【作者】 刘勇；

【导师】 亢建民；

【作者基本信息】 天津医科大学 ， 外科学 神经外科（专业学位）， 2017， 硕士

【摘要】 目的:1.SD大鼠开腹暴露肝门静脉,经肝门静脉注射髓鞘碱性蛋白(Myelin Basic Protein,MBP)诱导机体建立针对此抗原的特异性免疫耐受,10d后经外周静脉(尾静脉)注入MBP致敏。探讨经肝门静脉注射脑抗原诱导机体建立特异性免疫耐受以及肝脏库普佛细胞(kupffer)在肝脏诱导脑抗原免疫耐受中的作用。2.建立SD大鼠手术脑损伤(Surgical Brain Injury,SBI)模型,通过开腹经肝门静脉注入自体脑细胞匀浆、髓鞘碱性蛋白建立机体针对其特异性免疫耐受。探讨肝脏免疫耐受治疗手术脑损伤的效果。方法:1.32只雄性SD大鼠随机分为4组,每组8只,A1组:假手术组,B1组:肝门静脉注射生理盐水对照组,C1组:肝门静脉注射MBP实验组,D1组:肝门静脉注射MBP~+鼠尾静脉注射氯化钆(GdCl3)预处理组(门静脉注射前24h经鼠尾静脉注射GdCl3溶液)。10d后经鼠尾静脉注射MBP致敏,各组大鼠于鼠尾静脉注射MBP致敏后24h、48h行酶联免疫吸附实验法(ELISA)检测外周血抗MBP抗体浓度;流式细胞术检测外周血CD4~+/CD8~+T细胞比值;ELISA法检测转化生长因子-β1浓度;鼠尾静脉注射MBP致敏后48h处死大鼠,定量PCR检测肝组织FasL表达含量。2.32只雄性SD大鼠随机分为4组,每组8只:A2组:假手术组;B2组:手术脑损伤~+肝门静脉注射生理盐水对照组;C2组:手术脑损伤~+肝门静脉注射MBP实验组;D2组:手术脑损伤~+肝门静脉注射自体脑细胞匀浆实验组。B2、C2、D2组行标准手术脑损伤(SBI)模型,A2组只开颅不破坏硬脑膜。依据分组,A2组开腹游离暴露肝门静脉不予注射;B2组经肝门静脉注入生理盐水0.5mL;C2组经肝门静脉注入脑髓鞘碱性蛋白溶液0.5mL;D2组肝门静脉注入自体脑细胞匀浆0.5mL。术后1、3、7、14、21d行改良神经功能缺陷评分(MNSS);流式细胞技术检测外周血CD4~+/CD8~+T细胞比值;酶联免疫吸附实验法(ELISA)检测血清白介素-2(IL-2)、白介素-4(IL-4)表达水平;免疫组织化学方法检测损伤周围脑组织神经细胞凋亡率。结果:1.与A1组、B1组、D1组比较,C1组鼠尾静脉注射MBP致敏后24h、48h外周血抗MBP抗体浓度显著降低(P<0.05)、外周血CD4~+/CD8~+T细胞比值降低(P<0.05);C1组鼠尾静脉注射MBP致敏后48h肝组织FasL表达含量升高(P<0.05)。与A1、B1组比较,C1组鼠尾静脉注射MBP致敏后24h、48h外周血TGF-β1浓度升高(P<0.05);D1组鼠尾静脉注射MBP致敏后24h、48h外周血TGF-β1浓度升高(P<0.05);A1组与B1组比较,同一指标间差异无统计学意义(P>0.05);2.SBI术后1、3、7、14d,C2、D2组改良神经功能缺陷评分均高于A2组,B2组各时间点均高于A2组,差异有统计学意义(P<0.05),且术后7、14、21d,C2、D2组改良神经功能缺陷评分低于B2组(P<0.05);SBI术后7、14d,C2、D2组IL-2表达水平低于A2、B2组,且D2组IL-2表达水平低于C2组,差异均有统计学意义(P<0.05);C2、D2组IL-4表达水平高于A2、B2组,且D2组IL-4表达水平高于C2组,差异均有统计学意义(P<0.05);C2、D2组CD4~+/CD8~+T细胞比值低于A2、B2组,且术后14d D2组CD4~+/CD8~+T细胞比值低于C2组,差异均有统计学意义(P<0.05);术后21d,C2、D2组IL-4表达数量高于A2、B2组,且D2组IL-4表达数量高于C2组,差异均有统计学意义(P<0.05);C2、D2组损伤周围神经细胞凋亡率较B2组减少(P<0.05)。结论:1.经肝门静脉注射MBP可诱导机体建立针对MBP的特异性免疫耐受,降低机体免疫系统针对MBP抗原的继发免疫攻击,而MBP占脑髓鞘30%,因此具有保护受损脑组织的可能性;肝脏Kupffer细胞在肝脏免疫耐受中发挥重要作用。2.经肝门静脉注射自体脑抗原及MBP可建立针对其的特异性免疫耐受,减轻SBI术后神经炎症反应、减少神经细胞凋亡,有利于神经功能恢复,对手术脑损伤有较好的治疗作用,为手术脑损伤提供了新的治疗途径。3.肝门静脉注射自体脑抗原及MBP均可建立特异性免疫耐受,对手术脑损伤具有治疗作用,混合脑抗原优于单一脑抗原MBP。考虑两方面因素,第一,混合脑抗原诱导机体建立对多种抗原的免疫耐受,从而优于单一抗原建立的免疫耐受;第二,“旁观者抑制效应”[1]可能在其中发挥一定的作用,即一种抗原诱导免疫耐受对另外的抗原起耐受作用。具体原因及机制仍需要进一步研究,也为我们后续的研究指明方向。更多还原

