【作者】 瞿冀琛；

【导师】 沈振亚；

【作者基本信息】 苏州大学 ， 心胸外科， 2010， 博士

【摘要】 背景肺移植是终末期肺病唯一有效的治疗方法。尽管目前肺移植的术后一年生存率可达71%,约46%的患者术后生存期可超过5年,但真正限制肺移植患者长期生存的原因仍然是移植术后的慢性排异,其最终结果即闭塞性细支气管炎(Obliterans Bronchiolitis, OB)的发生。国外的大宗资料统计表明,在生存期超过3月的病人中,有28%以上的患者存在着不同程度的OB;移植术后第一年OB发生率为50%。更长期的随访资料提示,在肺移植术后5到10年后,OB的发生率将高达60-80%。它不单可以直接导致死亡,同时也可以通过继发感染、以及相关治疗所引起的各类不良反应等间接导致患者死亡。匹兹堡医学中心的经验认为近25-40%的患者直接或者间接病死于OB。目前预防和治疗移植后排斥反应的主要手段是应用免疫抑制药物。这些药物除需终生服用外,其毒副作用也非常明显,且价格昂贵,部分患者甚至因药物的毒副作用或经济负担被迫停药;同时此类免疫抑制治疗OB的方法往往疗效不理想,严重影响移植患者的长期存活。因此,研究免疫抑制或耐受的有效方法,对预防和治疗慢性排异的发生、进一步延长移植物存活具有至关重要的意义。近年来随着研究的不断深入,临床免疫学家发现器官移植后通过向受体移植供体造血干细胞或骨髓来源的间充质干细胞(bone marrow mesenchymal stem cells, BM-MSCs)建立混合嵌合体,对免疫耐受的诱导有帮助。这一理论的提出解决了困绕移植学者多年的免疫排斥难题,成为目前移植领域研究的新前沿。BM-MSCs是成体干细胞的一种,存在于骨髓中,具有干细胞的共性和很强的自我增殖能力及多向分化潜能,能表达多种表面抗原和分泌多种细胞因子,是一种较原始的骨髓基质细胞。研究发现经非清髓处理后,骨髓间充质干细胞( Bone marrow mesenchymal stem cells, BM-MSCs )输注受体后,髓系树突状细胞(DC)会定向迁移至胸腺并向T细胞提供阴性选择信号,有可能产生外周免疫耐受,这为抑制慢性排异提供了可能。综上所述,我们构想将骨髓细胞移植用于诱导受体嵌合,并进一步诱导肺移植慢性排异的免疫抑制或免疫耐受,并对肺移植慢性排异的机理进行探讨。方法1、实验分两组:实验组(I组)采用10只SD大鼠为供体,10只Wistar大鼠为受体;对照组(II组)10只供体、受体均采用SD大鼠。均行异位气管移植。术后3,14,28天,处死大鼠,取出移植物。镜下观察:气道闭塞情况;气道上皮完整性;气道粘膜下炎细胞浸润、纤维组织增生的情况。2、实验分7组( n=10 ):A组:在既定实验日早晨全身辐照。然后经全身辐照大鼠于2天后给予腹腔内注射CTX(50mg/kg)一次,7天后直接行大鼠气管异位移植,移植后4h内经尾静脉输注生理盐水0.8mL。B组:在既定实验日早晨全身辐照。然后经全身辐照大鼠于2天后给予腹腔内注射CTX(50mg/kg)一次。7天后直接行大鼠气管异位移植,移植后4h内经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓细胞(BMC)的细胞悬液0.8ml。C组:在既定实验日早晨全身辐照。然后经全身辐照大鼠于2天后给予腹腔内注射CTX(50mg/kg)一次。7天后直接行大鼠气管异位移植,移植后4h内经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓间充质干细胞(BM-MSCs)的细胞悬液0.8ml。D组:移植前5天开始给予腹腔内注射CTX(50mg/kg)一次,移植前2天大剂量FK506腹腔注射,3 mg x 2 d;术前一天受体Wistar大鼠腹腔内注射抗淋巴细胞血清1ml。2天后直接行大鼠气管异位移植,移植后4h内经尾静脉输注生理盐水0.8mL。移植当天起至术后第6天每天腹腔注射FK506 0.5mg/kg/d。E组:移植前5天开始给予腹腔内注射CTX(50mg/kg)一次,移植前2天大剂量FK506腹腔注射,3 mg x 2 d;术前一天受体Wistar大鼠腹腔内注射抗淋巴细胞血清1ml。2天后直接行大鼠气管异位移植,移植后4h内经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓细胞(BMC)的细胞悬液0.8ml。移植当天起至术后第6天每天腹腔注射FK506 0.5mg/kg/d。F组:移植前5天开始给予腹腔内注射CTX(50mg/kg)一次,移植前2天大剂量FK506腹腔注射,3 mg x 2 d;术前一天受体Wistar大鼠腹腔内注射抗淋巴细胞血清1ml。2天后直接行大鼠气管异位移植,移植后4h内经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓间充质干细胞(BM-MSCs)的细胞悬液0.8ml。移植当天起至术后第6天每天腹腔注射FK506 0.5mg/kg/d。G组:移植前5天开始给予腹腔内注射CTX(50mg/kg)一次,移植前2天大剂量FK506腹腔注射,3 mg x 2 d;术前一天受体Wistar大鼠腹腔内注射抗淋巴细胞血清1ml。2天后直接行大鼠气管异位移植,移植后4h内经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓细胞(BMC)的细胞悬液0.8ml。移植当天起至术后第6天每天腹腔注射FK506 0.5mg/kg/d。移植后6天经大鼠尾静脉输注浓度为2×108/ml SD大鼠骨髓间充质干细胞(BM-MSCs)的细胞悬液0.8ml。随后测定异基因嵌合体量的变化及单向混合淋巴细胞培养的刺激效应,流式细胞仪检测了T淋巴细胞亚群的变化,检测细胞移植后受体内IL-2、IL-10细胞因子的表达和气管的阻塞率。检测移植物病理组织学,并进行统计学分析。结果1、两组移植气管及支气管的闭塞性细支气管炎发生率实验组闭塞性细支气管炎的发生率为100%,对照组未发生闭塞。对照组与实验组移植气管及支气管闭塞性细支气管炎发生率差异具有显著性意义2、B、C组动物接受60Coγ射线全身照射经环磷酰胺预处理后行BMC/BM-MSCs移植, E、F组动物接受FK506腹腔注射后行BMC/BM-MSCs移植,在外周血、脾脏中均检测到嵌合的存在。