 times increase of insulin secretion compared with low concentration glucose stimulation.
3. Pharmacological diabetes on SD rats were induced successfully by single intraperitoneal STZ injection (56mg/kg·weight) with an average model-performed ratio of 94%.
4. The survival time of islet grafts of diabetic SD rats who were pretreated with donor apoptotic cells were dramatically prolonged, compared with who are pretreated with Hank's, normal donor cells and necrotic donor cells (P<0.050), with an average survival time of 32.00 days and the longest survival time of 42 days, the normal donor cells group also has a prolonged survival time compared with the A ,D group. The A, D groups had no prolonged effect.
5. The insulin level in serum of the diabetic SD rats in C group had a notable increase at 7d, 14d, compared with the time of pre-transplantation (P<0.050), and the insulin level in C group at 14d after transplantation was higher than the B group (T=3.439 ,P=0.009) . The A, D groups have no significant changes.
6. Grafts from groups who are pretreated with Hank's, normal donor cells and necrotic donor cells, harvested at the time of return to the diabetic state, revealed the complete absence of insulin-positive cells. Compared with control grafts, the grafts from STZ SD rats being pretreated with donor apoptotic cells, harvested from euglycemic animals, showed intact islets immunohistochemically stained for insulin. In the contrast, the grafts harvested from the same group at the time return to hyperglycemia, showed no staining for islets.
7. After the stimulation by ConA , the Proliferative index of lymphocytes before transplantation and at 7d after transplantation in C group was lowest compared with other groups (F=29.943, P=0.000) . and the C group had a lowest proliferative index before transplantation(P<0.050). the suppressing effect existed until at 14 days after transplantation and disappeares after the rejection.
Conclusion
1. Irradiation by X rays from electron linear accelerator is a convenient, safe and effective way to induce apoptosis of Rat spleen cells.
2. A large quantity of islets which still have good functions can be acquired by isolation by intraductal collagenase P and purification by Ficoll-400 discontinuous density gradients after culturing 1 -2d .
3. The transfusion of donor apoptotic cells can prolong the survival time of islet grafts and suppress transplantation rejection; therefore, the utility of donor apoptotic cells will be a feasible pathway to induce islets transplantation tolerance.
4. We have demonstrated that the transfusion of donor apoptotic cells can have an inhibitive effect on the proliferation response of the recipient's lymphocytes to the stimulation of Con A.
引文
[1] Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med, 2000,343(4):230-238.
[2] Seung E, Mordes JP, Greiner DL, et al. Induction of tolerance for islet transplantation for type 1 diabetes. Curr Diab Rep, 2003,3(4):329-335.
[3] Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med, 2006,355(13):1372-1374.
[4] Truong W, Shapiro AM. Progress in islet transplantation in patients with type 1 diabetes mellitus. Treat Endocrinol., 2006,5(3): 147-158.
[5] Balamurugan AN, Bottino R, Giannoukakis N, et al. Prospective and challenges of islet transplantation for the therapy of autoimmune diabetes. Pancreas, 2006, 32(3):231-243.
[6] Posselt AM, Barker CF, Tomaszewski JE, et al. Induction of donor-specific unresponsiveness by intrathymic islet transplantation. Science, 1990,249(4974): 1293-1295.
[7] Voll RE, Herrmann M, Roth EA, et al. Immunosuppressive effects of apoptotic cells. Nature, 1997, 390(6658):350-351.
[8] Kitajima T, Ariizumi K, Bergstresser PR, et al. Ultraviolet B radiation sensitizes a murine epidermal dendritic cell line(XS52) to undergo apoptosis upon antigen presentation to cells. J Immunol, 1996, 157(8): 3312-3318.
[9] Fadok VA, Bratton DL, Rose DM, et al. A Receptor for phosphatidylserine- specific clearance of apoptotic cells. Nature, 2000, 4059(6782): 85-90.
[10]Lauber K, Bohn E, Krober SM, et al. Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal. Cell, 2003,113(6):717-725.
[11] Fadok VA, Bratton DL, Konowal A, et al. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAR J Clin Invest, 1998,101(4):890-898.
[12]Sun EW,Shi YF.Apoptosis:the quite death silences the immune system.Pharmaco Ther,2001,92(2-3):135-145.
[13]Tanigawa T,Gotoh M,Nagano H,et al.Injection of mitomycin-c-treated spleen cells induces donor specific unresponsiveness to cardiac allografts in rats.Transplantation,1999,67(5):653-658.
[14]Sun E,Zhang L,Zeng Y,et al.Apoptotic cells actively inhibit the expression of CD69 on Con A activated T lymphocytes.Scand J Immunol,2000,51(3):231-236.
[15]Bittencourt MC,Penuche S,Contassot E,et al.Intravenous injection of apoptotic leukocytes enhances bone marrow engraftment across major histocompatibility barriers.Blood,2001,98(1):224-230.
[16]Sun E,Gao Y,Chen J,et al.Transfusion of donor apoptotic splenocytes prolong cardiac allograft survival in absence of immunosuppression.Cell Research,2002,12(3-4):272-273.
[17]Gao Y,Wang S,Shi YF,et al.Permanent liver allograft survival induced by transfusion of apoptotic donor splenocyte without immunosuppression.Cell Research,2002,12(3-4):284-285.
[18]Sun E,Gao Y,Chen J,et al.Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient.Cell Death Differ,2004,11(12):1258-1264.
[19]陈建锋,高毅,孙尔维,等.供者凋亡细胞预输注诱导同种器官移植免疫抑制的研究.中华实验外科杂志,2003,20(7):608-610.
[20]高毅,刘静,汪爽,等.预输注供者凋亡的脾细胞诱导大鼠肝移植免疫耐受的研究.中国肝胆外科杂志,2005,11(4):261-264.
[21]Sakata K,Sakata A,Kong L,et al.Role of Fas/FasL interaction in physiology and pathology:the good and the bad.Clin Immunol Immunopath,1998,87(1):1-7.
[22]Vermes I,Haanen C,Steffens-Nakken H,et al.A novel assay for apoptosis.Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.J Immunol Methods,1995,184(1):39-51.
[23]Nicoletti I,Migliorati G,Pagliacci MC.A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.J Immunot Method,1991,139(2):271-276.
