Kupffer细胞功能对大鼠肝移植急性排斥反应的影响
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摘要
目的 (1) 探讨建立稳定大鼠原位肝移植模型的方法,观察Wistar→SD大鼠肝移植后急性排斥反应的发生,以选择适宜、可靠的大鼠原位肝移植急性排斥模型。(2) 氯化钆、非损伤剂量脂多糖(LPS)及门静脉输血预处理改变Kupffer细胞功能,研究其对移植后Kupffer细胞FasL表达、脾淋巴细胞增殖与杀伤作用的影响。(3) 研究Kupffer细胞功能改变对大鼠肝移植急性排斥反应的影响,探讨其可能的机制,进一步探索Kupffer细胞在同种异基因大鼠肝移植免疫耐受诱导中的作用。通过以上三部分实验,试图进一步阐明Kupffer细胞在大鼠原位肝移植免疫耐受诱导中的确切作用,为进一步利用Kupffer细胞特异性地诱导肝移植免疫耐受的实验研究及临床应用提供依据和基础。
方法 (1) 参照Kamada“二袖套法”,加以适当改进,建立Wistar→SD大鼠原位肝移植动物模型,并以Wistar→Wistar大鼠原位肝移植为对照,观察两组受鼠生存时间,全自动生化分析仪测定术后3天、5天、7天血浆谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBIL)和白蛋白(Alb)水平变化,观察肝脏组织病理改变。(2) 氯化钆、非损伤剂量脂多糖(LPS)及门静脉输血1ml预处理供体Wistar大鼠以改变Kupffer细胞功能,未处理组为对照。肝移植后7天分离上述四组动物肝脏Kupffer细胞,对分离的Kupffer细胞进行计数,流式细胞技术检测Kupffer细胞的FasL表达。各组Kupffer细胞与激活SD脾淋巴细胞混合培养,MTT法检测淋巴细胞增殖,流式细胞技术检测淋巴细胞凋亡百分率。(3) 按上述方法建立Wistar→SD大鼠肝移植动物模型和进行动物分组,观察各组生存时间;观察术后7天肝脏功能和组织病理改变,双抗体夹心ABC-ELISA法测定血清细胞因子白介素-10(IL-10)、转化生长因子β1(TGF-β1)和肿瘤坏死因子
Objective (1) To investigate the technique for performing orthotopic liver transplantation .(OLT) stably in Wistar→SD rats and observe the allogrft rejection occurred after OLT. (2) To study the effects of FasL expression , suppression on spleen lymphocyte proliferation and inducing lymphocyte apoptosis of Kupffer cells in different functional states, Which were induced by the pretreatment of gadolinium chloride (GdCl_3), lipopolysaccharide (LPS) and portal venous transfusion(PVT). (3) To investigate the hepatic allograft rejection affected by the functions of Kupffer cells in rats , explore its possible mechanisms and find out the exact roles of Kupffer cells in inducing the liver allograft tolerance .Methods (1) Orthotopic liver transplantation in Wistar→SD rats was performed with the two-cuff method that described by Kamada. OLT in Wistar→Wistar rats were used as control. The rats were killed at 3d, 5d and 7d after operation , liver tissues and blood samples were collected. Recipient survival, histopathological characteristics of hepatic allogfrats were investigated. Plasm levels of alanine aminotransferase (ALT), aspartase aminotransferase(AST), total bilirubin (TBIL), and albumin (Alb) were measured with an automatic biochemical analyser. (2) Pretreatment donor with gadolinium chloride (GdCl_3), lipopolysaccharide (LPS) and portal venous transfusion(PVT) respectively, OLT in Wistar → SD rats were performed. Kupffer cells in liver grafts were isolated 7days after OLT using the technique of Isopyknic gradient centrifugation with Percall. Isolated Kupffer cells were counted. Expression of Fas ligand protein in Kupffer
cells ,lymphocytes apoptosis in coculture with Kupffer cells were evaluated by flow cytometric assay (FCM) and the proliferation of lymphocytes was detected using MTT. (3) Another study was conducted to investigate the survival of recipients in which the function of Kupffer cells were altered. The liver function and histopathological characteristics of hepatic allogfrats were investigated. IL-10, TGF- |3 1, and TNF-a cytokine levels in plasma were also measured with enzyme linked immunosorbent assay (ELISA), respectively. Expression of FasL on Kupffer cells and apoptosis of lympocytes in liver grafts were detected using immunohistochemical staining and TUNEL.Data were expressed as means±SEM. Statistical analysis was performed using the Student t test. P value of O.05 was considered statistically significant.Results (1) Duration of operation is not significantly different between the two groups, 1 week survival were 87.5%. Liver transplantation from Wistar—SD result in acute rejection of hepatic graft and the survival of recipient rats were 9.63± 2. 