【摘要】：目的 在供肝資源稀缺、無血型相合供肝的情況下,急診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。但傳統(tǒng)的血型不合肝移植圍手術(shù)期免疫抑制方案過于復(fù)雜,需要術(shù)前數(shù)周開始進(jìn)行特殊的準(zhǔn)備,不適用于急診肝移植及危重癥患者。因此,我們探索并嘗試了一種簡化的圍手術(shù)期跨越血型屏障方案(以利妥昔單抗和免疫球蛋白為基礎(chǔ),不采用血漿置換、脾臟切除和肝臟灌注),觀察其是否可以有效預(yù)防血型不合肝移植術(shù)后發(fā)生血型抗體介導(dǎo)的排斥反應(yīng)；并通過與同期開展的血型相合肝移植治療急性肝衰竭病例在生存率、并發(fā)癥等各方面進(jìn)行比較,探討其在急診ABO血型不合肝移植治療急性肝衰竭中應(yīng)用的可行性、有效性和安全性。 同時(shí),我們還嘗試通過動(dòng)物實(shí)驗(yàn)建立ABO血型不合肝移植受者人PBMC-NOD/SCID小鼠嵌合體模型,以供體血型紅細(xì)胞抗原致敏,檢測小鼠血型抗體水平,以此來驗(yàn)證并闡明ABO血型不合肝移植術(shù)后可以產(chǎn)生針對供體血型抗原的特異性免疫耐受。并通過比較健康人群、血型不合患者術(shù)后早期和術(shù)后晚期的CD19+CD24hiCD38hi調(diào)節(jié)性B細(xì)胞水平,初步探討了調(diào)節(jié)性B細(xì)胞在血型抗原特異性免疫耐受中可能發(fā)揮的作用。 材料與方法 第一部分： 急診ABO血型不合肝移植治療急性肝衰竭---利妥昔單抗聯(lián)合免疫球蛋白簡化方案的應(yīng)用 1.病人資料：分析2010年1月-2013年7月,我中心(浙江大學(xué)醫(yī)學(xué)院附屬第一醫(yī)院肝移植中心)共為101例急性肝衰竭患者進(jìn)行急診肝移植治療(尸體供肝),血型相合組66例,血型不合組35例； 2.圍手術(shù)期免疫抑制方案：血型不合組患者手術(shù)當(dāng)天使用1劑利妥昔單抗(375mg/m2),免疫球蛋白手術(shù)當(dāng)天開始使用并連續(xù)使用10天(0.4g/kg*10d)。兩組術(shù)后基礎(chǔ)免疫抑制方案包括巴利昔單抗、腎上腺糖皮質(zhì)激素、他克莫司和嗎替麥考酚酯； 3.術(shù)后抗感染治療方案； 4.他克莫司血藥濃度檢測：酶聯(lián)免疫吸附分析法； 5.血型不合組患者圍手術(shù)期抗供體血型抗體(IgM)效價(jià)檢測：鹽水稀釋法； 6.血型不合組患者圍手術(shù)期CD20+B淋巴細(xì)胞檢測：直接免疫熒光標(biāo)記流式細(xì)胞術(shù)； 7.統(tǒng)計(jì)學(xué)分析：所有統(tǒng)計(jì)分析均使用SPSS17.0統(tǒng)計(jì)軟件包進(jìn)行統(tǒng)計(jì)學(xué)處理。連續(xù)變量以平均數(shù)和標(biāo)準(zhǔn)差描述,非連續(xù)變量以數(shù)字和百分比描述,以Students't檢驗(yàn)或卡方檢驗(yàn)比較組間差異�；颊呃鄯e生存率與移植肝存活率使用Kaplan-Meier法統(tǒng)計(jì),組間生存曲線比較使用log-rank檢驗(yàn)。P0.05為差異有統(tǒng)計(jì)學(xué)意義。 第二部分： ABO血型不合肝移植誘導(dǎo)供體血型抗原特異性免疫耐受的實(shí)驗(yàn)研究 1.研究對象基本數(shù)據(jù)：2例接受ABO血型不合肝移植長期生存受者作為實(shí)驗(yàn)組,并根據(jù)其供受者血型設(shè)立相應(yīng)陽性對照與陰性對照,共6例研究對象。分別編號(hào)為1A(血型不合受者AB→A),陽性對照1B(血型相合受者A→A),陰性對照1C(血型相合受者AB→AB);2A(血型不合受者AB→),陽性對照2B(血型相合受者O→O),陰性對照2C(血型相合受者AB→AB)。另入組18例健康志愿者和6例血型不合肝移植術(shù)后早期受者作為CD19+CD24hiCD38hi調(diào)節(jié)性B細(xì)胞檢測的對照組； 2.主要試劑及其配方； 3.主要儀器與設(shè)備； 4.血型不合受者(1A、2A)外周血抗供體血型抗體(IgM)檢測：鹽水稀釋法； 5.人外周血單個(gè)核細(xì)胞(Peripheral blood mononuclear cells, PBMCs)的分離：葡聚糖-泛影葡胺密度梯度離心法,(1A、1B、1C、2A、2B、2C); 6. NOD/SCID小鼠飼養(yǎng)； 7.構(gòu)建人PBMC-NOD/SCID小鼠嵌合體模型及紅細(xì)胞致敏：第一天,PBMCs腹腔注射小鼠；第五天,AB型紅細(xì)胞免疫小鼠；第十四天,獲取小鼠血清； 8. PBMC-NOD/SCID嵌合小鼠血型抗體(IgM)檢測：酶聯(lián)免疫吸附分析法； 9.