肝臟免疫耐受治療手術腦損傷的實驗研究
發(fā)布時間：2018-08-19 17:55
【摘要】：目的:1.SD大鼠開腹暴露肝門靜脈,經(jīng)肝門靜脈注射髓鞘堿性蛋白(Myelin Basic Protein,MBP)誘導機體建立針對此抗原的特異性免疫耐受,10d后經(jīng)外周靜脈(尾靜脈)注入MBP致敏。探討經(jīng)肝門靜脈注射腦抗原誘導機體建立特異性免疫耐受以及肝臟庫普佛細胞(kupffer)在肝臟誘導腦抗原免疫耐受中的作用。2.建立SD大鼠手術腦損傷(Surgical Brain Injury,SBI)模型,通過開腹經(jīng)肝門靜脈注入自體腦細胞勻漿、髓鞘堿性蛋白建立機體針對其特異性免疫耐受。探討肝臟免疫耐受治療手術腦損傷的效果。方法:1.32只雄性SD大鼠隨機分為4組,每組8只,A1組:假手術組,B1組:肝門靜脈注射生理鹽水對照組,C1組:肝門靜脈注射MBP實驗組,D1組:肝門靜脈注射MBP~+鼠尾靜脈注射氯化釓(GdCl3)預處理組(門靜脈注射前24h經(jīng)鼠尾靜脈注射GdCl3溶液)。10d后經(jīng)鼠尾靜脈注射MBP致敏,各組大鼠于鼠尾靜脈注射MBP致敏后24h、48h行酶聯(lián)免疫吸附實驗法(ELISA)檢測外周血抗MBP抗體濃度;流式細胞術檢測外周血CD4~+/CD8~+T細胞比值;ELISA法檢測轉化生長因子-β1濃度;鼠尾靜脈注射MBP致敏后48h處死大鼠,定量PCR檢測肝組織FasL表達含量。2.32只雄性SD大鼠隨機分為4組,每組8只:A2組:假手術組;B2組:手術腦損傷~+肝門靜脈注射生理鹽水對照組;C2組:手術腦損傷~+肝門靜脈注射MBP實驗組;D2組:手術腦損傷~+肝門靜脈注射自體腦細胞勻漿實驗組。B2、C2、D2組行標準手術腦損傷(SBI)模型,A2組只開顱不破壞硬腦膜。依據(jù)分組,A2組開腹游離暴露肝門靜脈不予注射;B2組經(jīng)肝門靜脈注入生理鹽水0.5mL;C2組經(jīng)肝門靜脈注入腦髓鞘堿性蛋白溶液0.5mL;D2組肝門靜脈注入自體腦細胞勻漿0.5mL。術后1、3、7、14、21d行改良神經(jīng)功能缺陷評分(MNSS);流式細胞技術檢測外周血CD4~+/CD8~+T細胞比值;酶聯(lián)免疫吸附實驗法(ELISA)檢測血清白介素-2(IL-2)、白介素-4(IL-4)表達水平;免疫組織化學方法檢測損傷周圍腦組織神經(jīng)細胞凋亡率。結果:1.與A1組、B1組、D1組比較,C1組鼠尾靜脈注射MBP致敏后24h、48h外周血抗MBP抗體濃度顯著降低(P0.05)、外周血CD4~+/CD8~+T細胞比值降低(P0.05);C1組鼠尾靜脈注射MBP致敏后48h肝組織FasL表達含量升高(P0.05)。與A1、B1組比較,C1組鼠尾靜脈注射MBP致敏后24h、48h外周血TGF-β1濃度升高(P0.05);D1組鼠尾靜脈注射MBP致敏后24h、48h外周血TGF-β1濃度升高(P0.05);A1組與B1組比較,同一指標間差異無統(tǒng)計學意義(P0.05);2.SBI術后1、3、7、14d,C2、D2組改良神經(jīng)功能缺陷評分均高于A2組,B2組各時間點均高于A2組,差異有統(tǒng)計學意義(P0.05),且術后7、14、21d,C2、D2組改良神經(jīng)功能缺陷評分低于B2組(P0.05);SBI術后7、14d,C2、D2組IL-2表達水平低于A2、B2組,且D2組IL-2表達水平低于C2組,差異均有統(tǒng)計學意義(P0.05);C2、D2組IL-4表達水平高于A2、B2組,且D2組IL-4表達水平高于C2組,差異均有統(tǒng)計學意義(P0.05);C2、D2組CD4~+/CD8~+T細胞比值低于A2、B2組,且術后14d D2組CD4~+/CD8~+T細胞比值低于C2組,差異均有統(tǒng)計學意義(P0.05);術后21d,C2、D2組IL-4表達數(shù)量高于A2、B2組,且D2組IL-4表達數(shù)量高于C2組,差異均有統(tǒng)計學意義(P0.05);C2、D2組損傷周圍神經(jīng)細胞凋亡率較B2組減少(P0.05)。結論:1.經(jīng)肝門靜脈注射MBP可誘導機體建立針對MBP的特異性免疫耐受,降低機體免疫系統(tǒng)針對MBP抗原的繼發(fā)免疫攻擊,而MBP占腦髓鞘30%,因此具有保護受損腦組織的可能性;肝臟Kupffer細胞在肝臟免疫耐受中發(fā)揮重要作用。2.經(jīng)肝門靜脈注射自體腦抗原及MBP可建立針對其的特異性免疫耐受,減輕SBI術后神經(jīng)炎癥反應、減少神經(jīng)細胞凋亡,有利于神經(jīng)功能恢復,對手術腦損傷有較好的治療作用,為手術腦損傷提供了新的治療途徑。3.肝門靜脈注射自體腦抗原及MBP均可建立特異性免疫耐受,對手術腦損傷具有治療作用,混合腦抗原優(yōu)于單一腦抗原MBP�？紤]兩方面因素,第一,混合腦抗原誘導機體建立對多種抗原的免疫耐受,從而優(yōu)于單一抗原建立的免疫耐受;第二,“旁觀者抑制效應”[1]可能在其中發(fā)揮一定的作用,即一種抗原誘導免疫耐受對另外的抗原起耐受作用。具體原因及機制仍需要進一步研究,也為我們后續(xù)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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R651.15
本文編號：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.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R651.15
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