Promotion of pancreatic islet allograft survival by intrathymic transplantation of bone marrow.

An important goal in the treatment of insulin-dependent diabetes by pancreatic islet transplantation is the development of strategies that allow permanent survival of islet allografts without continuous host immunosuppression. In this study, we demonstrate that inoculation of allogeneic bone marrow into the thymus of adult rats treated with a single dose of anti-lymphocyte serum induces an unresponsive state that permits survival of subsequent pancreatic islet allografts transplanted to an extrathymic site. This effect is donor specific, cannot be reproduced by systemic administration of bone marrow, and is associated with persistence of chimeric cells in the thymus of the recipient. In addition, lymph node cells from long-term recipients of intrathymic bone marrow display markedly reduced proliferative responses to donor alloantigens in mixed lymphocyte culture. Interaction of maturing thymocytes with foreign alloantigens may produce the unresponsiveness. This model offers a potential approach for establishing donor-specific allograft acceptance in adult recipients.     

Skin allograft survival following intrathymic injection of donor bone marrow.

BACKGROUND: Success has been reported using intrathymic injection in the preconditioning regimen to induce allograft tolerance. Although long-term stable tolerance has been achieved in numerous rodent vascularized solid organ allograft models, tolerance to skin transplants has only been achieved across minor antigenic or concordant species disparities. This study sought to induce tolerance across an allogeneic barrier in a rat model with a major genetic disparity. MATERIALS AND METHODS: Lewis rats were injected intrathymically with 1 x 10(8) Brown-Norway (BN) bone marrow cells and intraperitoneally with 1.0 cc of rabbit anti-rat anti-lymphocyte serum (ALS). Twenty-one days later, BN skin grafts were placed on the injected animals. Control groups were included to isolate the effect of technique, thymic manipulation, strain specificity, and ALS. RESULTS: Animals receiving both intrathymic bone marrow cells and ALS had a skin graft median survival time of 24 days versus 8 days for the control group (P = 0.003). Groups receiving anti-lymphocyte serum alone or intrathymic bone marrow cell injection alone exhibited no skin graft survival prolongation. Mixed lymphocyte reactions revealed normal responsiveness of tolerant animal lymphocytes to donor strain lymphocytes. CONCLUSION: This protocol utilizing the intrathymic injection of donor bone marrow cells along with short-term immunosuppression with anti-lymphocyte serum produced markedly prolonged survival of skin allografts transplanted across a major histocompatibility barrier. Although tolerance was incomplete, significant prolongation has not previously been reported in genetic disparities of this degree. These results suggest that the application of this technique for central immune modulation may be beneficial for allograft tolerance induction and deserves further study in large animals models.     

Suppressed alloreactivity and mixed chimerism in rats with accepted cardiac allografts by intrathymic injection of donor bone marrow cells

Intrathymic injection of donor bone marrow cells (ITBMCs) at the time of transplantation and treatment with antilymphocyte serum (ALS) permitted the indefinite survival of Brown Norway (BN, RT1ⁿ) rat heart grafts in 6 out of 8 Lewis (LEW, RT1^l) rat recipients. LEW recipients with long-surviving BN heart grafts (LSGs) also accepted additional BN heart grafts without further immunosuppression, though they rejected Piebald Virol Glaxo (PVG, RT1^c) rat heart grafts in the usual fashion. In the in vitro study, the proliferative response of the lymphocytes from LEW recipients with LSGs remained suppressed when they were stimulated by BN spleen cells, but not when stimulated by PVG cells. Bone marrow cells (BMCs) from LEW rats with LSGs showed strong, nonspecific, suppressive effects on the proliferative response in the mixed lymphocyte culture reaction, suggesting that one of the possible explanations for tolerance might be the involvement of a suppressor mechanism. Received: 7 August 1996 Received after revision: 12 February 1997 Accepted: 17 February 1997     

重组腺相关病毒载体介导CTLA4Ig转染延长同种大鼠心脏移植存活时间

目的　研究重组腺相关病毒载体介导 CTLA4Ig(rAAV CTLA4Ig)全身转染对大鼠同种心脏移植的影响。方法　以BN大鼠为供者,Lewis大鼠为受者,建立心脏移植模型。实验分为两组。对照组:供、受者不给予任何处理;转染组:受者于心脏移植前30 d,通过尾静脉全身注射 1×109斑点形成单位(PFU)的 rAAV CTLA4Ig。观察移植心存活时间;ELISA法检测血清 CTLA4Ig、干扰素 γ(IFN γ)和白细胞介素 4(IL 4)水平;免疫组织化学法(SABC法)检测移植心组织中 CD4 和CD8 T细胞的浸润。对移植心存活时间明显延长的受者,用其脾细胞与供者脾细胞进行混合淋巴细胞培养,观察受者对供者的免疫反应状态。结果　转染组移植心存活时间较对照组明显延长(P<0.05);转染组血清CTLA4Ig蛋白一直维持在26.67~35.47 mg/L,移植后转染组血清 IFN γ水平下调,血清 IL 4水平上调(P<0.05);对照组出现典型的急性排斥反应表现,转染组心肌组织基本正常,间质内无炎性细胞浸润或血管外周及心肌间质内有局灶性炎性细胞浸润,未见坏死;对照组移植心组织中浸润的CD4 和CD8 T细胞数量明显高于转染组(P<0.01);转染组受者对供者的脾细胞增殖反应明显低于对照组(P<0.05)。结论　重组腺相关病毒载体可以介导 CTLA4Ig基因的持续表达,通过全身途径转染受者可以明显延长     