【Abstract】 Objective: 1.To explore the induction of immune tolerance by the injection of MBP into hepatic portal vein and the role of Kupffer cells in this process.We value this result by detected some inflammation factors.2.SD rat model of SBI was established by injecting auto-brain cell homogenate and MBP into the portal vein to establish the specific immune tolerance.To investigate the effect of liver immune tolerance on brain injury.Methods: 1.32 male SD rats were divided into four groups according to random number table,8 in each group: A1 group: sham group,B1 group: normal saline control group,C1 group: MBP experiment group,D1 group: Gd Cl3 were treatment group(injected Gd Cl3 into rat tail vein 24 h before the portal vein injection).Inject MBP into rat tail vein 10 days after the portal vein injection.MBP antibody 、 TGF-β1 were detected by ELISA,CD4~+/CD8~+ T cells ratio in peripheral blood detected by FCM 24 h and 48 h after the rat tail vein injection.48 h after rat tail vein injection,Fas L were measured by Real-time Polymerase Chain Reaction(RT-PCR).2.32 males SD rats were divided into four groups according to random number table,8 in each group: A2 group: sham group,B2 group: normal saline control group,C2 group: MBP treatment group,D2 group: auto-cerebral cells treatment group.A standardized SBI model was used in B2、C2 and D2 groups,as described in previous reports,A2 group no durotomy.After SBI and sham surgery,all rats developed laparotomy to exposed hepatic portal vein and injected into 0.5ml normal saline、MBP and auto-cerebral cells separately in B2、C2 and D2 groups.Function outcomes was evaluated using MNSS;IL-2,IL-4were detected by ELISA;CD4~+/CD8~+ T ratio in peripheral blood detected by FCM;apoptosis of nerve cells around the surgery wound detected by immunofluorescence at 1,3,7,14,21 d after SBI.Results: 1.Compared with group A1,B1 and D1,the concentration of MBP antibody were extraordinarily lower in group C1(P<0.05),meanwhile CD4~+/CD8~+ T ratio were obviously lower in group C1(P<0.05),at 24 h and 48 h after rat tail vein injection,the gene expression of Fas L were notably higher than that in group C1(P<0.05),at 48 h after rat tail vein injection.Compared with group A1 and B1,the concentration of TGF-β1 were obviously higher than that in group C1 and D1(P<0.05),and there were no difference between group C1 and D1 at 24 h and 48 h after rat tail vein injection.There was no difference about the same indicator,at different time points,between group A1 and B1.2.The modified neurological severity score in C2,D2 group higher than that in A2 group at 1,3,7,14 d,and B2 group higher than A2 group at all the time points,moreover the MNSS in C2 and D2 group lower than that in B2 group(P<0.05)at 7,14,21 d after SBI.D2 and C2 groups show significant lower IL-2 cytokine level and CD4~+/CD8~+ T ratio and higher IL-4 cytokine level compare with A2 and B2 group and at the same time;D2 group better than C2 group at 7,14 d after SBI(P<0.05).At 21 d after SBI,C2,D2 group have higher IL-4 cytokine level than A2,B2 group moreover D2 group better than C2 group(P<0.05),C2 and D2 group have lower nerve cells apoptosis than B2 group(P<0.05).Conclusion:1.Through hepatic portal vein injection of MBP can induce the establishment of specific immune tolerance of MBP,reduce the body’s immune system secondary immune attack against MBP antigen,and MBP accounted for 30% of the brain antigen,therefore has the possibility to protect the damaged brain tissue;liver Kuppffer cells play an important role in liver immune tolerance.2.The establishment of specific immune tolerance by its own brain antigen and MBP of hepatic portal vein injection,nerve inflammation,reduce the apoptosis of nerve cells reduced after SBI,is conducive to the recovery of neurological function,has a good curative effect on surgical brain injury and provide a new way for treatment of brain injury surgery.3.The specific immune tolerance can be established by injecting autologous brain antigen and MBP into the portal vein,and the therapeutic effect of the mixed brain antigen is better than that of the single brain antigen MBP.Considering the two factors,first,the establishment of a variety of antigens induce immune tolerance to self antigen mixed brain,is better than a single antigen immune tolerance;second,“bystander suppression” was activated by tolerogen and resulted in the suppression of immune responses to other antigens Specific reasons and mechanisms still need further study,but also for our follow-up research direction.更多还原