重型β-地中海贫血患儿异基因造血干细胞移植后的免疫恢复

发布时间：2018-05-23 08:21

  本文选题：重型地中海贫血 + 儿童　； 参考：《南方医科大学》2012年硕士论文

【摘要】：异基因造血干细胞移植(allogenic hematopoietic stem cell transplantation, allo-HSCT)仍然是治愈遗传性血红蛋白病如重型β地中海贫血(Thalassemia Major,TM)患儿的唯一手段,但其临床应用受到以下因素的限制,如预处理的相关毒性,供者细胞对受者细胞抗原的免疫反应引起的移植物抗宿主病(graft versus host disease,GVHD)及延迟的免疫重建或免疫缺陷导致感染,复发及偶尔的排斥等等。造血干细胞移植后的免疫重建在成人已有较为深入的研究,但对于儿童研究很少。经过大剂量的化疗后,受者免疫细胞的大量减少,但受者浆细胞往往可能持续存在至移植后数月到数年。移植前许多因素也可影响移植后的免疫状态及其恢复,如年龄,干细胞来源,组织相容性抗原(histocompatibility antigen,HLA)相合的程度等等。移植后的急性GVHD,巨细胞病毒感染(cytomegalovirus,CMV),免疫抑制剂应用、抗菌素,免疫球蛋白的应用及供者的淋巴细胞输注(donor lymphocyte infusions,DLI)等均可影响移植后免疫的恢复。提高移植后的免疫重建是决定其临床治疗效果的重要因素。我们研究在TM的儿童在清髓性的allo-HSCT后2年内的T细胞亚群及体液免疫的恢复及其相关影响因素。 本课题旨在探讨TM儿童在异基因外周血造血干细胞移植后2年内的免疫恢复情况,评估CMV感染对T细胞亚群的影响,评估不同的供者年龄和受者年龄对体液免疫的恢复的影响,从而评估免疫细胞在造血干细胞移植中可能的作用,期望通过该研究为移植后免疫重建的快速恢复,减少移植后感染死亡提供指导意义。 我们回顾性的分析在南方医科大学南方医院儿科层流病房进行外周血造血干细胞移植的地贫患儿免疫恢复情况。第一部分移植后T细胞亚群的研究对象和方法第一节：移植后半年的T细胞亚群的研究对象和方法 1.1)对于半年内的T细胞亚群的研究对象,入选研究范围的标准是：2009年3月至2011年6月内所有在我中心的地中海贫血患儿行非亲缘外周血造血干细胞移植的对象,在移植后半年的随访期间内未发生死亡或排斥,半年时完成T细胞亚群8项(包括CD3+、CD4+、CD8+、NK、CD4+CD45RA+、CD4+CD29+、 CD8+CD28+、CD8+CD28-这八项)和血常规的评估,移植后截止于2012年1月仍无病存活的病例。共有40例患儿,其中CMV感染有20例。评估其免疫恢复情况和CMV对其的影响。 1.2)预处理方案 患儿均给予清髓性的预处理方案,包括环磷酰胺(cyclosphosphamide,Cy)100-120mg/kg,d-10至d-9分两天静脉滴注,氟达拉滨(fludarabine,Flu),200mg/m2,-6至-2天分5天静脉滴注,塞替哌(thiotepa,TT),10mg/kg,-5天1天分两次静脉滴注,白消安(buslfan, BU),9.6-17.6mg/kg,分3-4天静脉注射(-8至-6天)。所有患儿给予环孢素A(cyclosporine A),霉酚酸酯(mycophenolate mofetil, MMF)和ATG(-3—-1天)预防GVHD。短疗程的甲氨蝶呤(methotrexate、MTX)在移植后第1,3,6天分别给予15,10and10mg/m2治疗。肝素钠预防肝静脉阻塞综合症(hepatic veno-occlusive disease,VOD)。 1.3)移植前及移植后检测及预防巨细胞病毒定量 移植前常规进行更昔洛韦(10mg/kg)治疗,疗程8-10天。在allo-HSCT后中性粒细胞数0.5×109/L开始每周监测CMV-DNA复制数,直到100天,移植后1月开始,常规进行每月7天的或每2月10天的预防性更昔洛韦治疗。CMV感染定义为在外周血中可监测到病毒的复制数,即500/ul。更昔洛韦的剂量或改用磷甲酸钠会依据患儿的肾功能和外周血中性粒细胞数进行调整,如外周血中性粒细胞数连续2天小于0.5×109/L停止更昔洛韦。 1.4)植入的标准：移植后28天,第二个月,适当时甚至更长时间的检测植入证据。 间接指标：移植后临床存活21天；各系统造血恢复正常,包括红系,粒系,淋巴系,巨核系等,中性粒细胞数在0.5×109/L,血小板数维持在20×109/L；临床上出现GVHD。肿大的肝脾回缩至正常大小；乳酸脱氢酶降至正常水平。 直接指标：供受者性别不同采用免疫荧光原位杂交法(fluorescence in situ hybridization,FISH)检测性染色体的转变,供受者性别相同采用短串联重复序列(variable number of tandem repeat, VNTR)(HLA抗原转变成供者型,或供者基因占受者的比例)。 1.5)统计分析 应用SPSS13.0软件进行统计分析,移植后巨细胞病毒感染与否与半年内的T细胞亚群的恢复的影响。先采用单个样本T检验对各个T细胞亚群数量与设定的参考值进行比较,然后将患儿按有无CMV感染分组,采用独立two-samples t Test检验方法比较。组间的基础指标的分类变量采用卡方比较,等级资料可采用独立样本的非参数检验(Mann-Whitney),定量变量采用2个样本的t检验比较。采用线性相关分析分析T细胞亚群CD8+细胞之间的相关性。P值小于0.05为有统计学意义。第二节移植后1年的T细胞亚群的研究对象和方法 对于1年内的T细胞亚群的研究对象,入选研究范围的标准是：2009年3月至2011年1月内在我中心的地中海贫血患儿行非亲缘外周血造血干细胞移植的对象,在移植后1年以上的随访期间内未发生死亡或排斥,移植后截止于2012年1月仍无病存活的病例,移植后1年完成T细胞亚群4项(至少包括CD3+、CD4+、CD8+、NK这四项)及血常规的评估。病人共有38例。对其病毒的检测与植入检测同上。应用SPSS13.0软件进行统计分析,采用单个样本t检验对各个T细胞亚群数量与设定的参考值进行比较。P值小于0.05为有统计学意义。