Prolonged skin allograft survival in Scid mice reconstituted with isogeneic bone marrow stem cell antigen-1-positive cells and thymus tissue

INTRODUCTION: Many studies indicate that tolerance induction is much more dependent on the maturation status of lymphocytes than the age of the animal. We hypothesized that direct persistent contact of bone marrow stem cells with graft alloantigen will result in tolerance to that antigen in the adult animal. MATERIAL AND METHODS: Severe combined immunodeficient mice (CB-17-Scid, H-2b) were reconstituted with isogeneic bone marrow stem cell antigen-1 (Sca-1)-positive cells and grafted with fetal thymus (BMSC-T), followed by transplant of allogeneic skin grafts from C57BL/6 (H-2d) mice. The control group include CB-17 non-Scid mice, CB-17-Scid mice, and CB-17 Scid mice pretransplanted with nonmodified isogeneic bone marrow cells and fetal thymus (BMC-T). RESULTS AND DISCUSSION: Skin allograft survival was significantly prolonged in the group pretransplanted with isogeneic BMSC-T compared the group of non-Scid mice and the group of Scid mice pretransplanted with BMC-T (59.6 days vs 7.1 days vs 11.7 days). In 2 of 10 mice pretransplanted with BMSC-T, the skin allografts transplanted immediately after BMSC-T survived for more than 100 days, but the third-party skin allografts transplanted at 100 days after BMSC-T transplant were rejected. The results suggest direct persistent contact of bone marrow Sca-1-positive cells with graft alloantigen may be a feasible approach to prolong allograft survival and induce tolerance in a small fraction of adult animals.     

白茅苷对去卵巢大鼠骨髓间充质干细胞功能和骨量的影响

目的观察白茅苷治疗去卵巢大鼠骨髓间充质干细胞功能和骨量的影响并初步探索可能机制。方法通过双侧去卵巢建立骨质疏松大鼠模型;随后随机分为假手术组(Sham)、去卵巢组(OVX)以及白茅苷组(BMG),每组10只;其中BMG组去卵巢大鼠每天给予白茅苷(20 mg/kg)灌胃治疗;待12周治疗结束后分离培养各组大鼠骨髓间充质干细胞(BMSCs),使用碱性磷酸酶(ALP)和茜素红(ARS)染色并使用蛋白质印迹检测BMP-2、Runx2、OPN、OCN、ALP和Col1蛋白表达;进一步使用Micro-CT和骨生物力学检测观察治疗效果。结果 OVX组大鼠BMSCs向成骨细胞分化后ALP和ARS染色阳性面积以及BMP-2、Runx2、OPN、OCN、ALP和Col1表达较Sham组明显降低(P0.05);而经过BMG治疗,BMG组大鼠BMSCs向成骨细胞分化后ALP和ARS染色阳性面积以及BMP-2、Runx2、OPN、OCN、ALP和Col1表达较OVX组明显增加(P0.05)。OVX组股骨最大载荷和弹性模量、BMD、BV/TV、Tb.N和Tb.Th较Sham组明显降低,而Tb.Sp则明显升高(P0.05)。BMG组左侧股骨最大载荷和弹性模量、BMD、BV/TV、Tb.N和Tb.Th均明显高于OVX组(P0.05),而Tb.Sp明显低于OVX组(P0.05)。结论白茅苷通过促进BMSCs诱导成骨分化来减少去卵巢大鼠骨骨密度、骨量和骨强度下降。     

小鼠胸腺内注射异基因抗原对异体神经移植免疫反应的影响

目的　探讨胸腺内注射异基因抗原对异体鼠坐骨神经移植存活的影响。　方法　 70只Balb/c小鼠随机分为 5组 :自体神经移植组 (A组 )、异体神经移植组 (B组 )、冷冻后异体神经移植组 (C组 )、应用免疫抑制剂异体神经移植组 (D组 )和胸腺内注射供体组织相容性 (MHC)抗原后移植组 (E组 )。 3周后进行神经电生理检查、组织学检查、混合淋巴细胞培养及迟发性超敏反应的检测。　结果　运动神经传导速度E组为 3 8 2 3 (m /s) ,与D组相比差异无显著性 (P >0 0 5 ) ,且E组优于C组、B组。混合淋巴细胞培养E组为 2 668 3 7(cpm ) ,迟发性超敏反应E组为 0 5 10 (cpm ) ,组织学及电镜检查均证实A组优于E组、E组分别优于C组、D组、B组。　结论　胸腺内注射异基因抗原可诱导对异体坐骨神经移植的特异性免疫耐受。     

