ABO血型不合肝移植临床创新和免疫学基础研究
发布时间：2019-07-07 19:33
【摘要】：目的 在供肝资源稀缺、无血型相合供肝的情况下,急诊ABO血型不合肝移植是挽救急性肝功能衰竭患者生命的有效手段。但传统的血型不合肝移植围手术期免疫抑制方案过于复杂,需要术前数周开始进行特殊的准备,不适用于急诊肝移植及危重症患者。因此,我们探索并尝试了一种简化的围手术期跨越血型屏障方案(以利妥昔单抗和免疫球蛋白为基础,不采用血浆置换、脾脏切除和肝脏灌注),观察其是否可以有效预防血型不合肝移植术后发生血型抗体介导的排斥反应；并通过与同期开展的血型相合肝移植治疗急性肝衰竭病例在生存率、并发症等各方面进行比较,探讨其在急诊ABO血型不合肝移植治疗急性肝衰竭中应用的可行性、有效性和安全性。 同时,我们还尝试通过动物实验建立ABO血型不合肝移植受者人PBMC-NOD/SCID小鼠嵌合体模型,以供体血型红细胞抗原致敏,检测小鼠血型抗体水平,以此来验证并阐明ABO血型不合肝移植术后可以产生针对供体血型抗原的特异性免疫耐受。并通过比较健康人群、血型不合患者术后早期和术后晚期的CD19+CD24hiCD38hi调节性B细胞水平,初步探讨了调节性B细胞在血型抗原特异性免疫耐受中可能发挥的作用。 材料与方法 第一部分： 急诊ABO血型不合肝移植治疗急性肝衰竭---利妥昔单抗联合免疫球蛋白简化方案的应用 1.病人资料：分析2010年1月-2013年7月,我中心(浙江大学医学院附属第一医院肝移植中心)共为101例急性肝衰竭患者进行急诊肝移植治疗(尸体供肝),血型相合组66例,血型不合组35例； 2.围手术期免疫抑制方案：血型不合组患者手术当天使用1剂利妥昔单抗(375mg/m2),免疫球蛋白手术当天开始使用并连续使用10天(0.4g/kg*10d)。两组术后基础免疫抑制方案包括巴利昔单抗、肾上腺糖皮质激素、他克莫司和吗替麦考酚酯； 3.术后抗感染治疗方案； 4.他克莫司血药浓度检测：酶联免疫吸附分析法； 5.血型不合组患者围手术期抗供体血型抗体(IgM)效价检测：盐水稀释法； 6.血型不合组患者围手术期CD20+B淋巴细胞检测：直接免疫荧光标记流式细胞术； 7.统计学分析：所有统计分析均使用SPSS17.0统计软件包进行统计学处理。连续变量以平均数和标准差描述,非连续变量以数字和百分比描述,以Students't检验或卡方检验比较组间差异。患者累积生存率与移植肝存活率使用Kaplan-Meier法统计,组间生存曲线比较使用log-rank检验。P0.05为差异有统计学意义。 第二部分： ABO血型不合肝移植诱导供体血型抗原特异性免疫耐受的实验研究 1.研究对象基本数据：2例接受ABO血型不合肝移植长期生存受者作为实验组,并根据其供受者血型设立相应阳性对照与阴性对照,共6例研究对象。分别编号为1A(血型不合受者AB→A),阳性对照1B(血型相合受者A→A),阴性对照1C(血型相合受者AB→AB);2A(血型不合受者AB→),阳性对照2B(血型相合受者O→O),阴性对照2C(血型相合受者AB→AB)。另入组18例健康志愿者和6例血型不合肝移植术后早期受者作为CD19+CD24hiCD38hi调节性B细胞检测的对照组； 2.主要试剂及其配方； 3.主要仪器与设备； 4.血型不合受者(1A、2A)外周血抗供体血型抗体(IgM)检测：盐水稀释法； 5.人外周血单个核细胞(Peripheral blood mononuclear cells, PBMCs)的分离：葡聚糖-泛影葡胺密度梯度离心法,(1A、1B、1C、2A、2B、2C); 6. NOD/SCID小鼠饲养； 7.构建人PBMC-NOD/SCID小鼠嵌合体模型及红细胞致敏：第一天,PBMCs腹腔注射小鼠；第五天,AB型红细胞免疫小鼠；第十四天,获取小鼠血清； 8. PBMC-NOD/SCID嵌合小鼠血型抗体(IgM)检测：酶联免疫吸附分析法； 9.健康志愿者、血型不合肝移植术后早期受者和术后长期存活受者(1A、2A)外周血CD19+CD24hiCD38hi调节性B细胞检测：直接免疫荧光标记流式细胞术； 10.统计学分析：用SPSS17.0软件计算各组数据的平均数和标准差。用Students't检验比较组间差异。p0.05为差异有统计学意义。 结果 第一部分 1.两组患者肝移植术前基本资料比较无统计学差异； 2.两组患者肝移植术后他克莫司血药浓度比较无统计学差异； 3.血型不合组供受者血型为AB→O最常见,占34%； 4.血型不合组患者术前抗供体血型抗体(IgM)效价1：4-1：1024(平均1：192),术后迅速下降,术后第1天和第7天分别为1：1-1：32(平均1：16)和1：4-1：128(平均1：28),并在术后数月维持较低水平。CD20+B淋巴细胞计数在术后迅速下降至小于1%并维持数月； 5.血型不合组共发生2例(5.7%)抗体介导的排斥反应(antibody-mediated rejection, AMR),术前抗体效价1：8-1：64,术后2周左右上升至1：256-1：512,分别表现为肝脏多发坏死和肝内胆管并发症,1例经血浆置换和免疫球蛋白治疗好转,1例治疗无效后于术后6月接受再次肝移植； 6.血型不合与血型相合组患者3年累积生存率分别为83%与86%(lok-rank, p=0.596),两组移植肝3年存活率分别为80%与86%(lok-rank,p=0.417),无统计学差异； 7.血型不合与血型相合组患者术后并发症(感染、排斥反应、胆道并发症、血管并发症、腹腔出血、肾功能不全、颅内出血、移植物抗宿主病等)发生率比较无统计学差异。 第二部分 1.血型不合受者抗供体血型抗体(IgM)效价：受者1A(血型不合AB→A)外周血抗B IgM长期维持较低水平甚至测不出0-1：4；受者2A(血型不合AB→O)外周血抗A和抗B IgM长期维持较低水平甚至测不出：抗A效价1：1-1：4,抗B效价0-1：4； 2. PBMC-NOD/SCID嵌合小鼠血型抗体(IgM) ELISA测定：受者1A(血型不合AB→A)嵌合小鼠接受AB型红细胞刺激后,测抗B OD值为0.09,明显低于阳性对照组(1B),与阴性对照组(1C)比较无明显差异；受者2A(血型不合AB→O)嵌合小鼠接受AB型红细胞刺激后,测抗A和抗B OD值分别为0.08和0.07,明显低于阳性对照组(2B),与阴性对照组(2C)比较无明显差异； 3.外周血CD19+CD24hiCD38hi调节性B细胞比例：两例血型不合受者1A和2A术后4年的外周血CD19+CD24hiCD38hi调节性B细胞比例分别为5.95%和4.51%,明显高于健康人群和血型不合肝移植患者术后短期水平。 结论 1.以利妥昔单抗联合免疫球蛋白为基础的简化方案用于急诊ABO血型不合肝移植治疗急性肝衰竭安全有效,较传统方案更具可行性,AMR发生率低,增加了肝衰竭患者进行肝移植的机会,是在供体短缺、无血型相合供肝的情况下,挽救危重症肝衰竭患者生命的合适选择； 2.ABO血型不合肝移植术后可以诱导产生针对供体血型抗原的特异性免疫耐受,CD19+CD24hiCD38hi调节性B细胞的免疫抑制功能可能参与了血型抗原特异性免疫耐受的发生。
文内图片：
图片说明：血型不合肝移植围手术期免疫抑制方案