[24]徐岚,周迎会,史宁,等,电子直线加速器照射对NIH 3T3细胞的细胞周期及凋亡的影响。苏州大学学报(医学版),2002,22(2):130-132.
[25]吴伟康.细胞凋亡与疾病.见:金惠铭,王建枝主编.病理生理学.第6版.北京:人民卫生出版社,2004.131-137.
[26]Mathieu J,Richards S,Ballester B,et al.Apoptosis and gamma rays.Ann Pharm Fr,1999,57(4):314-323.
[27]Olive PL,Durand RE.Apoptosis:an indication of radiosensitivity in vitro? Int J Radial Biol Phys,1997,71(6):695-707.
[28]Henriksson C,Bergmark J,Claes G.Use of trypsin for isolation of islets of Langerhans in the rat.Eur Surg Res,1977,9(6):427-431.
[29]Moskalewski S.Isolation and culture of the islets of Langerhans of the guinea pig.Gen Comp Endocrinol,1965,44:342-353.
[30]Gotoh M,Maki T,Kiyoizumi T,et al.An improved method for isolation of mouse pancreatic islets.Transplantation,1985,40(4):437-438.
[31]张桦,蔡德鸿,徐春生,等.一种简易高效的大鼠胰岛分离纯化方法.第一军医大学学报,2001,21(2):129-131.
[32]赵德明,蔡德鸿,张桦,等.试管法纯化人胰岛细胞的初步研究.广东医学,2004,25(2):136-138.
[33]Brandhorst H,Brandhorst D,Hering BJ,et al.Significant progress in porcine islet mass isolation utilizing liberase HI for enzymatic low-temperature.Transplantation,1999,68(3):355-367.
[34]Ricordi C,Lacy PE,Finke EH,et al.Automated method for isolation of human pancreatic islets.Diabetes,1988,37(4):413-420.
[35]Lakey JR,Warnock GL,Shapiro AM,et al.Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion.Cell Transplantation,1999,8(3):285-292.
[36]Arita S,Smith CV,Nagai T,et al.Improved human islet isolation by a tube method for collagenase infusion.Transplantation,1999,68(5):705-707.
[37]Murdoch TB,McGhee-Wilson D,Shapiro AM,et al.Methods of Human Islet Culture for Transplantation.Cell Transplantation,2004,13(6):605-617.
[38]Agern A,Mandel TE,Kennedy MM,et al.Human pancreas culture and function in vitro.Diabetes,1980,29(suppl 1):64.
[39]Jahr H,Hussmann B,Eckhardt T,et al.Successful single donor islet allotransplantation in the streptozotocin diabetes rat model.Cell Transplantation,2002,11(6):513-518.
[40]Tze WI,Wong FC,Tingle AJ.The use of hypaque-ficoll in the isolation of pancreatic islets in rats.Transplantation,1976,22(2):201-205.
[41]Vargas F,Vives-Pi M,Somoza N,et al.Advantages of using a cell separator and metrizamide gradients for human islet purification.Transplantation,1996,61(11):1562-1566.
[42]Frisch SM,Francis H.Disruption of epithelial cell matrix interactions induces apoptosis.J Cell Biol,1994,124(4):619-626.
[43]Thomas FT,Contreras JL,Bilbao G,et al.Anoikis,extracellular matrix and apoptosis factors in isolated cell transplantation.Surgery,1999,126(2):229-304.
[44]White SA,James RF,Swift SM,et al.Human islet cell transplantation—future prospects.Diabet Med,2001,18(2):78-103
[45]张学光.适应性免疫:T淋巴细胞对抗原的识别及免疫应答.见:陈慰峰 主编.医学免疫学.第4版.北京:人民卫生出版社,2004.155.
[46]Meyer D,Baumgardt S,Loeffeler S,et al.Apoptosis of T lymphocytes in liver and/or small bowel allografis during tolerance induction.Transplantation,1998,66(11):1530-1536.
[47]Bellgrau D,Guld D,Selawry H,et al.A role for CD95 ligand in preventing graft rejection.Nature,1995,377(6550):630-632.
[48]Opelz G,Terasaki PI.Poor kidney-transplant survival in recipients with frozen-blood transfusions or no transfusions.Lancet,1974,2(7882):696-698.
[49]Frabetti F, Tazzari PL, Musiani D, et al. White cell apoptosis in platelet concentrates. Transfusion, 2000,40(2):160-168.
[50]Frabetti F, Musiani D, Marini M, et al. White cell apoptosis in packed red cells. Transfusion, 1998,38 (11-12):1082-1089
[51] Kelly DA, Young A, McGregor JM, et al. Sensitivity to sunburn is associated with susceptibility to ultraviolet radiation-induced suppression of cutaneous cell-mediated immunity. J Exp Med, 2000,191(3):561-566.
[52]Ozawa M, Ferenczi K, Kikuchi T, et al. 312-nanometer ultraviolet B light (narrow-band UVB) induces apoptosis of T cells within psoriatic lesions. J Exp Med, 1999,189(4):711-718.
[53]Ponner BB, Stach C, Zoller 0, et al. Induction of apoptosis reduces immunogenicity of human T-cell lines in mice. Scand J Immunol, 1998, 47(4):343-347.
[54]Ronchetti A, Rovere P, Iezzi G. et al. Immunogenicity of apoptotic cells in vivo: role of antigen load, antigen-presenting cells, and cytokines. J Immunol, 1999,163(1):130-136.
[55]Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: endogenous activators of dendritic cells. Nat Med, 1999,5(11):1249-1255.
[56]Reiter I, Krammer B, Schwamberger G. Cutting edge: differential effect of apoptotic versus necrotic tumor cells on macrophage antitumor activities. J Immunol, 1999,163(4):1730-1741.
[57] Simon JC, Cruz PD, Bergstresser PR, et al. Low dose ultraviolet B-irradiated Langerhans cells preferentially activate CD4+ cells of the T helper 2 subset. J Immunol, 1990,145(7):2087-2093
[58] Young JW, Baggers J, Soergel SA. High-dose UV-B radiation alters human dendritic cell costimulatory activity but dose not allow dendritic cells to tolerize T lymphocytes to alloantigen in vitro. Blood, 1993,81(11):2987-2991.
[59]Stumbles PA, Thomas JA, Pimm CL, et al. Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2(Th2) responses and require obligatory cytokine signals for induction of Th1 immunity. J Exp Med, 1998, 188(11):2019-2031.
