Objectives: Heparin is mainly used as an anticoagulant in clinic, and it also has a certain anti-inflammatory effect. At present, after portal vein islet transplantation in diabetic patients, heparin is mainly infused through the peripheral veins of the limbs to achieve the purpose of anticoagulation and protection of the graft, rather than through the portal vein. In this study, animal experiments were conducted to investigate the effect of heparin infusion via the portal vein and marginal ear vein on the instant blood-mediated inflammatory reaction (IBMIR) after portal vein islet transplantation, which is the choice of anticoagulation methods for clinical islet transplantation to provide a basis for decision-making.
Methods: A total of 50 neonatal pigs (Xeno-1 type, 3-5 days) were selected. Islets were isolated and purified from the pancreas of neonatal pigs. Ten non-diabetic Landrace pigs (1.5-2.0 months) served as recipients, and 12 000 IEQ/kg neonatal porcine islets were transplanted into the liver through the portal vein. All recipients received bolus injection of 50 U/kg of heparin 10 minutes before transplantation. After the bolus injection of heparin, the experimental group received heparin via the portal vein [10 U/(kg·h), 5 recipients], and the control group received heparin via the marginal ear vein [10 U/(kg·h), 5 recipients]. The superior vena cava blood was collected from the 2 groups pre-operation at 1, 3, 24 h post-operation of the transplantation. The portal vein blood was collected from the experimental group at 1 and 3 h after the transplantation as well. The levels of complement C3a, C5a, thrombin-antithrombin complex (TAT), β-thromboglobulin (β-TG), and D-dimer as well as activated partial thromboplastin time (APTT) in superior vena cava blood from 1 and 3 h post-transplantation were detected in the 2 groups, and the levels of anti-Xa and anti-IIa in the portal vein and superior vena cava blood from 1 and 3 h post-transplantation in the experimental group were detected. Twenty four hours after the transplantation, the liver tissues in the 2 groups were collected for pathological examination to observe the inflammatory cell infiltration and peripheral thrombosis around the islets graft in liver.
Results: Before transplantation, there was no statistically significant difference in C3a, C5a, TAT, β-TG, D-dimer levels and APTT between the 2 groups (all P>0.05). At 1 and 3 h after transplantation, the C3a, TAT, and D-dimer levels in the experimental group were significant decreased than those in the control groups (all P<0.05), and at 3 h after transplantation the C5a was significant decreased than that in the control group (P<0.05). At 1 and 3 h after transplantation, the anti-Xa and anti-IIa levels in the portal vein blood were significantly increased than those in the superior vena cava blood in the experimental group (all P<0.05). Pathological results showed the presence of islet cell clusters in the liver blood vessels. The thrombus formation and neutrophil infiltration around islet graft was not obvious in the experimental group, while massive thrombus formation and neutrophil infiltration in the control group.
Conclusions: Compared with marginal ear vein infusion of heparin, the direct infusion of heparin in the portal vein has a certain inhibitory effect on complement system, coagulation system activation and inflammatory cell infiltration in portal vein islet transplantation, which may attenuate the occurrence of IBMIR.
目的: 临床上肝素主要作为抗凝药物使用，也具有一定的抗炎作用。目前糖尿病患者门静脉胰岛移植术后主要通过四肢的外周静脉而不是门静脉输注肝素，达到抗凝、保护移植物的目的。本研究采用动物实验探讨经门静脉与耳缘静脉输注肝素对门静脉胰岛移植术后立即经血液介导炎症反应(instant blood mediated inflammatory reaction，IBMIR)的影响，为临床胰岛移植手术抗凝方式的选择提供决策依据。方法: 选择日龄3~5 d新生猪(Xeno-1型)50只，取出胰腺后分离并提纯胰岛。选择1.5~2.0 月龄非糖尿病模型长白猪10只作为受体，外科手术暴露门静脉，将新生猪胰岛(12 000 IEQ/kg)通过门静脉移植于长白猪肝脏。移植术前10 min所有受体均团注肝素50 U/kg，团注肝素后实验组通过门静脉输注肝素[10 U/(kg·h)，5只]、对照组通过耳缘静脉输注肝素[10 U/(kg·h)，5只]。分别于术前、术后1、3及24 h采集两组动物上腔静脉血液，实验组于术后1、3 h采集门静脉血液。检测实验组与对照组术前、术后1、3 h上腔静脉血液补体C3a、C5a、凝血酶-抗凝血酶复合物(thrombin-antithrombin complex，TAT)、β-血小板球蛋白(β-thromboglobulin，β-TG)和D-二聚体(D-dimer)水平，以及活化部分凝血活酶时间(activated partial thromboplastin time，APTT)；检测术后1、3 h实验组门静脉、上腔静脉血液抗Xa(anti-Xa)、抗IIa(anti-IIa)水平。术后24 h取两组动物肝组织行病理检查，观察肝内胰岛炎性细胞浸润和周围血栓的形成情况。结果: 实验组与对照组术前C3a、C5a、TAT、β-TG、D-dimer水平及APTT的组间差异均无统计学意义(均P>0.05)。术后1、3 h实验组C3a、TAT及D-Dimer水平均低于对照组，术后3 h实验组C5a低于对照组(均P<0.05)。术后1、3 h实验组门静脉血液anti-Xa、anti-IIa水平均高于上腔静脉(均P<0.05)。病理结果显示肝血管内存在胰岛细胞团。实验组胰岛周围少量血栓形成、少量中性粒细胞浸润；对照组胰岛周围明显有血栓形成和较多的中性粒细胞浸润。结论: 门静脉输注肝素与耳缘静脉输注肝素比较，门静脉直接输注肝素对补体系统、凝血系统激活及炎性细胞浸润具有一定的抑制作用，在一定程度上可以缓解IBMIR的发生。.
Keywords: diabetes mellitus; heparin; instant blood mediated inflammatory reaction; islet; portal vein.