[Abstract]:Purpose In the case of the lack of liver resources and the non-blood group-consistent supply of the liver, the emergency ABO blood group incompatibility is the effective hand to save the life of patients with acute liver failure However, the traditional blood group is too complex to be used in the perioperative period of liver transplantation. It is necessary to start a special preparation for several weeks before operation. It is not suitable for emergency liver transplantation and critical illness. As a result, we have explored and attempted a simplified perioperative cross-type barrier regimen (for the basis of brentuximab and immunoglobulin, without plasma replacement, spleen resection, and liver perfusion ) To observe whether it can effectively prevent blood group-type antibody-mediated rejection after liver transplantation; and compare the patients with acute liver failure with blood group-consistent liver transplantation in the same period to compare the survival rate, the complication and so on To explore the feasibility, effectiveness and safety of its application in the treatment of acute liver failure in the treatment of acute liver failure Sex. At the same time, we also tried to establish a human PBMC-NOD/ SCID mouse chimera model by animal experiments to test the blood group resistance of the mice with the donor blood group red blood cell antigen. The level of the body is used to verify and clarify that the ABO blood group does not have specific immunity to the donor blood group antigen after the liver transplantation. By comparing the level of CD19 + CD24hiCD38hi regulatory B cells in the early and late stages of the post-operative and post-operation of the patient, the potential of the regulatory B cells in the specific immune tolerance of the blood group was discussed. The effect of the present invention. material and party The first part of the method: the combination of emergency ABO blood group and non-liver transplantation in the treatment of acute hepatic failure--rituximab Application of the Simplified Scheme of the Globulin 1. Patient Data: Analysis 2010 From January to July,2013, a total of 101 patients with acute liver failure were treated with emergency liver transplantation (cadavers for liver) and blood type combination group 6 for 101 patients with acute liver failure (liver transplantation center of the First Affiliated Hospital of Zhejiang University Medical College). in 6 case,35 of that group of the blood group were not group; 2. Immunosuppression in the perioperative period:1 dose of rituximab (375 mg/ m2) was used on the day of operation of the patients with the blood group and 10 days were used for continuous use on the same day of the operation of the immunoglobulin. (0.4 g/ kg * 10 d). The two-group post-operative basic immunosuppression regimen consisted of Baliximab, Adrenocortical, Cystatin, Tacrolimus, and? Esters;3. Postoperative anti-infective therapy;4. Serum concentration test: enzyme-linked immunosorbent assay;5. Perioperative anti-donor for patients with blood group incompatibility group Blood group antibody (IgM) titer detection: saline dilution method;6. Perioperative CD20 + for patients with blood group incompatibility group. B-lymphocyte detection: Direct immunofluorescence labeling flow cytometry;7. Statistical analysis: all statistical analysis Statistical processing was performed using the SPSS17.0 statistical software package. The continuous variables are described by mean and standard deviation, and the non-continuous variables are described in numbers and percentages, Difference between groups between the study 't test or the chi-square test. The cumulative survival rate of the patient was consistent with the use of the Kaplan-Meier method