[60]Martzinger P.Tolerance,danger and the extended family.Annu Rev Immunol,1994,12:991-1045.
[61]Segal BM,Dwger BK,Shevach EM.An interleukin(IL)-10/IL-12immunoregulatory circuit controls susceptibility to autoimmune disease.J ExP Med,1998,187(4):537-54I.
[62]Shinozaki K,Yahata H,Tanji H,et al.Allograft transduction of IL-10 prolongs survival following orthotopic liver transplantation.Gene Therapy,1999,6(5):816-822.
[63]Minguela A,Torio A,Marin L,et al.Implication of soluble and membrane HLA class Ⅰ and serum IL-10 in liver graft acceptance.Hum lmmunol,1999,60(6):500-509.
[64]Torio AM,Martin L,Sanchez F,et al.Implication ofThl,Th2 and Th3 cytokines in liver graft acceptance.Transplant Proc,1999,31(1-2):519-520.
[65]Hotchkiss RS,Chang KC,Grayson MH,et al.Adoptive transfer of apoptotic splenocytes worsens survival,whereas adoptive transfer of necrotic splenocytes improves survival in sepsis.Proc Natl Acad Sci USA,2003,100(11):6724-6729.
[66]Hotchkiss RS,Tinsley KW,Swanson PE,et al.Sepsis-induced apoptosis cause progressive profound depletion of B and CD4~+ T lymphocytes in humans.J Immunol,2001,166(11):6952-6963.
[67]汪艳,高毅,孙尔维,等.肝脏抗原递呈细胞对同种异基因供体凋亡细胞的吞噬作用.中华肝脏病杂志,2006,14(5):370-374.
[68]Chen W,Frank ME,Jin W,et al.TGF-beta released by apoptotic T cells contributes to an immunosuppressive milieu.Immunity,2001,14(6):715-725.
[69]Douglas RG,Helen MB.Apoptosis:Gone but not forgotten.Nature,2000,405(6782):28-29.
[70]Huynh ML,Fadok VA,Henson PM.Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-betal secretion and the resolution of inflammation.J Clin Invest,2002,109(1):41-50.
[71]Steinman RM,Hawiger D,Nussenzweig MC.Tolerogenic dendritic cells.Annu Rev Immunol,2003,21:685-711.
[72]Sauter B,Albert ML,Francisco L,et al.Consequences of cell death:exposure to necrotic tumor cells,but not primary tissue cells or apoptotic cells,induces the maturation of immunostimulatory dendritic cells.J Exp Med,2000,191(3):423-434.
[73]Basu S,Binder RJ,Suto R,et al.Necrotic but not apoptotic cell death,releases heat shock proteins,which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway.Int Immunol,2000,12(11):1539-1946.
[74]Belz GT,Behrens GM,Smith CM,et al.The CD8alpha(+) dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens.J Exp Med,2002,196(8):1099-1104.
[75]Kurts C,Kosaka H,Carbone FR,et al.Class Ⅰ-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells.J Exp Med,1997,186(2):239-245.
[76]孙尔维.凋亡细胞诱导免疫耐受的机制及意义.见:陈实 主编.移植学前沿.武汉:湖北科学技术出版社.2002.26-45.
[77]Gujar SA,Michalak TI:Flow cytometric quantification of T cell proliferation and division kinetics in woodchuck model of hepatitis B.Immunol Invest,2005,34:215-236.
[78]Givan AL,Fisher JL,Waugh MG,Bercovici N,Wallace PK:Use of cell-tracking dyes to determine proliferation precursor frequencies of antigen-specific T cells.Methods Mol Biol,2004,263:109-124.
[79]Lyons AB:Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution.J Immunol Methods,2000,243:147-154.
[80]Lyons AB,Hasbold J,Hodgkin PD:Flow cytometric analysis of cell division history using dilution of carboxyfluorescein diacetate succinimidyl ester,a stably integrated fluorescent probe.Methods Cell Biol,2001,63:375-398.
[1]Posselt AM,Barker CF,Tomaszewski JE,et al.Induction of donor-specific unresponsiveness by intrathymic islet transplantation.Science,1990,249(4974):1293-1295.
[2] Turvey SE, Hara M, Morris PJ, et al. Mechanisms of tolerance induction after intrathymic islet injection: determination of the fate of alloreactive thymocytes. Transplantation, 1999,68(1):30-39.
[3] Ali A, Garrovillo M, Jin MK, et al. Major histocompatibility complex class I peptide-pulsed host dendritic cells induces antigen-specific acquired thymic tolerance to islet cells. Transplantation, 2000,69(2):221-226.
[4] Benhamou PY. Immunomodulation with CTLA4-Ig in islet transplantation. Transplantation, 2002,73(1 Suppl):S40-2.
[5] Nabeyama K, Yasunami Y, Toyofuku A, et al. Beneficial effects of costimulatory blockade with anti-inducible costimulator antibody in conjunction with CTLA4Ig on prevention of islet xenograft rejection from rat to mouse. Transplantation, 2004,78(11):1590-1596.
[6] Kenyon NS, Fernandez LA, Lehmann R, et al. Long-term survival and function of intrahepatic islet allografts in baboons treated with humanized anti-CD 154. Diabetes, 1999,48(7): 1473-1479.
[7] Grochowiecki T, Gotoh M, Dono K, et al. Induction of unresponsiveness to islet xenograft by MMC treatment of graft and blockage if LFA-1/ICAM-1 pathway. Transplantation, 2000,69(8): 1567-1571.
[8] Nanji SA, Hancock WW, Luo B, et al. Costimulation blockade of both inducible costimulator and CD40 ligand induces dominant tolerance to islet allografts and prevents spontaneous autoimmune diabetes in the NOD mouse. Diabetes, 2006,55(1):27-33.
[9] Malm H, Pahlman C, Veress B,et al. Combined costimulation blockade prevents rejection of allogeneic islets in mice. Scand J Immunol, 2006,64(4):398-403.
[10]Rayat GR, Gill RG. Indefinite survival of neonatal porcine islet xenografts by simultaneous targeting of LFA-1 and CD154 or CD45RB. Diabetes, 2005, 54(2): 443-451.
