肥大细胞在移植供肝保护、受体急性排斥中的作用

发布时间：2018-05-06 18:14

本文选题：肥大细胞 + 色甘酸钠　；参考：《上海交通大学》2014年博士论文

【摘要】：近年来，肥大细胞在移植免疫中的作用越来越被人们所重视。肥大细胞在其中的功能仍然有很多问题需要解决，如肥大细胞脱颗粒在肝脏缺血再灌注损伤中的作用、调控肥大细胞获得的供肝前置状态对受体生存的影响和移植后受体肥大细胞脱颗粒在急性排斥反应中的作用等。本研究通过大鼠肝脏缺血再灌注损伤模型、大鼠原位肝移植模型、大鼠异位心脏移植模型三个移植研究中的经典模型，分别探讨了以上三个问题。为比较全面地研究肥大细胞在器官移植免疫中的作用，最终改善移植预后提供理论依据。第一部分肥大细胞脱颗粒对肝脏缺血再灌注损伤作用的研究研究背景：肥大细胞脱颗粒参与了许多器官的缺血再灌注损伤。但是在最近的一篇研究中却发现肥大细胞不参与大鼠肝脏的缺血再灌注损伤。我们对此结果提出异议。材料和方法：我们使用大鼠在体热缺血1小时然后灌注24小时的模型观察肥大细胞在肝脏缺血再灌注损伤中的作用。用了两种方法研究肥大细胞脱颗粒所产生的效应。第一种是用肥大细胞膜稳定剂色甘酸钠稳定肝脏肥大细胞；另外一种是用肥大细胞脱颗粒剂化合物48/80“耗竭”肝脏肥大细胞。使用肝脏甲苯胺蓝组织学染色和ELISA检测血清中肥大细胞特异性蛋白肥大细胞胰蛋白酶的水平对两种调控方法的效果进行评估。使用肝脏组织HE染色下的Suzuki评分、血清中的谷丙转氨酶（ALT）和谷草转氨酶（AST）、肝脏组织裂解液丙二醛（MDA）等指标对肝脏损伤严重程度进行评估。结果：第一，肥大细胞脱颗粒在再灌注2h时达到高峰，而ALT和AST反应的肝脏损害在再灌注6h后达到高峰；第二，之前“耗竭”皮肤肥大细胞的方法，也能达到所谓的“耗竭”肝脏的肥大细胞；第三，通过色甘酸钠稳定肝脏肥大细胞和化合物48/80耗竭肝脏肥大细胞的方法能够减轻Suzuki评分、血清中的ALT和AST、肝脏组织裂解液MDA等反映的肝脏缺血再灌注损伤程度。结论：肥大细胞脱颗粒促进了大鼠肝脏的缺血再灌注损伤。第二部分调控供肝肥大细胞获得的前置状态对大鼠原位肝移植后受体生存的影响及其机制研究研究背景：移植后的免疫抑制治疗会导致机会菌的感染和肿瘤的生成等副反应。我们前面的研究表明，通过色甘酸钠稳定肥大细胞和化合物48/80“耗竭”肥大细胞后能减轻肝脏的缺血再灌注损伤。本部分旨在探讨这两种调控肥大细胞的方法处理供肝获得的前置状态对受体生存的影响及其机制。材料和方法：用色甘酸钠稳定供肝肥大细胞（CRM组）和化合物48/80（CMP48/80组）耗竭供肝肥大细胞的方法预处理供肝，，获得相应的前置状态。然后用雄性DA大鼠作为供体，雌性Lewis大鼠作为受体建立大鼠原位肝移植模型。用PBS预处理供肝作为阳性对照（PBS组）。雄性Lewis作为供体，雌性Lewis作为受体的同基因移植作为阴性对（Syn组）。结果：PBS组的受鼠快速发生急性排斥反应并在7.40±1.14天死亡。与PBS组相比，CRM和CMP48/80组的生存期明显延长，分别为17.40±1.67和14.20±2.28天，排斥活性指数和肝功能也明显改善(p 0.05)。 Th1的代表细胞因子IFN-γ在肝脏组织中的mRNA表达和血清中的蛋白水平均明显降低；而Th2代表细胞因子IL-10在肝脏组织中的mRNA表达和血清中的蛋白水平均明显升高。