13days. Banff RAI(rejection activity index) was 7. 1 ± 0. 85. (2) By pretreatment with GdCl3 ,LPS and PVT, the number and expression of FasL of Kupffer cells isolated from LPS or PVT pretreatments allografts 7d after OLT were much higher than the control (P<0.05 ) . The effects of suppression on spleen lymphocyte proliferation and inducing lymphocyte apoptosis by Kupffer cells were stronger also (P<0.05 ) .These effects could be abrogated by pretreatments with GdCb which can inhibit the function of Kupffer cells.(3) The survival of the GdCl3 group was 6.87+1. 25d, but prolonged in PVT group(25.75 + 5.44d) .Allograft rejection was enhanced by pretreatments with GdCb. IL-10, TGF- P 1, TNF-a cytokine levels in plasma and FasL expression of Kupffer cells were decreased together with the apoptosis of lymphocytes in the allografts(P<0. 05) .This effects were improved by pretreatments with PVT significantly.
These results suggested that Kupffer cells-dependent T-cell deletion via the Fas/FasL pathway and immunoregualtion might play an important role in liver transplantation acceptance. Although pretreatments with LPS could bring some benefit as PVT, there was a decrease of survival. Probably it was related to the damage of high TNF-a level.Conclusions (1) Liver transplantation from donor Wistar rats to recipient SD rats can result in acute rejection of hepatic graft. It is a reliable combination for the research of hepatic allograft rejection and tolerance inducing. (2) Apoptosis of infiltrating lymphocytes, immunoregulation of cytokines IL-10/ TGF-P l could play an important role in mechanism of liver transplantation acceptance. Change the function of Kupffer cells by pretreatment could affect the Fas/FasL pathway and immunoregulation to induce liver transplantation tolerance. (3) Although enhanced Kupffer cells function could help to induce tolerance of hepatic allograft , there is still need to explore the effective, safe and applicable stimulation.
方法 (1) 参照Kamada“二袖套法”,加以适当改进,建立Wistar→SD大鼠原位肝移植动物模型,并以Wistar→Wistar大鼠原位肝移植为对照,观察两组受鼠生存时间,全自动生化分析仪测定术后3天、5天、7天血浆谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆红素(TBIL)和白蛋白(Alb)水平变化,观察肝脏组织病理改变。(2) 氯化钆、非损伤剂量脂多糖(LPS)及门静脉输血1ml预处理供体Wistar大鼠以改变Kupffer细胞功能,未处理组为对照。肝移植后7天分离上述四组动物肝脏Kupffer细胞,对分离的Kupffer细胞进行计数,流式细胞技术检测Kupffer细胞的FasL表达。各组Kupffer细胞与激活SD脾淋巴细胞混合培养,MTT法检测淋巴细胞增殖,流式细胞技术检测淋巴细胞凋亡百分率。(3) 按上述方法建立Wistar→SD大鼠肝移植动物模型和进行动物分组,观察各组生存时间;观察术后7天肝脏功能和组织病理改变,双抗体夹心ABC-ELISA法测定血清细胞因子白介素-10(IL-10)、转化生长因子β1(TGF-β1)和肿瘤坏死因子
Objective (1) To investigate the technique for performing orthotopic liver transplantation .(OLT) stably in Wistar→SD rats and observe the allogrft rejection occurred after OLT. (2) To study the effects of FasL expression , suppression on spleen lymphocyte proliferation and inducing lymphocyte apoptosis of Kupffer cells in different functional states, Which were induced by the pretreatment of gadolinium chloride (GdCl_3), lipopolysaccharide (LPS) and portal venous transfusion(PVT). (3) To investigate the hepatic allograft rejection affected by the functions of Kupffer cells in rats , explore its possible mechanisms and find out the exact roles of Kupffer cells in inducing the liver allograft tolerance .Methods (1) Orthotopic liver transplantation in Wistar→SD rats was performed with the two-cuff method that described by Kamada. OLT in Wistar→Wistar rats were used as control. The rats were killed at 3d, 5d and 7d after operation , liver tissues and blood samples were collected. Recipient survival, histopathological characteristics of hepatic allogfrats were investigated. Plasm