[11]Nanji SA, Hancock WW, Anderson CC, et al. Multiple combination therapies involving blockade of ICOS/B7RP-1 costimulation facilitate long-term islet allograft survival. Am J Transplant, 2004,4(4):526-536.
[12]Lau HT, Yu M, Fontana A, et al. Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice. Science, 1996,273(5271):109-112.
[13]Takeda Y, Gotoh M, Dono K, et al. Protection of islet allografts transplanted together with Fas ligand expressing testicular allografts. Diabetologia, 1998,41(3):315-321.
[14]Korbutt GS, Elliott JF, Rajotte RV. Cotransplantation of allogeneic islets with allogeneic testicular cell aggregates allows long-term graft survival without systemic immunosuppression. Diabetes, 1997, 46(2): 317-322.
[15] Li XC, Ima A, Li Y, et al. Blocking the common gamma-chain of cytokine receptors induces T cell apoptosis and long term islet allograft survival. J Immunol, 2000,164(3): 1193-1199.
[16] Sharif S, Guillermo A, Peter Z, et al. Activation of NKT cells by alpha-galactosylceramide treatment prevents the onset and recurrence of autoimmune type I diabetes. Nature Med, 2001,7(9): 1057-1062.
[17]Toyofuku A, Yasunami Y, Nabeyama K, et al. Natural killer T-cells participate in rejection of islet allografts in the liver of mice. Diabetes, 2006,55(1):34-39.
[18] Yasunami Y, Kojo S, Kitamura H, et al. Valphal4 NKT cell-triggered IFN-gamma production by Gr-1+CD11b+ cells mediates early graft loss of syngeneic transplanted islets. J Exp Med, 2005,202(7):913-918.
[19]Chatenoud L, Primo J, Bach J F. CD3 antibody-induced dominated self tolerance in overtly diabetic NOD mice. J Immunol, 1997,158(6):2947-2954.
[20] Philips JM, Harach SZ, Parish NM, et al. Nondepleting anti-CD4 has an immediate action on diabetogenic effector cells halting their destruction of pancreatic beta cells. J Immunol, 2000,165(4):1949-1955.
[23]Basadonna GP, Auersvald I,Khuong CQ, et al. Antibody-mediated targeting of CD45 isoforms: a novel immunotherapeutic strategy. Proc Natl Acad Sci USA, 1998,95(7):3821-3826.
[22]Visser L, Poppema S, de Haan B, et al. Prolonged survival of rat islet xenografts in mice after CD45RB monotherapy. Transplantation, 2004,77(3):386-391.
[23]Molano RD, Pileggi A, Berney T, et al. Prolonged islet allograft survival in diabetic NOD mice by targeting CD45RB and CD154. Diabetes, 2003, 52(4): 957-963.
[24]Gregori S, Mangia P, Bacchetta R, et al. An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells. J Exp Med, 2005,201(8):1293-1305.
[25] Seung E, Iwakoshi N, Woda BA, et al. Allogeneic hematopoietic chimerism in mice treated with sublethal myeloablation and anti-CD154 antibody: absence if graft-versus-host disease,induction of skin allograft tolerance, and prevention of recurrent autoimmunity inislet-allografted NOD/Lt mice. Blood, 2000,95(6):2175-2182.
[26] Lee BW, Lee JI, Oh SH, et al. A more persistent tolerance to islet allografts through bone marrow transplantation in minimal nonmyeloablative conditioning therapy. Transplant Proc, 2005,37(5):2266-2269.
[27]Xu H, Exner BG, Chilton PM, et al. A delay in bone marrow transplantation after partial conditioning improves engraftment. Transplantation, 2004,77(6):819-826.
[28]Horton PJ, Hawthorne WJ, Walers SN, et al. Induction of allogeneic islet tolerance in a large-animal model. Cell Transplantation, 2000,9(6):877-887.
[29] Seung E, Mordes JP, Greiner DL, et al. Induction of tolerance for islet transplantation for type 1 diabetes. Curr Diab Rep, 2003,3(4):329-335.
[30]Gaur LK, Kennedy E, Nitta Y, et al. Induction of donor-specific tolerance to islet allografts in nonhuman primates. Ann N Y Acad Sci, 2002,958:194-198.
[31] Akashi S, Sho M, Kashizuka H,et al. A novel small-molecule compound targeting CCR5 and CXCR3 prevents acute and chronic allograft rejection. Transplantation, 2005,80(3):378-384.
[32] Yi S, Ouyang L, Ha H, et al. Involvement of CCR5 signaling in macrophage recruitment to porcine islet xenografts. Transplantation, 2005,80(10):1468-1475.
[33]Fernandes JR, Duvivier-Kali VF, Keegan M, et al.Transplantation of islets transduced with CTLA4-Ig and TGFbeta using adenovirus and lentivirus vectors. Transpl Immunol, 2004,13(3): 191-200.
[34]Londrigan SL, Sutherland RM, Brady JL, et al. Prolonged local expression of anti-CD4 antibody by adenovirally transduced allografts can promote long-term graft survival. J Gene Med, 2006,8(1):42-52.
[35] Carter JD, Ellett JD, Chen M, et al. Viral IL-10-mediated immune regulation in pancreatic islet transplantation. Mol Ther, 2005,12(2):360-368.
[36]Machen J, Bertera S, Chang Y, et al. Prolongation of islet allograft survival following ex vivo transduction with adenovirus encoding a soluble type 1 TNF receptor-Ig fusion decoy. Gene Ther, 2004,11(20): 1506-1514.
[37]Ikemoto T, Tashiro S, Yasutomo K, et al. Donor specific tolerance induced by simultaneous allogeneic islet transplantation with CD4~+CD25~+ T cells into hepatic parenchyma in mice. J Med Invest, 2004,51 (3-4): 178-185.
[38]Wekerle T, Kurtz J, Ito H, et al. Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat Med, 2000,6(4):464-469.
[39]Zheng XX, Markees TG, Hancock WW, et al. CTLA4 Signals are required to optimally induce allograft toleramce with combined donor-specific transfusion and anti-CD 154 monoclonal antibody treatment. J Immunol, 1999,162(8):4983-4990.
[40]Masaki H, Appel MC, Leahy L, et al. Anti-mouse CD154 antibody treatment facilitates generation of mixed xenogeneic rat hematopoietic chimerism, prevents wasting disease and prolongs xenograft survival in mice. Xenotransplantation, 2006,13(3):224-232.