第二部分移植后体液免疫的研究对象和方法第一节移植后半年的体液免疫的研究对象和方和 对于半年内的体液免疫的研究对象,入选研究范围的标准是：2010年3月至2011年6月内所有在我中心的地中海贫血患儿行外周血造血干细胞移植的对象,在移植后半年的随访期间内未发生死亡或排斥,半年时完成体液免疫的免疫评估,移植后截止于2012年1月仍无病存活的病例,病人共有35例。对其病毒的检测与植入证据检测同上。应用SPSS13.0软件进行统计分析,先采用单个样本t检验对各个年龄段的体液免疫与设定的参考值进行比较,患儿年龄,供者年龄与移植后半年内体液免疫的恢复的影响,采用双变量的Pearson相关分别分析。P值小于0.05为有统计学意义第二节移植后1年的体液免疫的研究对象和方法 对于1年内的体液免疫的研究对象,入选研究范围的标准是：2009年3月至2011年1月内所有在我中心的地中海贫血患儿行外周血造血干细胞移植的对象,在移植后1年的随访期间内未发生死亡或排斥,移植后1年完成体液免疫评估,移植后截止于2012年1月仍无病存活的病例。病人共有35例。对其病毒的检测与植入证据检测同上。应用SPSS13.0软件进行统计分析,采用单个样本t检验对各个年龄段的体液免疫与设定的参考值进行比较。P值小于0.05为有统计学意义。 第一部分.T细胞亚群的情况 第一节移植后半年的T细胞亚群的恢复 (1)半年的T细胞亚群内CD3+细胞的均值1.46×109/L,NK细胞均值0.33×109/L,CD4+细胞均值0.25×109/L,CD8+细胞均值1.14×109/L,其中CD4+细胞低于设定参考值0.5×109/L水平(t=-6.874,P0.001) (2)CMV感染组的半年CD3+、CD8+、CD8+CD28-的细胞数分别为1.87±1.47×109/L、1.51±1.27×109/L、1.20±1.13×109/L,明显高于巨细胞病毒未感染组,其细胞数分别是1.05±0.60×109/L、0.78±0.50×109/L、0.51±0.41×109/L(分别为t=-2.315,P=0.029；t=-2.414,P=0.024；t=-2.540,P=0.018；),其两组的CD8+CD28-比率分别是46.17%±19.68%,33.32%±12.50%(t=-2.465,P=0.018)。 (3)在CMV感染组内,CD8+细胞与CD8+CD28-细胞呈线性正相关(r=0.980,P0.001),而与CD8+CD28+T细胞数负相关,但无直线线性相关(r=0.585,P=0.007)。 第二节移植后1年以上的T细胞亚群的恢复 地中海贫血患儿在造血干细胞移植后1年以上T细胞亚群内CD3+细胞的均值2.22×109/L,NK细胞均值0.27×109/L,CD4+细胞均值0.63×109/L。 第二部分.体液免疫的情况 第一节移植后半年的体液免疫恢复 (1)地中海贫血患儿移植后半年的免疫球蛋白水平,3-6岁的IgA、IgM, IgG的平均值和标准差分别是0.40±0.22g/L、0.71±0.35g/L、7.17±2.30g/L,其中IgA、IgM恢复低于正常同龄儿童水平(分别t=-8.467,P0.001；t=-2.305,P=0.042),7岁的IgA、IgM,IgG的平均值和标准差分别是0.81±0.71g/L、1.02±0.40g/L、8.13±3.28g/L,均低于正常同龄儿童水平(分别t=-2.305,P=0.042：t=-6.161,P0.001；t=-2.733,P=0.019)。 (2)移植后的抗体水平和供者年龄无关,但患儿年龄分别与IgA、C3,C4的水平成正相关关系,相关系数分别是0.667、0.511、0.341,P值分别0.001、0.002、0.045。 (3)2例脾切除后的患儿在造血干细胞移植后半年内的IgA、IgM, IgG的平均值分别1.58g/L,0.76g/L,14.25g/L第二节移植后1年以上的体液免疫恢复 1年后地贫患儿的3-6岁的IgA、IgG, IgM的均值和标准差分别是0.83±0.40g/L、9.81±2.92g/L、0.86±0.27g/L,7岁的IgA, IgG, IgM的均值和标准差分别是1.35±0.74g/L、11.45±4.85g/L、1.05±0.53g/L。两年龄段IgM低于正常同龄儿童(t分别为-8.982,-5.380,P均为0.001)。 (1)TM儿童allo-HSCT后半年时NK细胞数比率及CD4+细胞数和比率不受CMV感染与否的影响,除了CD4+细胞数低于0.5×109/L外,其余均恢复正常。但一年后的患儿的T细胞各亚群处于正常参考值水平。 (2)TM儿童allo-HSCT后的巨细胞病毒感染会导致高的CD8+细胞中的CD8+CD28-细胞的单克隆扩增,从而改变T细胞亚群的之间的转化率,且CD8+细胞升高比率与CD8+CD28-比率呈直线线性相关。 (3)对于TM儿童allo-HSCT后半年时免疫球蛋白水平均低于正常同龄儿童,除了3-6岁的IgG水平外；同时患儿年龄与IgA,C3,C4的恢复成正相关。 (4)对于TM儿童allo-HSCT后1年时免疫球蛋白水平均恢复至正常同龄儿童,除了IgM水平低于正常同龄儿童的外。 (5)脾切除的TM患儿allo-HSCT后半年内有低的IgM水平和更高的IgG和IgA水平的趋势。
[Abstract]:Allogeneic hematopoietic stem cell transplantation (allogenic hematopoietic stem cell transplantation, allo-HSCT) is still the only cure for hereditary hemoglobinemia, such as severe beta thalassemia (Thalassemia Major, TM), but its clinical application is limited by the following factors, such as the related toxicity of pretreated, donor cells to the recipient. The immune response to cellular antigens caused by graft versus host disease (GVHD) and delayed immune reconstruction or immunodeficiency caused