levels of alanine aminotransferase (ALT), aspartase aminotransferase(AST), total bilirubin (TBIL), and albumin (Alb) were measured with an automatic biochemical analyser. (2) Pretreatment donor with gadolinium chloride (GdCl_3), lipopolysaccharide (LPS) and portal venous transfusion(PVT) respectively, OLT in Wistar → SD rats were performed. Kupffer cells in liver grafts were isolated 7days after OLT using the technique of Isopyknic gradient centrifugation with Percall. Isolated Kupffer cells were counted. Expression of Fas ligand protein in Kupffer
cells ,lymphocytes apoptosis in coculture with Kupffer cells were evaluated by flow cytometric assay (FCM) and the proliferation of lymphocytes was detected using MTT. (3) Another study was conducted to investigate the survival of recipients in which the function of Kupffer cells were altered. The liver function and histopathological characteristics of hepatic allogfrats were investigated. IL-10, TGF- |3 1, and TNF-a cytokine levels in plasma were also measured with enzyme linked immunosorbent assay (ELISA), respectively. Expression of FasL on Kupffer cells and apoptosis of lympocytes in liver grafts were detected using immunohistochemical staining and TUNEL.Data were expressed as means±SEM. Statistical analysis was performed using the Student t test. P value of O.05 was considered statistically significant.Results (1) Duration of operation is not significantly different between the two groups, 1 week survival were 87.5%. Liver transplantation from Wistar—SD result in acute rejection of hepatic graft and the survival of recipient rats were 9.63± 2. 13days. Banff RAI(rejection activity index) was 7. 1 ± 0. 85. (2) By pretreatment with GdCl3 ,LPS and PVT, the number and expression of FasL of Kupffer cells isolated from LPS or PVT pretreatments allografts 7d after OLT were much higher than the control (P<0.05 ) . The effects of suppression on spleen lymphocyte proliferation and inducing lymphocyte apoptosis by Kupffer cells were stronger also (P<0.05 ) .These effects could be abrogated by pretreatments with GdCb which can inhibit the function of Kupffer cells.(3) The survival of the GdCl3 group was 6.87+1. 25d, but prolonged in PVT group(25.75 + 5.44d) .Allograft rejection was enhanced by pretreatments with GdCb. IL-10, TGF- P 1, TNF-a cytokine levels in plasma and FasL expression of Kupffer cells were decreased together with the apoptosis of lymphocytes in the allografts(P<0. 05) .This effects were improved by pretreatments with PVT significantly.
These results suggested that Kupffer cells-dependent T-cell deletion via the Fas/FasL pathway and immunoregualtion might play an important role in liver transplantation acceptance. Although pretreatments with LPS could bring some benefit as PVT, there was a decrease of survival. Probably it was related to the damage of high TNF-a level.Conclusions (1) Liver transplantation from donor Wistar rats to recipient SD rats can result in acute rejection of hepatic graft. It is a reliable combination for the research of hepatic allograft rejection and tolerance inducing. (2) Apoptosis of infiltrating lymphocytes, immunoregulation of cytokines IL-10/ TGF-P l could play an important role in mechanism of liver transplantation acceptance. Change the function of Kupffer cells by pretreatment could affect the Fas/FasL pathway and immunoregulation to induce liver transplantation tolerance. (3) Although enhanced Kupffer cells function could help to induce tolerance of hepatic allograft , there is still need to explore the effective, safe and applicable stimulation.