[41]Koulmanda M, Smith RN, Qipo A, et al. Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term anti-CD154-based therapy: nonimmunologic graft failure? Am J Transplant, 2006,6(4):687-696.
[42]Hirakawa E, Yasunami Y, Nakano M, et al. Amelioration of hyperglycemia in streptozotocin-induced diabetic mice with fetal pancreatic allografts: prevention of rejection by donor specific transfusion in conjunction with CTLA4Ig. Pancreas, 2004,28(2):146-152.
[43] Appel MC, Banuelos SJ, Greiner DL, et al. Prolonged survival of neonatal porcine islet xenografts in mice treated with a donor-specific transfusion and anti-CD 154 antibody. Transplantation, 2004,77(9):1341-1349.
[2] Seung E, Mordes JP, Greiner DL, et al. Induction of tolerance for islet transplantation for type 1 diabetes. Curr Diab Rep, 2003,3(4):329-335.
[3] Shapiro AM, Ricordi C, Hering BJ, et al. International trial of the Edmonton protocol for islet transplantation. N Engl J Med, 2006,355(13):1372-1374.
[4] Truong W, Shapiro AM. Progress in islet transplantation in patients with type 1 diabetes mellitus. Treat Endocrinol., 2006,5(3): 147-158.
[5] Balamurugan AN, Bottino R, Giannoukakis N, et al. Prospective and challenges of islet transplantation for the therapy of autoimmune diabetes. Pancreas, 2006, 32(3):231-243.
[6] Posselt AM, Barker CF, Tomaszewski JE, et al. Induction of donor-specific unresponsiveness by intrathymic islet transplantation. Science, 1990,249(4974): 1293-1295.
[7] Voll RE, Herrmann M, Roth EA, et al. Immunosuppressive effects of apoptotic cells. Nature, 1997, 390(6658):350-351.
[8] Kitajima T, Ariizumi K, Bergstresser PR, et al. Ultraviolet B radiation sensitizes a murine epidermal dendritic cell line(XS52) to undergo apoptosis upon antigen presentation to cells. J Immunol, 1996, 157(8): 3312-3318.
[9] Fadok VA, Bratton DL, Rose DM, et al. A Receptor for phosphatidylserine- specific clearance of apoptotic cells. Nature, 2000, 4059(6782): 85-90.
[10]Lauber K, Bohn E, Krober SM, et al. Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal. Cell, 2003,113(6):717-725.
[11] Fadok VA, Bratton DL, Konowal A, et al. Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanisms involving TGF-beta, PGE2, and PAR J Clin Invest, 1998,101(4):890-898.
[12]Sun EW,Shi YF.Apoptosis:the quite death silences the immune system.Pharmaco Ther,2001,92(2-3):135-145.
[13]Tanigawa T,Gotoh M,Nagano H,et al.Injection of mitomycin-c-treated spleen cells induces donor specific unresponsiveness to cardiac allografts in rats.Transplantation,1999,67(5):653-658.
[14]Sun E,Zhang L,Zeng Y,et al.Apoptotic cells actively inhibit the expression of CD69 on Con A activated T lymphocytes.Scand J Immunol,2000,51(3):231-236.
[15]Bittencourt MC,Penuche S,Contassot E,et al.Intravenous injection of apoptotic leukocytes enhances bone marrow engraftment across major histocompatibility barriers.Blood,2001,98(1):224-230.
[16]Sun E,Gao Y,Chen J,et al.Transfusion of donor apoptotic splenocytes prolong cardiac allograft survival in absence of immunosuppression.Cell Research,2002,12(3-4):272-273.
[17]Gao Y,Wang S,Shi YF,et al.Permanent liver allograft survival induced by transfusion of apoptotic donor splenocyte without immunosuppression.Cell Research,2002,12(3-4):284-285.
[18]Sun E,Gao Y,Chen J,et al.Allograft tolerance induced by donor apoptotic lymphocytes requires phagocytosis in the recipient.Cell Death Differ,2004,11(12):1258-1264.
[19]陈建锋,高毅,孙尔维,等.供者凋亡细胞预输注诱导同种器官移植免疫抑制的研究.中华实验外科杂志,2003,20(7):608-610.
[20]高毅,刘静,汪爽,等.预输注供者凋亡的脾细胞诱导大鼠肝移植免疫耐受的研究.中国肝胆外科杂志,2005,11(4):261-264.
[21]Sakata K,Sakata A,Kong L,et al.Role of Fas/FasL interaction in physiology and pathology:the good and the bad.Clin Immunol Immunopath,1998,87(1):1-7.
[22]Vermes I,Haanen C,Steffens-Nakken H,et al.A novel assay for apoptosis.Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.J Immunol Methods,1995,184(1):39-51.
[23]Nicoletti I,Migliorati G,Pagliacci MC.A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry.J Immunot Method,1991,139(2):271-276.
[24]徐岚,周迎会,史宁,等,电子直线加速器照射对NIH 3T3细胞的细胞周期及凋亡的影响。苏州大学学报(医学版),2002,22(2):130-132.
[25]吴伟康.细胞凋亡与疾病.见:金惠铭,王建枝主编.病理生理学.第6版.北京:人民卫生出版社,2004.131-137.
[26]Mathieu J,Richards S,Ballester B,et al.Apoptosis and gamma rays.Ann Pharm Fr,1999,57(4):314-323.
[27]Olive PL,Durand RE.Apoptosis:an indication of radiosensitivity in vitro? Int J Radial Biol Phys,1997,71(6):695-707.
[28]Henriksson C,Bergmark J,Claes G.Use of trypsin for isolation of islets of Langerhans in the rat.Eur Surg Res,1977,9(6):427-431.
[29]Moskalewski S.Isolation and culture of the islets of Langerhans of the guinea pig.Gen Comp Endocrinol,1965,44:342-353.
[30]Gotoh M,Maki T,Kiyoizumi T,et al.An improved method for isolation of mouse pancreatic islets.Transplantation,1985,40(4):437-438.
[31]张桦,蔡德鸿,徐春生,等.一种简易高效的大鼠胰岛分离纯化方法.第一军医大学学报,2001,21(2):129-131.
[32]赵德明,蔡德鸿,张桦,等.试管法纯化人胰岛细胞的初步研究.广东医学,2004,25(2):136-138.