骨髓细胞移植改善宿主肝细胞功能研究

目的 观察骨髓问充质细胞(BMCs)移植能否提高严重肝功能损害合并再生障碍的同种先天性无白蛋白大鼠(F344alb)肝再生和修复能力.方法 F344大鼠为供体,F344alb受体鼠接受Retrorsine(RS)1次/2周腹腔注射2次后4周行2/3肝切除(PH).正常F344alb为组Ⅰ(n=5);BMCs移植为组Ⅱ(n=8);RS/PH预处理为组Ⅲ(n=8);RS/PH预处理后BMCs移植为组Ⅳ(n=8);RS/PH预处理后肝实质细胞移植为组V(n=8).4周后行各组大鼠肝脏形态学和组化染色研究,检测肝功能,及肝组织和骨髓基因检测.结果 (1)生存率:组Ⅳ75%,组Ⅴ 50%,组Ⅲ37.5%,组Ⅰ和组Ⅱ 100%.(2)4周后组肝再生率(67.38±8.66)%显著高于组Ⅳ、Ⅲ[(55.31±8.69)%,(44.27±6.51)%].(3)PH后1 d,组Ⅲ、Ⅳ、V血清TB、ALT显著升高;PH后2 d,组Ⅳ血清,rB、ALT显著下降.(4)组Ⅳ、Ⅴ肝组织切片白蛋白免疫组织化学染色显示白蛋白染色阳性肝细胞呈簇状分布.(5)F344来源白蛋白基因片段出现在组Ⅳ、Ⅴ大鼠肝组织内.(6)PH后2、4周,组Ⅳ、Ⅴ血清白蛋白显著升高.结论 BMCs移植可提高严重肝损合并再生障碍受体鼠肝再生能力,保护肝功能,促进肝修复.     

供体特异性输血和骨髓胸腺内注入在大鼠肝移植中的作用

目的 探讨供体特异性输血（DST）和骨髓细胞胸腺内注入(IBMC)在大鼠肝移植中的作用。方法 在建立稳定的大鼠肝移植模型的基础上，在移植当天进行DST 2ml，在移植前7d行供体胸腺内MBC注入了解能否延长移植物的存活状况。结果 移植当天行DST或(和)移植前7d BMC胸腺内注入，可将移植肝脏存活时间中位数从对照组的7d分别有效延长12d和16d(P＜0．01)，DST和IBMC联合应用时延长至33d(P＜0．01)。结论 DST和IBMC能延长大鼠肝移植的存活时间，两者联合应用有协同作用。     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

目的 观察大鼠胸腺内注射异基因抗原在同种异体异基因股静脉移植免疫耐受中的作用.方法 将48只SD大鼠随机分为4组:自体股静脉移植组(A组)、异体股静脉移植组(B组)、异体股静脉移植免疫抑制剂组(C组)、胸腺内注射供体组织相容性(MHC)抗原后移植组(D组).于2周后进行影像学、组织学、免疫学检测.结果 组织学检测结果显示:D组、C组急性排斥反应损伤较轻,B组血管壁的各层结构破坏最重,可见大量炎性细胞浸润.B组受体大鼠血清干扰素(IFN)-γ浓度为(86.707±10.928)ng/L,显著高于A、C、D组[(29.328±4.170)、(69.076±8.059)、(63.355±4.895)ng/L,P<0.05];B组受体大鼠血清白细胞介素(IL)-4浓度为(23.656±3.369)ng/L,显著低于C、D组[(29.425±4.174)、(31.000±4.659)ng/L,P<0.05].结论 胸腺内注射异基因MHC抗原可诱导大鼠对同种异体血管移植的特异性免疫耐受.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.     

胸腺修饰诱导同种异体静脉移植的免疫耐受研究

Objective To investigate the possibility of inducing immune tolerance to vein trans-plantation in rats by intrathymic injection of allogene. Methods MHC antigen extracted from splenic cells of donor Wistar rats was intrathymically injected to recipients SD rats, and donor Wistar femur vein was transplanted after 2 weeks. Forty-eight SD rats were randomly divided into 4 groups: group A ( femur vein autograft) ,group B ( femur vein allograft) ,group C ( femur vein allograft with use of immunosuppressant), group D (intrathymus injection). Imaging, histology and immunological assays of these groups were carried out 2 weeks after the transplantation. Results Histologic parameters that were tested were better in group D than those in group B. The concentration of serum IFN-γ in group B was significantly higher than that in groups A, C and D [ ( 86. 707±10.928 ) ng/L vs (29.328±4. 170), (69.076±8.059) and (63.355± 4.895) ng/L respectively, P < 0.05 ]. The concentration of serum IL-4 in group B was obviously lower than in groups C and D [ (23.656±3.369) ng/L vs (29.425±4.174) and ( 31.000±4.659) ng/L re-spectively,P < 0.05 ]. Conclusion Intrathymus injection of allogenic antigen might induce specific im-mune tolerance to femur vein transplantation in rats.