结论：通过色甘酸钠稳定供肝肥大细胞和化合物48/80耗竭供肝肥大细胞获得的前置状态能够减轻急性排斥反应和延长移植物生存。这结果可能是通过使Th1/Th2免疫平衡向Th2方向倾斜达到的。第三部分移植后调控受体的肥大细胞对急性排斥反应的影响及其机制研究研究背景：受体体内肥大细胞在急性移植排斥中的作用还不十分清楚。本部分研究旨在探讨移植后受体肥大细胞脱颗粒与否及其对急性排斥反应的影响。材料和方法：在体外实验部分中，我们分离获得了大鼠原代皮肤肥大细胞（RSMC）和腹腔肥大细胞（RPMC）。然后分别检测肥大细胞表面受体C5a的表达情况及其在C5a作用下能否发生脱颗粒；在体内实验中，我们建立DA大鼠到Lewis大鼠的异位心脏移植模型，按照受体处理方式设计分组：注射PBS的PBS组、注射色甘酸钠的CRM组、注射3mg/kg体重CsA的CsA3组、联合注射3mg/kg体重CsA和色甘酸钠的CsA3+CRM组、注射5mg/kg体重的CsA5组、联合注射5mg/kg体重CsA和色甘酸钠的CsA5+CRM组以及同基因移植组。观察各组的生存期。分别在术后第5、10、15天取移植受体静脉血进行C5a、IL-10和IFN-γ的检测；肥大细胞脱颗粒试剂盒检测肥大细胞脱颗粒水平；取移植心脏进行HE染色，观察急性排斥情况；移植心脏组织提取mRNA，检测IL-10和IFN-γ的转录水平。结果：在体外研究中，58.69%~67.64%的RSMC表达C5a受体，仅1.23%~1.89%的RPMC表达C5a受体。经过脂多糖（LPS）刺激的RPMC表达C5a受体的细胞占73.52%~81.14%。C5a可以引起RSMC和经过LPS刺激的RPMC脱颗粒，但不能引起不经刺激的RPMC脱颗粒。C5a诱发的肥大细胞脱颗粒可以被肥大细胞膜稳定剂色甘酸钠所抑制。在体内实验中，大鼠异位心脏移植后的受体C5a和肥大细胞脱颗粒水平较同基因移植组高，而且排斥越重C5a和肥大细胞脱颗粒水平也越高。色甘酸钠能降低受体肥大细胞脱颗粒水平。但是，CRM组和PBS组比、CsA3+CRM组和CsA3组比、CsA5+CRM组和CsA5组比，生存期均不能显著延长。心脏排斥评分、心脏组织IL-10、IFN-γ的mRNA和血清中的IL-10、IFN-γ之间的水平也均无显著差异。结论：在异位心脏移植的大鼠体内的确有C5a和肥大细胞脱颗粒水平的上升；C5a也能促进肥大细胞脱颗粒；色甘酸钠能够降低肥大细胞脱颗粒程度，但最终无法减轻急性排斥反应和延长移植物生存。
[Abstract]:In recent years , the role of mast cells in transplantation immunity has been paid more and more attention . The function of mast cells still has many problems to be solved , such as the role of mast cell degranulation in liver ischemia - reperfusion injury , the effect of regulatory mast cells on the survival of receptors and the role of receptor mast cell degranulation in acute rejection .