输注BMC组较BM-MSCs组嵌合体水平维持更久。A、D组动物未接受BMC/BM-MSCs移植的未检测到嵌合。但嵌合体模型随着时间推移,其嵌合会渐渐消失。3、于气管移植后21天后取雌性Wistar大鼠脾淋巴细胞作为效应细胞,用雄性SD脾淋巴细胞经丝裂霉素处理后作为刺激细胞将两者行单向混合细胞培养, A、B、C、D、E、F、G组与O组相比均呈增殖反应。计算刺激效应结果显示:C组较B组和A组的受体淋巴细胞增殖显著减弱(P<0.05);B组较A组也显著减弱(P<0.05)。G组较D、E、F组的受体淋巴细胞增殖显著减弱(P<0.05);F组较E组也显著减弱(P<0.05), E、F组较同期B、C组无明显差异。4、大鼠接受5Gy全身照射后外周淋巴细胞于照后一周左右降至最低水平,经辐照后白细胞中T细胞亚群CD3+细胞比例急剧减低降至5%左右,但各处理组中CD3+细胞中CD4+、CD8+含量比值并无大的变化,只是与正常大鼠相比CD4+有所降低,而CD8+的含量稍有上升。5、受体IL-2水平在无处理组(A、D组)气管移植后28天时较移植前明显升高(P<0.05),而在实验组(B、C、E、F、G组)移植术后IL-2水平较移植前也有升高,与A、D组比较有统计学差异(P<0.05)。无处理组受体IL-10水平在移植后28天时较移植前无明显差别(P>0.05),实验组(B、C、E、F、G组)在移植后28天时明显升高,与A组、D组比较有统计学差异(P<0.05)。E、F组较同期B、C组无明显差异。6、BMC移植组和BM-MSCs移植组气管移植后28天阻塞率较对照组减低。移植后28天阻塞率E、F组较同期B、C组减低。7、A、D组气管纤毛比例远低于正常气管和G组(P< 0.01),G组气管和正常气管相比无统计学意义。B组和E组相比无统计学意义, C组和F组相比无统计学意义。8、于术后28天分批处死动物,在对照组标本中可以观察到严重的结缔组织包裹。在低倍镜下观察,对照组标本的气管管腔内有肉芽组织向管腔内增生,造成管腔闭塞。镜下观察可见对照组组在术后28天移植气管内有大量炎性细胞浸润,上皮层有坏死、脱落,可见有大量肉芽组织向管腔内生长,气管管腔几乎完全闭塞。而在使用BMC/BM-MSCs干预下,上述情况有所改善。见管腔通畅,上皮细胞少量坏死、脱落,覆盖于移植气管内壁,气管壁内可见少量炎细胞浸润。但B、C、E、F、G各组差别不明显。结论1.大鼠气管异位移植模拟肺移植慢性排斥反应。2.新型的非破坏骨髓的预处理与传统的照射等非清髓性预处理方式比较同样诱导出了嵌合,并且在抑制慢性排斥反应方面同样有效。3.通过在非清髓性预处理后输注供体骨髓细胞或骨髓间充质干细胞,可以建立混合嵌合体,调节Th1/Th2型细胞因子的表达,使其向免疫耐受方向偏移,可以形成对肺移植慢性排斥反应的供体特异性的免疫抑制。更多还原

【Abstract】 Background:Lung transplantation is the most important method in healing the serious pulmonary disease. The advances in immunosuppressive drug have yielded improvement of transplantation; yet long-term survival recipient complications and graft loss continue to accompany chronic immunosuppression. Induction of tolerance may hold the key to improve transplant longevity and quality of life in recipient and increase the cost-effectiveness of transplant therapy at the same time. With further studies and developments of transplantation immunology, eliminating transplant rejection through inducing immune tolerance has become the main goal in organ transplantation. Obliterative bronchiolitis(OB) is the most important cause of graft dysfunction following lung transplantation.The purpose of this study was to induce chimera by donor bone marrrow and mesenchymal stem cells transplantation and observe its effects on obliterative bronchiolitis(OB) after lung transplantation, and investigate the pathogenesis of transplantation tolerance of chimera preliminarily in an animal model.Methods:1、SD and Wistar rat were, respectively, selected as donor and recipient In experimental group, while in control group, SD rat served as donors and recipients, respectively.All grafts were harvested 28 days after transplantation and stained with hematoxylin and eosin (HE) to observe the histopathology of the grafts. OB incidence in two groups was compared.2、SD and Wistar rat were, respectively, selected as donor and recipient.Donor