引文
1. Furkawa HK, Todo S, Imventurza O, et al. Effect of cold ischemia time on the early outcome of human hepatic allografts preserved with UW solution. Transplantation, 1991; 51: 1000-1004
2. Kojima N, Sato M, Suzuki A, et al. Enhanced expression of B7-1, B7-2, and intercellular adhension molecule 1 in sinusoidal endothelial cells by warm ischemia/reperfusion injury in rat liver. Hepatology, 2001; 34: 751-757
3. Kamada N, Calne RY: Orthotopic liver transplantation in the rat—technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 28: 47-50, 1979
4. Kamada N, Calne RY: A surgical experience with five hundred thirty five liver transplants in the rat. Surgery 93: 64-69, 1983.
5. Lee S, Charter AC, Chandler JB, Orloff MJ: A technique for orthotopic liver transplantation in the rat. Transplantation 16: 664-669, 1973
6. Miyata M, Fisher JH, Fuhs W, et al. A simple method for orthotopic liver transplantation in the rat. Transplantation, 1980; 30: 335
7. Delrivie RE, Gibbs P, Kobayashi E, et al. Technical details for safer venous and biliary anastomoses for liver transplantation in the rat. Microsurery, 1998; 18: 12-18
8. Delrivie RE, Gibbs P, Kobayashi E, et al. Detailed modified technique for safer harvesting and preparation of liver graft in the rat. Microsurery, 1996; 17: 690-69
9. Gorre K, Lee R G, Lewin K, et al. An International Panel. Banff schema for grading liver allograft rejection: an international consensus document. Hepatology, 1997, 25: 658-663
10.陈忠华,夏穗生.改进的大鼠原位肝移植术.中华器官移植杂,1984,5:50.
11.谢学羿,陈规划,黄洁夫。二袖套法建立大鼠原位肝移植模型的初步体会。肝胆外科杂志,1999;7:469-3
12. Tisone G, Vennarecci G, Bauicchi L, et al. Randomized study on insitu liver perfusion techniques: gravity perfusion vs high pressure perfusion. Transplant Proc, 1997; 29: 3460
13.张浩,王学浩,张峰。灌注压力对肝移植物活性的影响。中国普外继续与临床杂志,1999,6:140-142
14. Tokunaga Y, Ozaki N, Wakashiro S, et al. Effects of perfusion pressure during flushing on the viability of the procured liver using invasive fluorometry. Transplantation, 1988; 45: 1031
15. CalneR. Y., SmithD. P., Mcmaster. P, etal. Useofpartialcar-diopulmonarybypassduringth eanhepaticphaseoforthotopiclivergrafting. Lancet, 1979, 8143: 612.
16.王轩,杨甲梅,严以群,等.大鼠肝移植不同术式的探讨.中华器官移植杂志.1998,19:76-78
17. Wang C, Sheil AG, Sun J. Outcome of different models of multiorgan transplantation in rats Microsurgery. 1999; 19: 318-23
18.孙倍成,王学浩,胡建平等。大鼠肝移植技术改进及免疫排斥初步观察。南京医科大学学报,2001;4:281-283
19.梁健,刘利民,刘永锋等。大鼠肝移值排斥反应期一氧化氮合酶的动力学变化。免疫学杂志,2002:18:269-272
20. Sun ZL, Wada T, Maemura K. et al. Hepatic allograft-derived kupffer cells regulate T cell response in rats. Liver Transplantation, 2003; 9: 489-497
21. Mayer D, Thormarth W, Otto C, et al. Early T-cell inactivation and apoptosis—Critical events for tolerance induction after allogeneic liver transplantation Transplantation Proceedings, 2001; 33: 256-258
22.杜家文,黄林平,周雷,贾振庚雷公藤多甙对大鼠原位肝移植急性排斥反应的影响。中国中西医结合外科杂志,2003;9:74-75
23.许赤 黄洁夫 陈规划等。胆汁细胞学及细胞因子基因表达诊断大鼠肝移植急性排斥反应的实验研究。中华普通外科杂志,2001;16:602-604
2. Kojima N, Sato M, Suzuki A, et al. Enhanced expression of B7-1, B7-2, and intercellular adhension molecule 1 in sinusoidal endothelial cells by warm ischemia/reperfusion injury in rat liver. Hepatology, 2001; 34: 751-757
3. Kamada N, Calne RY: Orthotopic liver transplantation in the rat—technique using cuff for portal vein anastomosis and biliary drainage. Transplantation 28: 47-50, 1979
4. Kamada N, Calne RY: A surgical experience with five hundred thirty five liver transplants in the rat. Surgery 93: 64-69, 1983.