[33]Brandhorst H,Brandhorst D,Hering BJ,et al.Significant progress in porcine islet mass isolation utilizing liberase HI for enzymatic low-temperature.Transplantation,1999,68(3):355-367.
[34]Ricordi C,Lacy PE,Finke EH,et al.Automated method for isolation of human pancreatic islets.Diabetes,1988,37(4):413-420.
[35]Lakey JR,Warnock GL,Shapiro AM,et al.Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion.Cell Transplantation,1999,8(3):285-292.
[36]Arita S,Smith CV,Nagai T,et al.Improved human islet isolation by a tube method for collagenase infusion.Transplantation,1999,68(5):705-707.
[37]Murdoch TB,McGhee-Wilson D,Shapiro AM,et al.Methods of Human Islet Culture for Transplantation.Cell Transplantation,2004,13(6):605-617.
[38]Agern A,Mandel TE,Kennedy MM,et al.Human pancreas culture and function in vitro.Diabetes,1980,29(suppl 1):64.
[39]Jahr H,Hussmann B,Eckhardt T,et al.Successful single donor islet allotransplantation in the streptozotocin diabetes rat model.Cell Transplantation,2002,11(6):513-518.
[40]Tze WI,Wong FC,Tingle AJ.The use of hypaque-ficoll in the isolation of pancreatic islets in rats.Transplantation,1976,22(2):201-205.
[41]Vargas F,Vives-Pi M,Somoza N,et al.Advantages of using a cell separator and metrizamide gradients for human islet purification.Transplantation,1996,61(11):1562-1566.
[42]Frisch SM,Francis H.Disruption of epithelial cell matrix interactions induces apoptosis.J Cell Biol,1994,124(4):619-626.
[43]Thomas FT,Contreras JL,Bilbao G,et al.Anoikis,extracellular matrix and apoptosis factors in isolated cell transplantation.Surgery,1999,126(2):229-304.
[44]White SA,James RF,Swift SM,et al.Human islet cell transplantation—future prospects.Diabet Med,2001,18(2):78-103
[45]张学光.适应性免疫:T淋巴细胞对抗原的识别及免疫应答.见:陈慰峰 主编.医学免疫学.第4版.北京:人民卫生出版社,2004.155.
[46]Meyer D,Baumgardt S,Loeffeler S,et al.Apoptosis of T lymphocytes in liver and/or small bowel allografis during tolerance induction.Transplantation,1998,66(11):1530-1536.
[47]Bellgrau D,Guld D,Selawry H,et al.A role for CD95 ligand in preventing graft rejection.Nature,1995,377(6550):630-632.
[48]Opelz G,Terasaki PI.Poor kidney-transplant survival in recipients with frozen-blood transfusions or no transfusions.Lancet,1974,2(7882):696-698.
[49]Frabetti F, Tazzari PL, Musiani D, et al. White cell apoptosis in platelet concentrates. Transfusion, 2000,40(2):160-168.
[50]Frabetti F, Musiani D, Marini M, et al. White cell apoptosis in packed red cells. Transfusion, 1998,38 (11-12):1082-1089
[51] Kelly DA, Young A, McGregor JM, et al. Sensitivity to sunburn is associated with susceptibility to ultraviolet radiation-induced suppression of cutaneous cell-mediated immunity. J Exp Med, 2000,191(3):561-566.
[52]Ozawa M, Ferenczi K, Kikuchi T, et al. 312-nanometer ultraviolet B light (narrow-band UVB) induces apoptosis of T cells within psoriatic lesions. J Exp Med, 1999,189(4):711-718.
[53]Ponner BB, Stach C, Zoller 0, et al. Induction of apoptosis reduces immunogenicity of human T-cell lines in mice. Scand J Immunol, 1998, 47(4):343-347.
[54]Ronchetti A, Rovere P, Iezzi G. et al. Immunogenicity of apoptotic cells in vivo: role of antigen load, antigen-presenting cells, and cytokines. J Immunol, 1999,163(1):130-136.
[55]Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: endogenous activators of dendritic cells. Nat Med, 1999,5(11):1249-1255.
[56]Reiter I, Krammer B, Schwamberger G. Cutting edge: differential effect of apoptotic versus necrotic tumor cells on macrophage antitumor activities. J Immunol, 1999,163(4):1730-1741.
[57] Simon JC, Cruz PD, Bergstresser PR, et al. Low dose ultraviolet B-irradiated Langerhans cells preferentially activate CD4+ cells of the T helper 2 subset. J Immunol, 1990,145(7):2087-2093
[58] Young JW, Baggers J, Soergel SA. High-dose UV-B radiation alters human dendritic cell costimulatory activity but dose not allow dendritic cells to tolerize T lymphocytes to alloantigen in vitro. Blood, 1993,81(11):2987-2991.
[59]Stumbles PA, Thomas JA, Pimm CL, et al. Resting respiratory tract dendritic cells preferentially stimulate T helper cell type 2(Th2) responses and require obligatory cytokine signals for induction of Th1 immunity. J Exp Med, 1998, 188(11):2019-2031.
[60]Martzinger P.Tolerance,danger and the extended family.Annu Rev Immunol,1994,12:991-1045.
[61]Segal BM,Dwger BK,Shevach EM.An interleukin(IL)-10/IL-12immunoregulatory circuit controls susceptibility to autoimmune disease.J ExP Med,1998,187(4):537-54I.
[62]Shinozaki K,Yahata H,Tanji H,et al.Allograft transduction of IL-10 prolongs survival following orthotopic liver transplantation.Gene Therapy,1999,6(5):816-822.
[63]Minguela A,Torio A,Marin L,et al.Implication of soluble and membrane HLA class Ⅰ and serum IL-10 in liver graft acceptance.Hum lmmunol,1999,60(6):500-509.
[64]Torio AM,Martin L,Sanchez F,et al.Implication ofThl,Th2 and Th3 cytokines in liver graft acceptance.Transplant Proc,1999,31(1-2):519-520.
[65]Hotchkiss RS,Chang KC,Grayson MH,et al.Adoptive transfer of apoptotic splenocytes worsens survival,whereas adoptive transfer of necrotic splenocytes improves survival in sepsis.Proc Natl Acad Sci USA,2003,100(11):6724-6729.
