T淋巴细胞凋亡诱导大鼠肝移植自发性免疫耐受的实验研究

发布时间：2018-04-02 17:58

  本文选题：肝移植　切入点：近交系大鼠　出处：《广西医科大学》2008年博士论文

【摘要】： 第一部分近交系大鼠肝移植自发性免疫耐受模型的构建 目的:选择近交系BN大鼠和LEW(LEW)大鼠组合,拟建立大鼠肝移植自发性免疫耐受模型,为开展肝移植免疫耐受的实验研究提供模型及实验依据。 方法:采用Kamada二袖套法,利用封闭群Wistar大鼠进行建模技能训练,在此基础上建立近交系大鼠BN和LEW组合之间的肝移植模型。实验动物分三组,即A组:同系移植LEW→LEW(n=15);B组:同种移植LEW→BN(n=10)和C组同种移植BN→LEW(n=16)。根据临床表现和Banff标准判断是否发生排斥反应以及排斥反应发生的强度。以BN大鼠为供体,术后60天以上的C组受体(n=2)和A组受体(n=2)为受体,分别行颈部异位心脏移植,了解移植心是否被排斥。 结果:共完成171例次ROLT操作,总的手术成功率60.8%(104/171),其中模型训练的前期70例24h成活率仅17.1%(12/70),以后随着手术技巧成熟,手术成功率逐渐达到90%以上。大鼠肝移植手术死亡的主要原因为麻醉意外、出血性休克、肺水肿、腔血栓形成及狭窄和原发性移植物无功能等。A组同系移植,术后一周内出现肝功能受损,组织学检查发现肝脏间质水肿,但无排斥反应表现。B组同种移植自术后3d开始,出现Ⅰ级排斥反应,9d以后逐渐达到高峰,14d内全部死于Ⅲ级排斥反应。C组同种移植自术后5d开始,出现Ⅰ～Ⅱ级排斥反应,但无进行性加重,至术后28天左右,移植肝组织结构恢复正常。以BN大鼠为供体,术后60天以上的C组受体和A组受体为受体,进行心脏移植,结果发现移植后60d的C组受体并不排斥植入的心脏,但两例A组受体则分别于第7d和第9d排斥了供体的心脏。 结论:(1)良好的手术技巧以及对细节加以注意是大鼠肝移植模型构建成功的关键(2)BN→LEW肝移植组合能够产生稳定的自发性免疫耐受,其产生免疫耐受的时间估计在术后60天以上,是研究肝移植免疫耐受的较好动物模型。(3)近交系大鼠BN和LEW组合在组织结构上有其自身特点,手术耐受性相对较差,建模难度相对高于常用的远交系大鼠。 第二部分T淋巴细胞凋亡诱导大鼠肝移植自发性免疫耐受的实验研究 目的:探讨T淋巴细胞凋亡及其诱导的免疫调节在大鼠肝移植自发性免疫耐受中的作用,旨在为临床肝移植免疫耐受的诱导及其免疫抑制剂的合理应用提供理论依据。 方法:将健康雄性清洁的近交系BN和LEW大鼠随机分为4组,A、B和C组每组15对,D组12对。A组:BN→LEW同种肝移植(免疫耐受组);B组:LEW→BN同种肝移植(急性排斥组);C组:BN→LEW同种肝移植(甲基强的松龙干预组);D组:LEW→LEW同系肝移植(对照组)。C组甲基强的松龙干预剂量:术日12mg/kg(i.v.),术后第一天8mg/kg(i.m.)。采用Kamada二袖套法,建立上述BN和LEW组合之间的肝移植模型。A、B和C组受体分别于术后3d、5d及7d,三个时间点杀死三只大鼠,D组于这三个时间点杀死2只大鼠,收集肝组织和血清分别做相应检测,另留取部分新鲜肝组织立即置入液氮保存,供Fas、IL-10、TGF-β和Foxp3基因荧光定量PCR检测。剩余的大鼠用于观察肝移植术后长期生存率。 结果:(1)施行大鼠原位肝移植术57例,手术成功率91.2%。各组肝移植术后长期存活率分析,提示B组生存期最短,C组次之,而A与D组生存期明显较长(P＜0.01)。(2)A组大鼠术后3天移植肝内出现较多淋巴细胞凋亡,术后5天达高峰,然后开始下降。而B,C和D组大鼠术后移植肝内淋巴细胞较少凋亡。A组术后第3,5天移植肝内淋巴细胞凋亡率与B,C和D组相比,有显著性差异(P＜0.05)(3)A组术后第3d血清IL-2和IFN-γ水平升高十分明显,与B、C和D组相比较差异十分显著(P＜0.01),随后A组血清IL-2和IFN-γ水平逐渐下降。B组血清IL-2和IFN-γ水平于术后缓慢上升,于第7d达高峰。(4)术后3d和5d内,各组肝内CD4+CD25+Treg细胞的比例差异不明显,但术后7d A组肝内CD4+CD25+Treg细胞占CD4+的比例明显高于B,C和D组(p＜0.05)。(5)A组术后高表达Fas基因,于术后第5天达高峰,随后开始下降。经统计学分析,A组第3,5天Fas基因的相对表达量与B,C和D组比较,差异具有显著性(P＜0.05)。(6)A组术后3d和5d IL-10和TGF-β基因的相对表达量与B,C和D组比较,无显著性差异(P＞0.05),但术后第7d A组IL-10和TGF-β基因的相对表达量明显高于B,C和D组(P＜0.05)。(7)各组术后3d和5d Foxp3 mRNA的表达无明显差异,但术后7d A组Foxp3基因的相对表达量与B,C和D组比较,差异具有显著性(P＜0.05),表明术后7d开始A组大鼠Foxp3基因的表达明显增强。 结论:(1)T淋巴细胞凋亡在大鼠肝移植自发性免疫耐受中具有重要作用,这种T细胞凋亡的机制可能是通过Fas/FasL死亡受体途径实现的;(2)CD4+CD25+Treg细胞可能是诱导大鼠肝脏移植自发免疫耐受的因素之一,CD4+CD25+Treg细胞的免疫调节功能可能与Foxp3、IL-10和TGF-β等基因的表达量有关;(3)凋亡所诱导的免疫调节可能与肝移植自发性免疫耐受的长期维持有密切关系。(4)术中或术后早期大剂量类固醇激素的应用可能因为抑制移植肝内浸润淋巴细胞凋亡,而不利于长期免疫耐受的形成。
[Abstract]:Construction of spontaneous immune tolerance model of rat liver transplantation in the first part of inbred line
Objective: to establish a rat model of liver transplantation spontaneous immune tolerance by selecting inbred BN rats and LEW (LEW) rats, and provide a model and experimental basis for the experimental study of immune tolerance in liver transplantation.
