肝脏免疫耐受治疗手术脑损伤的实验研究
发布时间：2018-08-19 17:55
【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(P0.05)、外周血CD4~+/CD8~+T细胞比值降低(P0.05);C1组鼠尾静脉注射MBP致敏后48h肝组织FasL表达含量升高(P0.05)。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(P0.05);D1组鼠尾静脉注射MBP致敏后24h、48h外周血TGF-β1浓度升高(P0.05);A1组与B1组比较,同一指标间差异无统计学意义(P0.05);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(P0.05),且术后7、14、21d,C2、D2组改良神经功能缺陷评分低于B2组(P0.05);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(P0.05);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(P0.05);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(P0.05);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(P0.05);C2、D2组损伤周围神经细胞凋亡率较B2组减少(P0.05)。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。
[Abstract]:Objective: 1. SD rats were exposed to hepatic portal vein and injected with Myelin Basic Protein (MBP) through hepatic portal vein to induce specific immune tolerance. After 10 days, the rats were sensitized with MBP through peripheral vein (caudal vein). The role of Kupffer cells in liver-induced brain antigen immune tolerance. 2. To establish the model of Surgical Brain Injury (SBI) in SD rats, autologous brain cell homogenate was injected through hepatic portal vein through open abdomen, and myelin basic protein (myelin basic protein) was used to establish specific immune tolerance. Methods: 1.32 male SD rats were randomly divided into 4 groups, 8 rats in each group, A1 group: sham operation group, B1 group: normal saline group, C1 group: MBP experimental group, D1 group: gadolinium chloride (GdCl3) pretreatment group (GdCl3 solution by tail vein injection 24 hours before portal vein injection) After 10 days, the mice were sensitized by injecting MBP into the tail vein of rats. The concentration of anti-MBP antibody in peripheral blood was detected by enzyme-linked immunosorbent assay (ELISA) 24 hours and 48 hours after injecting MBP into the tail vein of rats. The ratio of CD4~+/CD8~+ T cells in peripheral blood was detected by flow cytometry, the concentration of transforming growth factor-beta 1 was detected by ELISA, and the concentration of transforming growth factor-beta 1 was detected 48 hours after injecting MBP into the tail vein of rats. 2.32 male SD rats were randomly divided into 4 groups: A2 group: sham operation group; B2 group: operation brain injury + portal vein injection normal saline control group; C2 group: operation brain injury + portal vein injection MBP experimental group; D2 group: operation brain injury + portal vein injection autologous brain cell homogenate Group B2, C2, D2 underwent standard surgical brain injury (SBI) model, group A2 only craniotomy did not destroy the dura mater. According to the grouping, group A2 was not injected with open hepatic portal vein, group B2 was injected with normal saline 0.5mL via hepatic portal vein, group C2 was injected with BMP 0.5mL via hepatic portal vein, group D2 was injected with autologous brain cell homogenate 0.5mL via hepatic portal vein. 5 ml. Modified neurological deficit score (MNSS) was performed 1,3,7,14,21 days after operation; peripheral blood CD4~+/CD8~+ T cell ratio was detected by flow cytometry; serum interleukin-2 (IL-2) and interleukin-4 (IL-4) levels were detected by enzyme linked immunosorbent assay (ELISA); neuronal apoptosis rate was detected by immunohistochemistry. Compared with A1 group, B1 group and D1 group, the concentration of anti-MBP antibody in peripheral blood of C1 group decreased significantly 24 hours and 48 hours after injection of MBP (P 0.05), and the ratio of CD4~+/CD8~+ T cells in peripheral blood decreased significantly (P 0.05); FasL expression in liver tissue of C1 group increased 48 hours after injection of MBP (P 0.05). The concentration of TGF-beta 1 in peripheral blood increased at 24 h and 48 h after MBP sensitization in D1 group (P 0.05); there was no significant difference between A1 group and B1 group in the same index (P 0.05); 2. The scores of improved neurological deficits in C2 and D2 groups were higher than those in A2 group at 1, 3, 7, 14 d after SBI, and B2 group at all time points than those in A2 group (P 0.05). There was statistical significance (P 0.05), and the improved neurological deficit score in group C2 and D2 was lower than that in group B2 (P 0.05) at 7, 14, 21 days after operation; the expression of IL-2 in group C2 and C2 was lower than that in group A2 and B2 at 7, 14 days after SBI, and the expression of IL-4 in group C2 and D2 was higher than that in group A2 and B2. The difference was statistically significant (P 0.05); the ratio of CD4~+/CD8~+ T cells in group C2 and D2 was lower than that in group A2 and B2, and the ratio of CD4~+/CD8~+ T cells in group D2 was lower than that in group C2 14 days after operation, the difference was statistically significant (P 0.05); the expression of IL-4 in group C2 and D2 was higher than that in group A2 and B2 21 days after operation, and the expression of IL-4 in group D2 was higher than that in group C2 (P 0.05). The apoptosis rate of injured peripheral nerve cells in group B2 was lower than that in group B2 (P 0.05). Conclusion: 1. Intrahepatic injection of MBP can induce specific immune tolerance to MBP and reduce the secondary immune attack of the immune system against MBP antigen. MBP accounts for 30% of the myelin sheath of the brain, so it is possible to protect the injured brain tissue. Intravenous injection of autologous brain antigen and MBP into the hepatic portal vein can establish specific immune tolerance, alleviate neuroinflammation, reduce neuronal apoptosis after SBI, facilitate the recovery of nerve function, have a better therapeutic effect on brain injury, and provide a new therapeutic approach for surgical brain injury. 3. Intravenous injection of autologous brain antigen and MBP into portal vein can establish specific immune tolerance, which has therapeutic effect on surgical brain injury. Mixed brain antigen is superior to single brain antigen MBP. Bystander inhibitory effect [1] may play a role in this process, i.e. one antigen induces immune tolerance to another antigen. The specific causes and mechanisms still need to be further studied, which also points out the direction for our follow-up study.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15