In this study , the model of rat liver ischemia - reperfusion injury , orthotopic liver transplantation model and ectopic heart transplantation model in rats were studied . The three problems were discussed . In order to study the role of mast cells in organ transplantation immunity , the theoretical basis for improving the prognosis of transplantation was provided .

Effect of first partial mast cell degranulation on ischemia - reperfusion injury in liver

Background : Mast cell degranulation was involved in the ischemia / reperfusion injury in many organs . However , in a recent study , it was found that mast cells did not participate in the ischemia / reperfusion injury in the liver of rats . We disputed the outcome .

Materials and Methods : The effects of mast cells on ischemia - reperfusion injury of liver were observed in rats after 1 hour ischemia and reperfusion for 24 hours . Two methods were used to study the effect of mast cell degranulation .
In addition , the liver mast cells were depleted by using a mast cell degranulation compound 48 / 80 . The effects of two methods were evaluated by histological stain and ELISA in serum . The severity of liver injury was assessed by using the Suzuki score under HE staining of liver tissue , alanine aminotransferase ( ALT ) and aspartate aminotransferase ( AST ) in serum , and malondialdehyde ( MDA ) in liver tissue .

Results : The first , mast cell degranulation peaked at 2 h reperfusion , while the liver injury of ALT and AST reaction reached the peak after 6 h reperfusion .
Secondly , the method of " depletion " of skin mast cells can also achieve the so - called " depletion " of the mast cells of the liver ;
Thirdly , the method of stabilizing liver mast cells and compound 48 / 80 depleted liver mast cells by sodium cromoglycate can reduce the degree of liver ischemia reperfusion injury , which is reflected by the Suzuki score , the ALT and AST in the serum , the MDA of the liver tissue lysate , and the like .

Conclusion : Mast cell degranulation promotes ischemia - reperfusion injury in rat liver .

The effect of the second part on the survival of the receptor after orthotopic liver transplantation in rats and its mechanism

Background : Immunosuppressive therapy after transplantation can lead to the infection of opportunistic bacteria and the formation of tumor . Our previous studies have shown that by stabilizing mast cells and compound 48 / 80 " depleted " mast cells by sodium crotalate , the ischemia - reperfusion injury of the liver can be reduced . This part is intended to investigate the effects of these two methods of regulating mast cells on the survival of the receptor and its mechanism .

Materials and Methods : A rat orthotopic liver transplantation model was established by stabilizing liver mast cells ( CRM group ) and compound 48 / 80 ( CMP48 / 80 ) with sodium cromoglycate . The model of orthotopic liver transplantation was established by using male DA rats as donor and female Lewis rats as donor and female Lewis rats as recipients . Male Lewis was used as donor and female Lewis as donor and female Lewis as negative pair ( Syn group ) .

Results : Compared with PBS group , the survival time of CRM and CMP48 / 80 group was significantly prolonged compared with PBS group ( 17.40 卤 1.67 and 14.20 卤 2.28 days respectively ) , and the rejection activity index and liver function were significantly improved ( p 0.05 ) . The expression of IFN - 纬 in liver tissue and the level of protein in serum decreased significantly in Th1 .
and Th2 represents the expression of IL - 10 in liver tissue and the level of protein in serum .

Conclusion : It is possible to stabilize the Th1 / Th2 immune balance in the Th2 direction by stabilizing the leading status of liver mast cells and compound 48 / 80 depleted donor liver mast cells by sodium crotalate . This results in a possible reduction in the Th1 / Th2 immune balance in the Th2 direction .

Study on the effect and mechanism of the third partial transplantation of the mast cells regulating the receptor on acute rejection

Background : The role of mast cells in acute rejection is not very clear in the study . The aim of this study is to investigate the effect of post - transplantation receptor mast cell degranulation and its effect on acute rejection .

Materials and Methods : In the in vitro experimental part , we isolated rat primary skin mast cells ( RSMC ) and peritoneal mast cells ( RPMC ) , then respectively detected the expression of the surface receptor C5a of mast cells and their ability to degranulation under the action of C5a .
In vivo experiments , we established a model of ectopic heart transplantation from DA rats to Lewis rats . The groups were designed according to the receptor treatment methods : the PBS group injected with PBS , the CRM group injected with sodium glycate , the CsA3 + CRM group injected with 3 mg / kg body weight CsA and the CsA3 + CRM group injected with 5 mg / kg body weight , and the same gene transplantation group . The survival time of each group was observed .
the mast cell degranulation kit is used for detecting the degranulation level of the mast cell ;
The transplanted heart was stained with HE and acute rejection was observed .
The mRNA was extracted from the transplanted heart tissue , and the transcription level of IL - 10 and IFN - 纬 was detected .

Results : In vitro study , the expression of C5a receptor was 58.69 % ~ 67.64 % . The C5a receptor was only 1.23 % ~ 1.89 % of RPMC . C5a could induce RSMC and LPS - stimulated release of RPMC . C5a could reduce the level of degranulation of receptor mast cells . However , there was no significant difference in the levels of IL - 10 , IL - 10 , IFN - 纬 mRNA and IL - 10 and IFN - 纬 in the heart rejection score , cardiac tissue IL - 10 , IFN - 纬 mRNA and serum .

Conclusion : C5a and mast cell degranulation levels increased in rats with ectopic heart transplantation .
C5a can also promote the degranulation of mast cells .
Sodium glycate can reduce the degree of degranulation of mast cells , but it is not possible to reduce acute rejection and prolong graft survival .

【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R657.3

【参考文献】