健康志愿者、血型不合肝移植術(shù)后早期受者和術(shù)后長期存活受者(1A、2A)外周血CD19+CD24hiCD38hi調(diào)節(jié)性B細(xì)胞檢測：直接免疫熒光標(biāo)記流式細(xì)胞術(shù)； 10.統(tǒng)計(jì)學(xué)分析：用SPSS17.0軟件計(jì)算各組數(shù)據(jù)的平均數(shù)和標(biāo)準(zhǔn)差。用Students't檢驗(yàn)比較組間差異。p0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果 第一部分 1.兩組患者肝移植術(shù)前基本資料比較無統(tǒng)計(jì)學(xué)差異； 2.兩組患者肝移植術(shù)后他克莫司血藥濃度比較無統(tǒng)計(jì)學(xué)差異； 3.血型不合組供受者血型為AB→O最常見,占34%； 4.血型不合組患者術(shù)前抗供體血型抗體(IgM)效價(jià)1：4-1：1024(平均1：192),術(shù)后迅速下降,術(shù)后第1天和第7天分別為1：1-1：32(平均1：16)和1：4-1：128(平均1：28),并在術(shù)后數(shù)月維持較低水平。CD20+B淋巴細(xì)胞計(jì)數(shù)在術(shù)后迅速下降至小于1%并維持?jǐn)?shù)月； 5.血型不合組共發(fā)生2例(5.7%)抗體介導(dǎo)的排斥反應(yīng)(antibody-mediated rejection, AMR),術(shù)前抗體效價(jià)1：8-1：64,術(shù)后2周左右上升至1：256-1：512,分別表現(xiàn)為肝臟多發(fā)壞死和肝內(nèi)膽管并發(fā)癥,1例經(jīng)血漿置換和免疫球蛋白治療好轉(zhuǎn),1例治療無效后于術(shù)后6月接受再次肝移植； 6.血型不合與血型相合組患者3年累積生存率分別為83%與86%(lok-rank, p=0.596),兩組移植肝3年存活率分別為80%與86%(lok-rank,p=0.417),無統(tǒng)計(jì)學(xué)差異； 7.血型不合與血型相合組患者術(shù)后并發(fā)癥(感染、排斥反應(yīng)、膽道并發(fā)癥、血管并發(fā)癥、腹腔出血、腎功能不全、顱內(nèi)出血、移植物抗宿主病等)發(fā)生率比較無統(tǒng)計(jì)學(xué)差異。 第二部分 1.血型不合受者抗供體血型抗體(IgM)效價(jià)：受者1A(血型不合AB→A)外周血抗B IgM長期維持較低水平甚至測不出0-1：4；受者2A(血型不合AB→O)外周血抗A和抗B IgM長期維持較低水平甚至測不出：抗A效價(jià)1：1-1：4,抗B效價(jià)0-1：4； 2. PBMC-NOD/SCID嵌合小鼠血型抗體(IgM) ELISA測定：受者1A(血型不合AB→A)嵌合小鼠接受AB型紅細(xì)胞刺激后,測抗B OD值為0.09,明顯低于陽性對照組(1B),與陰性對照組(1C)比較無明顯差異；受者2A(血型不合AB→O)嵌合小鼠接受AB型紅細(xì)胞刺激后,測抗A和抗B OD值分別為0.08和0.07,明顯低于陽性對照組(2B),與陰性對照組(2C)比較無明顯差異； 3.外周血CD19+CD24hiCD38hi調(diào)節(jié)性B細(xì)胞比例：兩例血型不合受者1A和2A術(shù)后4年的外周血CD19+CD24hiCD38hi調(diào)節(jié)性B細(xì)胞比例分別為5.95%和4.51%,明顯高于健康人群和血型不合肝移植患者術(shù)后短期水平。 結(jié)論 1.以利妥昔單抗聯(lián)合免疫球蛋白為基礎(chǔ)的簡化方案用于急診ABO血型不合肝移植治療急性肝衰竭安全有效,較傳統(tǒng)方案更具可行性,AMR發(fā)生率低,增加了肝衰竭患者進(jìn)行肝移植的機(jī)會(huì),是在供體短缺、無血型相合供肝的情況下,挽救危重癥肝衰竭患者生命的合適選擇； 2.ABO血型不合肝移植術(shù)后可以誘導(dǎo)產(chǎn)生針對供體血型抗原的特異性免疫耐受,CD19+CD24hiCD38hi調(diào)節(jié)性B細(xì)胞的免疫抑制功能可能參與了血型抗原特異性免疫耐受的發(fā)生。
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圖片說明：血型不合肝移植圍手術(shù)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，

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