for the survival of the transplanted liver Statistics, vs. survival curves between groups, lo g-ran k-test. P0.05 is of statistical significance for the difference. Part 2: AB An experimental study on the specific immune tolerance of donor blood group antigen induced by O-type non-liver transplantation (1). This data:2 cases of long-term survival recipients of ABO-incompatible liver transplantation were treated as experimental group, and The corresponding positive control and negative control were set up according to the blood group of the recipient,6 subjects were studied. The number of the positive control and the positive control 1B (A-A of the blood group-matched recipient) and the negative control 1C (AB-AB of the blood group-matched recipient) were the same as that of the negative control group. (blood type not in recipient AB), positive control 2B (blood O (O), negative control 2C (B-AB, B-AB),18 healthy volunteers and 6 patients with non-liver transplantation. CD19+CD24hi CD38hi Regulatory B control group for cell detection;2 . Main reagents and its formulation;3. Main instruments and equipment;4. Blood Detection of anti-donor blood group antibody (IgM) in peripheral blood of non-recipient (1A, 2A): saline dilution method;5. Peripheral blood mononuclear cells (PBMCs) Dislocation: Dextran-Pan-Famine Density Gradient Heart method (1A, 1B, 1C, 2A, 2B, 2C);6. NOD/ SCID mouse feeding;7. Construction of human PBMC-NOD/ SCID mouse chimera model and red blood cell sensitization: One day, PBMCs were intraperitoneally injected into mice; the fifth day, the AB-type red cell immune mice; and the fourteenth day to obtain the mouse serum ;8. PBMC-NOD/ SCID chimeric mouse blood group antibody (IgM) detection: enzyme-linked immunosorbent assay;9. healthy volunteers, early and post-operative long-term survival recipients (1A, 2A) Peripheral blood CD19 + CD24hiCD38hi regulatory B cell detection: directly Keywords Phytophthora capsici; Flow cytometry;10. Statistical analysis Analysis: use SPSS17.0 software to calculate each group level of data mean and standard deviation. Comparison groups were tested with the Student 't The difference was statistically significant for the difference. p0.05 was the difference. The first part 1. There was no statistical difference between the two groups before and after the liver transplantation. 2. There was no statistical difference in the plasma concentration of tacrolimus after liver transplantation in the two groups.3. The blood group of the blood group was the most common type of AB and O, accounting for 34%.4. The anti-donor blood group antibody (IgM) titer was 1:4-1:1024 (average 1:192) before the operation of the group. 1:1-1:32 (mean 1:16) and 1 on Day 1 and Day 7, respectively. 4-1:128 (mean 1:28) and remained low for several months. CD20 + B lymphocyte count decreased rapidly to less than 1% and maintained for several months after surgery;5.2 (5.7%) antibody-mediated rejection (antibo The pre-operative antibody titer was 1:8-1:64, and then increased to 1:256-1 at 2 weeks after the operation. 