本文编号：2192389
[Abstract]:Objective: 1. SD rats were exposed to hepatic portal vein and injected with Myelin Basic Protein (MBP) through hepatic portal vein to induce specific immune tolerance. After 10 days, the rats were sensitized with MBP through peripheral vein (caudal vein). The role of Kupffer cells in liver-induced brain antigen immune tolerance. 2. To establish the model of Surgical Brain Injury (SBI) in SD rats, autologous brain cell homogenate was injected through hepatic portal vein through open abdomen, and myelin basic protein (myelin basic protein) was used to establish specific immune tolerance. Methods: 1.32 male SD rats were randomly divided into 4 groups, 8 rats in each group, A1 group: sham operation group, B1 group: normal saline group, C1 group: MBP experimental group, D1 group: gadolinium chloride (GdCl3) pretreatment group (GdCl3 solution by tail vein injection 24 hours before portal vein injection) After 10 days, the mice were sensitized by injecting MBP into the tail vein of rats. The concentration of anti-MBP antibody in peripheral blood was detected by enzyme-linked immunosorbent assay (ELISA) 24 hours and 48 hours after injecting MBP into the tail vein of rats. The ratio of CD4~+/CD8~+ T cells in peripheral blood was detected by flow cytometry, the concentration of transforming growth factor-beta 1 was detected by ELISA, and the concentration of transforming growth factor-beta 1 was detected 48 hours after injecting MBP into the tail vein of rats. 2.32 male SD rats were randomly divided into 4 groups: A2 group: sham operation group; B2 group: operation brain injury + portal vein injection normal saline control group; C2 group: operation brain injury + portal vein injection MBP experimental group; D2 group: operation brain injury + portal vein injection autologous brain cell homogenate Group B2, C2, D2 underwent standard surgical brain injury (SBI) model, group A2 only craniotomy did not destroy the dura mater. According to the grouping, group A2 was not injected with open hepatic portal vein, group B2 was injected with normal saline 0.5mL via hepatic portal vein, group C2 was injected with BMP 0.5mL via hepatic portal vein, group D2 was injected with autologous brain cell homogenate 0.5mL via hepatic portal vein. 5 ml. Modified neurological deficit score (MNSS) was performed 1,3,7,14,21 days after operation; peripheral blood CD4~+/CD8~+ T cell ratio was detected by flow cytometry; serum interleukin-2 (IL-2) and interleukin-4 (IL-4) levels were detected by enzyme linked immunosorbent assay (ELISA); neuronal apoptosis rate was detected by immunohistochemistry. Compared with A1 group, B1 group and D1 group, the concentration of anti-MBP antibody in peripheral blood of C1 group decreased significantly 24 hours and 48 hours after injection of MBP (P 0.05), and the ratio of CD4~+/CD8~+ T cells in peripheral blood decreased significantly (P 0.05); FasL expression in liver tissue of C1 group increased 48 hours after injection of MBP (P 0.05). The concentration of TGF-beta 1 in peripheral blood increased at 24 h and 48 h after MBP sensitization in D1 group (P 0.05); there was no significant difference between A1 group and B1 group in the same index (P 0.05); 2. The scores of improved neurological deficits in C2 and D2 groups were higher than those in A2 group at 1, 3, 7, 14 d after SBI, and B2 group at all time points than those in A2 group (P 0.05). There was statistical significance (P 0.05), and the improved neurological deficit score in group C2 and D2 was lower than that in group B2 (P 0.05) at 7, 14, 21 days after operation; the expression of IL-2 in group C2 and C2 was lower than that in group A2 and B2 at 7, 14 days after SBI, and the expression of IL-4 in group C2 and D2 was higher than that in group A2 and B2. The difference was statistically significant (P 0.05); the ratio of CD4~+/CD8~+ T cells in group C2 and D2 was lower than that in group A2 and B2, and the ratio of CD4~+/CD8~+ T cells in group D2 was lower than that in group C2 14 days after operation, the difference was statistically significant (P 0.05); the expression of IL-4 in group C2 and D2 was higher than that in group A2 and B2 21 days after operation, and the expression of IL-4 in group D2 was higher than that in group C2 (P 0.05). The apoptosis rate of injured peripheral nerve cells in group B2 was lower than that in group B2 (P 0.05). Conclusion: 1. Intrahepatic injection of MBP can induce specific immune tolerance to MBP and reduce the secondary immune attack of the immune system against MBP antigen. MBP accounts for 30% of the myelin sheath of the brain, so it is possible to protect the injured brain tissue. Intravenous injection of autologous brain antigen and MBP into the hepatic portal vein can establish specific immune tolerance, alleviate neuroinflammation, reduce neuronal apoptosis after SBI, facilitate the recovery of nerve function, have a better therapeutic effect on brain injury, and provide a new therapeutic approach for surgical brain injury. 3. Intravenous injection of autologous brain antigen and MBP into portal vein can establish specific immune tolerance, which has therapeutic effect on surgical brain injury. Mixed brain antigen is superior to single brain antigen MBP. Bystander inhibitory effect [1] may play a role in this process, i.e. one antigen induces immune tolerance to another antigen. The specific causes and mechanisms still need to be further studied, which also points out the direction for our follow-up study.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R651.15
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