Methods: using Kamada two cuff technique using outbred Wistar rats model of skill training, on the basis of the establishment of liver transplantation model of inbred line between BN and LEW in rat. The experimental animal were divided into three groups: group A: LEW, LEW isotransplantation (n=15); group B, allograft, LEW BN (n=10) and C group, LEW BN transplantation (n=16). According to the clinical manifestations and Banff criteria to determine whether the occurrence of rejection and rejection strength. Using BN rats as donors and more than 60 days after the operation of group C receptor (n=2) and group A (n=2) receptor receptor, respectively. For cervical heterotopic heart transplantation, heart transplantation is understanding rejection.
Results: a total of 171 cases of ROLT operation, the overall success rate was 60.8% (104/171), the model in the early stage of training in 70 cases of 24h survival rate is only 17.1% (12/70) after surgery, with mature techniques, the surgical success rate has reached more than 90%. The main causes of death in rat liver transplantation for anesthesia accident, bleeding shock, pulmonary edema, cavity thrombosis and stenosis and primary graft non function.A group isotransplantation, appear to damage liver function within a week after operation, histological examination showed that liver interstitial edema, but no rejection in allograft group.B began 3D after operation, appear grade rejection 9D, then gradually reached the peak, 14d all died of grade rejection in group.C after 5D transplantation began to appear, I ~ II rejection, but no progressive increase, to 28 days after operation, liver transplantation structure return to normal. Using BN rats as donors, The recipients of C group and A group received cardiac transplantation for more than 60 days after operation. After heart transplantation, it was found that the C group receptor of 60d did not exclude the implanted heart, but two cases of A group rejected the donor heart at 7d and 9D respectively.
Conclusion: (1) excellent operation skills and attention to detail is the key to the success of the construction of rat liver transplantation model (2), BN LEW liver transplantation combined to produce stable spontaneous immune tolerance, the tolerance time is estimated at more than 60 days after the operation, is a good animal model for the study of liver transplantation immune tolerance. (3) inbred rat BN and LEW combination has its own characteristics in the organizational structure, the relatively poor surgical tolerance, the difficulty of modeling is relatively higher than the commonly used outbred rats.
Experimental study on spontaneous immune tolerance induced by second part of T lymphocyte apoptosis in rat liver transplantation
Objective: To explore the role of T lymphocyte apoptosis and its immunomodulation in the spontaneous immune tolerance of rat liver transplantation, in order to provide a theoretical basis for the induction of immune tolerance and the reasonable application of immunosuppressive agents in clinical liver transplantation.
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