512, with liver multiple necrosis and intra-hepatic bile duct complications,1 case of plasma replacement and immunoglobulin treatment, and 1 case of ineffective treatment. The 3-year cumulative survival rate of the group was 83% and 86%, respectively. Lok-rank, p = 0.596), the 3-year survival rate of the two groups was 80% and 86% (lok-rank, p = 0.417), respectively. No statistical difference;7. Blood type not consistent with blood group group-patient operation The incidence of post-complications (infection, rejection, biliary complications, vascular complications, abdominal hemorrhage, renal insufficiency, intracranial hemorrhage, graft versus host disease, etc.) was not statistically different. The second part 1. The anti-donor blood group antibody (IgM) titer in the blood group: the anti-B IgM in the peripheral blood of the recipient 1A (the blood group is not AB-A) is maintained at a low level for a long time, 0-1:4 can not be detected; the anti-A and anti-B IgM in the peripheral blood of the recipient 2A (the blood group is not AB-O) are long-term The maintenance was low or even impossible: the anti-A titer of 1:1-1:4, the anti-B titer of 0-1:4;2. PBMC-NOD/ SCID chimeric mouse blood group antibody (IgM) ELISA assay: the recipient The anti-B OD value was 0.09, which was significantly lower than that of the positive control group (1B), and there was no significant difference with the negative control group (1C). O) After the chimeric mice received AB-type red blood cell stimulation, the anti-A and anti-B OD values were 0, respectively. .08 and 0.07, significantly lower than the positive control group (2B), no significant difference compared to the negative control group (2C);3. Peripheral blood CD19 + CD24hiCD38hi regulatory B cell ratio: two blood group-mismatched recipients 1A and 2 A鏈，
本文编号：2511387
文内图片：
图片说明：血型不合肝移植围手术期免疫抑制方案
[Abstract]:Purpose In the case of the lack of liver resources and the non-blood group-consistent supply of the liver, the emergency ABO blood group incompatibility is the effective hand to save the life of patients with acute liver failure However, the traditional blood group is too complex to be used in the perioperative period of liver transplantation. It is necessary to start a special preparation for several weeks before operation. It is not suitable for emergency liver transplantation and critical illness. As a result, we have explored and attempted a simplified perioperative cross-type barrier regimen (for the basis of brentuximab and immunoglobulin, without plasma replacement, spleen resection, and liver perfusion ) To observe whether it can effectively prevent blood group-type antibody-mediated rejection after liver transplantation; and compare the patients with acute liver failure with blood group-consistent liver transplantation in the same period to compare the survival rate, the complication and so on To explore the feasibility, effectiveness and safety of its application in the treatment of acute liver failure in the treatment of acute liver failure Sex. At the same time, we also tried to establish a human PBMC-NOD/ SCID mouse chimera model by animal experiments to test the blood group resistance of the mice with the donor blood group red blood cell antigen. The level of the body is used to verify and clarify that the ABO blood group does not have specific immunity to the donor blood group antigen after the liver transplantation. By comparing the level of CD19 + CD24hiCD38hi regulatory B cells in the early and late stages of the post-operative and post-operation of the patient, the potential of the regulatory B cells in the specific immune tolerance of the blood group was discussed. The effect of the present invention. material and party The first part of