and recipient were divided randomly into 8 groups. Recipient was conditioned with sublethal whole body irradiation (WBI) in Group A, B and C. Group A infused with saline solution; Group B infused with bone marrow cells (BMC) of SD rat; Group C infused with bone marrow mesenchymal stem cells (BM-MSCs) of SD rat. Animals in other groups did not receive whole body irradiation, they received FK506 3 mg two days before transplantation. Then Group D infused with saline solution. Group E infused with bone marrow cells (BMC) of SD rat. Group F infused with bone marrow mesenchymal stem cells (BM-MSCs) of SD rat. Group G infused with bone marrow mesenchymal stem cells (BM-MSCs) and bone marrow cells (BMC) of SD rat. The tracheal segments of Wistar rat were then heterotopically transplanted into recipient rats in all groups. Grafts were harvested at day 3, 14 ,28 after transplantation. To explore immune depression mechanisms, the mixed lymphocyte reaction (MLR) was performed. The level of IL-2、IL-10 and the obliteration ratio were also measured. Recipient rats were detected for donor origin cells in the peripheral blood lymphocyte and spleen by polymerase chain reaction (PCR). All grafts were harvested 28 days after transplantation and stained with hematoxylin and eosin (HE) to observe the histopathology of the grafts.Results:1、The experimental group showed the histopathology of OB. The incidence of OB in the experimental group was significantly lower than in the control group.2、The results showed that donor lymphoid chimeras can be found in the immune depression Wistar rat (Group C > Group B). Wistar rat were specifically tolerant to the SD rat in MLR assay in Group B and Group C. The obliteration ratio of allograft in group A was higher than that in group B and C( P<0.05).3. Chimera can be found in the peripheral blood and spleen lymphocyte in the tolerance Wistar rats of Group B, C, E, F, and G.4. Allografts in Groups infused with bone marrow mesenchymal stem cells (BM-MSCs) / bone marrow cells (BMC) of SD rat expressed significantly less IL-2 expression than that in Group A. 5.At the same time, lymphocytic infiltration and luminal obliteration were lower in Groups infused with bone marrow mesenchymal stem cells (BM-MSCs) / bone marrow cells (BMC) of SD rat than that in Group A and D (p<0.01).Conclusions:1、Heterotopic tracheal allografting in rat as a model of chronic allograft rejection after lung transplantation displays many advantages. 2、These results suggest that non-myeloablative conditioning regimens for allogeneic BMC/BM-MSCs transplantation can successfully establish mixed chimeras model from SD to Wistar rat and induce a specific immune depression in Wistar rat .更多还原