[66]Hotchkiss RS,Tinsley KW,Swanson PE,et al.Sepsis-induced apoptosis cause progressive profound depletion of B and CD4~+ T lymphocytes in humans.J Immunol,2001,166(11):6952-6963.
[67]汪艳,高毅,孙尔维,等.肝脏抗原递呈细胞对同种异基因供体凋亡细胞的吞噬作用.中华肝脏病杂志,2006,14(5):370-374.
[68]Chen W,Frank ME,Jin W,et al.TGF-beta released by apoptotic T cells contributes to an immunosuppressive milieu.Immunity,2001,14(6):715-725.
[69]Douglas RG,Helen MB.Apoptosis:Gone but not forgotten.Nature,2000,405(6782):28-29.
[70]Huynh ML,Fadok VA,Henson PM.Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-betal secretion and the resolution of inflammation.J Clin Invest,2002,109(1):41-50.
[71]Steinman RM,Hawiger D,Nussenzweig MC.Tolerogenic dendritic cells.Annu Rev Immunol,2003,21:685-711.
[72]Sauter B,Albert ML,Francisco L,et al.Consequences of cell death:exposure to necrotic tumor cells,but not primary tissue cells or apoptotic cells,induces the maturation of immunostimulatory dendritic cells.J Exp Med,2000,191(3):423-434.
[73]Basu S,Binder RJ,Suto R,et al.Necrotic but not apoptotic cell death,releases heat shock proteins,which deliver a partial maturation signal to dendritic cells and activate the NF-kappa B pathway.Int Immunol,2000,12(11):1539-1946.
[74]Belz GT,Behrens GM,Smith CM,et al.The CD8alpha(+) dendritic cell is responsible for inducing peripheral self-tolerance to tissue-associated antigens.J Exp Med,2002,196(8):1099-1104.
[75]Kurts C,Kosaka H,Carbone FR,et al.Class Ⅰ-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells.J Exp Med,1997,186(2):239-245.
[76]孙尔维.凋亡细胞诱导免疫耐受的机制及意义.见:陈实 主编.移植学前沿.武汉:湖北科学技术出版社.2002.26-45.
[77]Gujar SA,Michalak TI:Flow cytometric quantification of T cell proliferation and division kinetics in woodchuck model of hepatitis B.Immunol Invest,2005,34:215-236.
[78]Givan AL,Fisher JL,Waugh MG,Bercovici N,Wallace PK:Use of cell-tracking dyes to determine proliferation precursor frequencies of antigen-specific T cells.Methods Mol Biol,2004,263:109-124.
[79]Lyons AB:Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution.J Immunol Methods,2000,243:147-154.
[80]Lyons AB,Hasbold J,Hodgkin PD:Flow cytometric analysis of cell division history using dilution of carboxyfluorescein diacetate succinimidyl ester,a stably integrated fluorescent probe.Methods Cell Biol,2001,63:375-398.
[1]Posselt AM,Barker CF,Tomaszewski JE,et al.Induction of donor-specific unresponsiveness by intrathymic islet transplantation.Science,1990,249(4974):1293-1295.
[2] Turvey SE, Hara M, Morris PJ, et al. Mechanisms of tolerance induction after intrathymic islet injection: determination of the fate of alloreactive thymocytes. Transplantation, 1999,68(1):30-39.
[3] Ali A, Garrovillo M, Jin MK, et al. Major histocompatibility complex class I peptide-pulsed host dendritic cells induces antigen-specific acquired thymic tolerance to islet cells. Transplantation, 2000,69(2):221-226.
[4] Benhamou PY. Immunomodulation with CTLA4-Ig in islet transplantation. Transplantation, 2002,73(1 Suppl):S40-2.
[5] Nabeyama K, Yasunami Y, Toyofuku A, et al. Beneficial effects of costimulatory blockade with anti-inducible costimulator antibody in conjunction with CTLA4Ig on prevention of islet xenograft rejection from rat to mouse. Transplantation, 2004,78(11):1590-1596.
[6] Kenyon NS, Fernandez LA, Lehmann R, et al. Long-term survival and function of intrahepatic islet allografts in baboons treated with humanized anti-CD 154. Diabetes, 1999,48(7): 1473-1479.
[7] Grochowiecki T, Gotoh M, Dono K, et al. Induction of unresponsiveness to islet xenograft by MMC treatment of graft and blockage if LFA-1/ICAM-1 pathway. Transplantation, 2000,69(8): 1567-1571.
[8] Nanji SA, Hancock WW, Luo B, et al. Costimulation blockade of both inducible costimulator and CD40 ligand induces dominant tolerance to islet allografts and prevents spontaneous autoimmune diabetes in the NOD mouse. Diabetes, 2006,55(1):27-33.
[9] Malm H, Pahlman C, Veress B,et al. Combined costimulation blockade prevents rejection of allogeneic islets in mice. Scand J Immunol, 2006,64(4):398-403.
[10]Rayat GR, Gill RG. Indefinite survival of neonatal porcine islet xenografts by simultaneous targeting of LFA-1 and CD154 or CD45RB. Diabetes, 2005, 54(2): 443-451.
[11]Nanji SA, Hancock WW, Anderson CC, et al. Multiple combination therapies involving blockade of ICOS/B7RP-1 costimulation facilitate long-term islet allograft survival. Am J Transplant, 2004,4(4):526-536.
[12]Lau HT, Yu M, Fontana A, et al. Prevention of islet allograft rejection with engineered myoblasts expressing FasL in mice. Science, 1996,273(5271):109-112.
[13]Takeda Y, Gotoh M, Dono K, et al. Protection of islet allografts transplanted together with Fas ligand expressing testicular allografts. Diabetologia, 1998,41(3):315-321.
[14]Korbutt GS, Elliott JF, Rajotte RV. Cotransplantation of allogeneic islets with allogeneic testicular cell aggregates allows long-term graft survival without systemic immunosuppression. Diabetes, 1997, 46(2): 317-322.
[15] Li XC, Ima A, Li Y, et al. Blocking the common gamma-chain of cytokine receptors induces T cell apoptosis and long term islet allograft survival. J Immunol, 2000,164(3): 1193-1199.
[16] Sharif S, Guillermo A, Peter Z, et al. Activation of NKT cells by alpha-galactosylceramide treatment prevents the onset and recurrence of autoimmune type I diabetes. Nature Med, 2001,7(9): 1057-1062.