the method: the combination of emergency ABO blood group and non-liver transplantation in the treatment of acute hepatic failure--rituximab Application of the Simplified Scheme of the Globulin 1. Patient Data: Analysis 2010 From January to July,2013, a total of 101 patients with acute liver failure were treated with emergency liver transplantation (cadavers for liver) and blood type combination group 6 for 101 patients with acute liver failure (liver transplantation center of the First Affiliated Hospital of Zhejiang University Medical College). in 6 case,35 of that group of the blood group were not group; 2. Immunosuppression in the perioperative period:1 dose of rituximab (375 mg/ m2) was used on the day of operation of the patients with the blood group and 10 days were used for continuous use on the same day of the operation of the immunoglobulin. (0.4 g/ kg * 10 d). The two-group post-operative basic immunosuppression regimen consisted of Baliximab, Adrenocortical, Cystatin, Tacrolimus, and? Esters;3. Postoperative anti-infective therapy;4. Serum concentration test: enzyme-linked immunosorbent assay;5. Perioperative anti-donor for patients with blood group incompatibility group Blood group antibody (IgM) titer detection: saline dilution method;6. Perioperative CD20 + for patients with blood group incompatibility group. B-lymphocyte detection: Direct immunofluorescence labeling flow cytometry;7. Statistical analysis: all statistical analysis Statistical processing was performed using the SPSS17.0 statistical software package. The continuous variables are described by mean and standard deviation, and the non-continuous variables are described in numbers and percentages, Difference between groups between the study 't test or the chi-square test. The cumulative survival rate of the patient was consistent with the use of the Kaplan-Meier method for the survival of the transplanted liver Statistics, vs. survival curves between groups, lo g-ran k-test. P0.05 is of statistical significance for the difference. Part 2: AB An experimental study on the specific immune tolerance of donor blood group antigen induced by O-type non-liver transplantation (1). This data:2 cases of long-term survival recipients of ABO-incompatible liver transplantation were treated as experimental group, and The corresponding positive control and negative control were set up according to the blood group of the recipient,6 subjects were studied. The number of the positive control and the positive control 1B (A-A of the blood group-matched recipient) and the negative control 1C (AB-AB of the blood group-matched recipient) were the same as that of the negative control group. (blood type not in recipient AB), positive control 2B (blood O (O), negative control 2C (B-AB, B-AB),18 healthy volunteers and 6 patients with non-liver transplantation. CD19+CD24hi CD38hi Regulatory B control group for cell detection;2 . Main reagents and its formulation;3. Main instruments and equipment;4. Blood Detection of anti-donor blood group antibody (IgM) in peripheral blood of non-recipient (1A, 2A): saline dilution method;5. Peripheral blood mononuclear cells (PBMCs) Dislocation: Dextran-Pan-Famine Density Gradient Heart method (1A, 1B, 1C, 2A, 2B, 2C);6. NOD/ SCID mouse feeding;7. Construction of human PBMC-NOD/ SCID mouse chimera model and red blood cell sensitization: One day, PBMCs were intraperitoneally injected