5. Lee S, Charter AC, Chandler JB, Orloff MJ: A technique for orthotopic liver transplantation in the rat. Transplantation 16: 664-669, 1973
6. Miyata M, Fisher JH, Fuhs W, et al. A simple method for orthotopic liver transplantation in the rat. Transplantation, 1980; 30: 335
7. Delrivie RE, Gibbs P, Kobayashi E, et al. Technical details for safer venous and biliary anastomoses for liver transplantation in the rat. Microsurery, 1998; 18: 12-18
8. Delrivie RE, Gibbs P, Kobayashi E, et al. Detailed modified technique for safer harvesting and preparation of liver graft in the rat. Microsurery, 1996; 17: 690-69
9. Gorre K, Lee R G, Lewin K, et al. An International Panel. Banff schema for grading liver allograft rejection: an international consensus document. Hepatology, 1997, 25: 658-663
10.陈忠华,夏穗生.改进的大鼠原位肝移植术.中华器官移植杂,1984,5:50.
11.谢学羿,陈规划,黄洁夫。二袖套法建立大鼠原位肝移植模型的初步体会。肝胆外科杂志,1999;7:469-3
12. Tisone G, Vennarecci G, Bauicchi L, et al. Randomized study on insitu liver perfusion techniques: gravity perfusion vs high pressure perfusion. Transplant Proc, 1997; 29: 3460
13.张浩,王学浩,张峰。灌注压力对肝移植物活性的影响。中国普外继续与临床杂志,1999,6:140-142
14. Tokunaga Y, Ozaki N, Wakashiro S, et al. Effects of perfusion pressure during flushing on the viability of the procured liver using invasive fluorometry. Transplantation, 1988; 45: 1031
15. CalneR. Y., SmithD. P., Mcmaster. P, etal. Useofpartialcar-diopulmonarybypassduringth eanhepaticphaseoforthotopiclivergrafting. Lancet, 1979, 8143: 612.
16.王轩,杨甲梅,严以群,等.大鼠肝移植不同术式的探讨.中华器官移植杂志.1998,19:76-78
17. Wang C, Sheil AG, Sun J. Outcome of different models of multiorgan transplantation in rats Microsurgery. 1999; 19: 318-23
18.孙倍成,王学浩,胡建平等。大鼠肝移植技术改进及免疫排斥初步观察。南京医科大学学报,2001;4:281-283
19.梁健,刘利民,刘永锋等。大鼠肝移值排斥反应期一氧化氮合酶的动力学变化。免疫学杂志,2002:18:269-272
20. Sun ZL, Wada T, Maemura K. et al. Hepatic allograft-derived kupffer cells regulate T cell response in rats. Liver Transplantation, 2003; 9: 489-497
21. Mayer D, Thormarth W, Otto C, et al. Early T-cell inactivation and apoptosis—Critical events for tolerance induction after allogeneic liver transplantation Transplantation Proceedings, 2001; 33: 256-258
22.杜家文,黄林平,周雷,贾振庚雷公藤多甙对大鼠原位肝移植急性排斥反应的影响。中国中西医结合外科杂志,2003;9:74-75
23.许赤 黄洁夫 陈规划等。胆汁细胞学及细胞因子基因表达诊断大鼠肝移植急性排斥反应的实验研究。中华普通外科杂志,2001;16:602-604