[17]Toyofuku A, Yasunami Y, Nabeyama K, et al. Natural killer T-cells participate in rejection of islet allografts in the liver of mice. Diabetes, 2006,55(1):34-39.
[18] Yasunami Y, Kojo S, Kitamura H, et al. Valphal4 NKT cell-triggered IFN-gamma production by Gr-1+CD11b+ cells mediates early graft loss of syngeneic transplanted islets. J Exp Med, 2005,202(7):913-918.
[19]Chatenoud L, Primo J, Bach J F. CD3 antibody-induced dominated self tolerance in overtly diabetic NOD mice. J Immunol, 1997,158(6):2947-2954.
[20] Philips JM, Harach SZ, Parish NM, et al. Nondepleting anti-CD4 has an immediate action on diabetogenic effector cells halting their destruction of pancreatic beta cells. J Immunol, 2000,165(4):1949-1955.
[23]Basadonna GP, Auersvald I,Khuong CQ, et al. Antibody-mediated targeting of CD45 isoforms: a novel immunotherapeutic strategy. Proc Natl Acad Sci USA, 1998,95(7):3821-3826.
[22]Visser L, Poppema S, de Haan B, et al. Prolonged survival of rat islet xenografts in mice after CD45RB monotherapy. Transplantation, 2004,77(3):386-391.
[23]Molano RD, Pileggi A, Berney T, et al. Prolonged islet allograft survival in diabetic NOD mice by targeting CD45RB and CD154. Diabetes, 2003, 52(4): 957-963.
[24]Gregori S, Mangia P, Bacchetta R, et al. An anti-CD45RO/RB monoclonal antibody modulates T cell responses via induction of apoptosis and generation of regulatory T cells. J Exp Med, 2005,201(8):1293-1305.
[25] Seung E, Iwakoshi N, Woda BA, et al. Allogeneic hematopoietic chimerism in mice treated with sublethal myeloablation and anti-CD154 antibody: absence if graft-versus-host disease,induction of skin allograft tolerance, and prevention of recurrent autoimmunity inislet-allografted NOD/Lt mice. Blood, 2000,95(6):2175-2182.
[26] Lee BW, Lee JI, Oh SH, et al. A more persistent tolerance to islet allografts through bone marrow transplantation in minimal nonmyeloablative conditioning therapy. Transplant Proc, 2005,37(5):2266-2269.
[27]Xu H, Exner BG, Chilton PM, et al. A delay in bone marrow transplantation after partial conditioning improves engraftment. Transplantation, 2004,77(6):819-826.
[28]Horton PJ, Hawthorne WJ, Walers SN, et al. Induction of allogeneic islet tolerance in a large-animal model. Cell Transplantation, 2000,9(6):877-887.
[29] Seung E, Mordes JP, Greiner DL, et al. Induction of tolerance for islet transplantation for type 1 diabetes. Curr Diab Rep, 2003,3(4):329-335.
[30]Gaur LK, Kennedy E, Nitta Y, et al. Induction of donor-specific tolerance to islet allografts in nonhuman primates. Ann N Y Acad Sci, 2002,958:194-198.
[31] Akashi S, Sho M, Kashizuka H,et al. A novel small-molecule compound targeting CCR5 and CXCR3 prevents acute and chronic allograft rejection. Transplantation, 2005,80(3):378-384.
[32] Yi S, Ouyang L, Ha H, et al. Involvement of CCR5 signaling in macrophage recruitment to porcine islet xenografts. Transplantation, 2005,80(10):1468-1475.
[33]Fernandes JR, Duvivier-Kali VF, Keegan M, et al.Transplantation of islets transduced with CTLA4-Ig and TGFbeta using adenovirus and lentivirus vectors. Transpl Immunol, 2004,13(3): 191-200.
[34]Londrigan SL, Sutherland RM, Brady JL, et al. Prolonged local expression of anti-CD4 antibody by adenovirally transduced allografts can promote long-term graft survival. J Gene Med, 2006,8(1):42-52.
[35] Carter JD, Ellett JD, Chen M, et al. Viral IL-10-mediated immune regulation in pancreatic islet transplantation. Mol Ther, 2005,12(2):360-368.
[36]Machen J, Bertera S, Chang Y, et al. Prolongation of islet allograft survival following ex vivo transduction with adenovirus encoding a soluble type 1 TNF receptor-Ig fusion decoy. Gene Ther, 2004,11(20): 1506-1514.
[37]Ikemoto T, Tashiro S, Yasutomo K, et al. Donor specific tolerance induced by simultaneous allogeneic islet transplantation with CD4~+CD25~+ T cells into hepatic parenchyma in mice. J Med Invest, 2004,51 (3-4): 178-185.
[38]Wekerle T, Kurtz J, Ito H, et al. Allogeneic bone marrow transplantation with co-stimulatory blockade induces macrochimerism and tolerance without cytoreductive host treatment. Nat Med, 2000,6(4):464-469.
[39]Zheng XX, Markees TG, Hancock WW, et al. CTLA4 Signals are required to optimally induce allograft toleramce with combined donor-specific transfusion and anti-CD 154 monoclonal antibody treatment. J Immunol, 1999,162(8):4983-4990.
[40]Masaki H, Appel MC, Leahy L, et al. Anti-mouse CD154 antibody treatment facilitates generation of mixed xenogeneic rat hematopoietic chimerism, prevents wasting disease and prolongs xenograft survival in mice. Xenotransplantation, 2006,13(3):224-232.
[41]Koulmanda M, Smith RN, Qipo A, et al. Prolonged survival of allogeneic islets in cynomolgus monkeys after short-term anti-CD154-based therapy: nonimmunologic graft failure? Am J Transplant, 2006,6(4):687-696.
[42]Hirakawa E, Yasunami Y, Nakano M, et al. Amelioration of hyperglycemia in streptozotocin-induced diabetic mice with fetal pancreatic allografts: prevention of rejection by donor specific transfusion in conjunction with CTLA4Ig. Pancreas, 2004,28(2):146-152.
[43] Appel MC, Banuelos SJ, Greiner DL, et al. Prolonged survival of neonatal porcine islet xenografts in mice treated with a donor-specific transfusion and anti-CD 154 antibody. Transplantation, 2004,77(9):1341-1349.