into mice; the fifth day, the AB-type red cell immune mice; and the fourteenth day to obtain the mouse serum ;8. PBMC-NOD/ SCID chimeric mouse blood group antibody (IgM) detection: enzyme-linked immunosorbent assay;9. healthy volunteers, early and post-operative long-term survival recipients (1A, 2A) Peripheral blood CD19 + CD24hiCD38hi regulatory B cell detection: directly Keywords Phytophthora capsici; Flow cytometry;10. Statistical analysis Analysis: use SPSS17.0 software to calculate each group level of data mean and standard deviation. Comparison groups were tested with the Student 't The difference was statistically significant for the difference. p0.05 was the difference. The first part 1. There was no statistical difference between the two groups before and after the liver transplantation. 2. There was no statistical difference in the plasma concentration of tacrolimus after liver transplantation in the two groups.3. The blood group of the blood group was the most common type of AB and O, accounting for 34%.4. The anti-donor blood group antibody (IgM) titer was 1:4-1:1024 (average 1:192) before the operation of the group. 1:1-1:32 (mean 1:16) and 1 on Day 1 and Day 7, respectively. 4-1:128 (mean 1:28) and remained low for several months. CD20 + B lymphocyte count decreased rapidly to less than 1% and maintained for several months after surgery;5.2 (5.7%) antibody-mediated rejection (antibo The pre-operative antibody titer was 1:8-1:64, and then increased to 1:256-1 at 2 weeks after the operation. 512, with liver multiple necrosis and intra-hepatic bile duct complications,1 case of plasma replacement and immunoglobulin treatment, and 1 case of ineffective treatment. The 3-year cumulative survival rate of the group was 83% and 86%, respectively. Lok-rank, p = 0.596), the 3-year survival rate of the two groups was 80% and 86% (lok-rank, p = 0.417), respectively. No statistical difference;7. Blood type not consistent with blood group group-patient operation The incidence of post-complications (infection, rejection, biliary complications, vascular complications, abdominal hemorrhage, renal insufficiency, intracranial hemorrhage, graft versus host disease, etc.) was not statistically different. The second part 1. The anti-donor blood group antibody (IgM) titer in the blood group: the anti-B IgM in the peripheral blood of the recipient 1A (the blood group is not AB-A) is maintained at a low level for a long time, 0-1:4 can not be detected; the anti-A and anti-B IgM in the peripheral blood of the recipient 2A (the blood group is not AB-O) are long-term The maintenance was low or even impossible: the anti-A titer of 1:1-1:4, the anti-B titer of 0-1:4;2. PBMC-NOD/ SCID chimeric mouse blood group antibody (IgM) ELISA assay: the recipient The anti-B OD value was 0.09, which was significantly lower than that of the positive control group (1B), and there was no significant difference with the negative control group (1C). O) After the chimeric mice received AB-type red blood cell stimulation, the anti-A and anti-B OD values were 0, respectively. .08 and 0.07, significantly lower than the positive control group (2B), no significant difference compared to the negative control group (2C);3. Peripheral blood CD19 + CD24hiCD38hi regulatory B cell ratio: two blood group-mismatched recipients 1A and 2 A鏈，
本文编号：2511387
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