infection, recurrence and occasional rejection, etc.. Immune reconstruction after hematopoietic stem cell transplantation has been studied in adults more deeply, but few studies have been made in children. After large doses of chemotherapy After that, the immune cells of the recipient are greatly reduced, but the recipient plasma cells tend to persist until several months to several years after transplantation. Many factors before transplantation also affect the immune state and its recovery after transplantation, such as age, stem cell origin, the degree of compatibility of histocompatibility antigen, HLA, and so on. The acute GV after transplantation. HD, cytomegalovirus infection (cytomegalovirus, CMV), immunosuppressant application, antibiotics, immunoglobulin application and donor lymphocyte infusion (donor lymphocyte infusions, DLI) can affect the recovery of post transplant immunity. Improving the immune reconstruction after transplantation is an important factor determining the effect of its clinical treatment. We study in TM The T cell subsets and humoral immunity of children in the 2 years after myeloablative allo-HSCT were restored.
The purpose of this study is to investigate the immune recovery of TM children within 2 years after allogeneic peripheral blood stem cell transplantation, to assess the effect of CMV infection on T cell subsets, to assess the effect of different donor age and age on the recovery of humoral immunity, and to evaluate the possible role of immune cells in hematopoietic stem cell transplantation. This study will provide guidance for the rapid restoration of immune reconstitution after transplantation and reduce the infection and death after transplantation.
We retrospectively analyzed the immune recovery of poor children with peripheral blood stem cell transplantation in the pediatric laminar flow ward of the Southern Hospital of Southern Medical University. The first part of the study and methods of T cell subsets after transplantation: the research objects and methods of the T cell subgroup in the second half of the transplant.
1.1) for the subjects of T cell subgroup within half a year, the criteria for the study were: all patients with thalassemia in my center from March 2009 to June 2011 were subject to non related peripheral blood stem cell transplantation. There was no death or rejection during the half year follow-up period, and 8 subsets of subgroups were completed half a year. (including the eight items of CD3+, CD4+, CD8+, NK, CD4+CD45RA+, CD4+CD29+, CD8+CD28+, CD8+CD28-) and blood routine assessment, after transplantation, the cases were still free from disease in January 2012. There were 40 cases of children, of which 20 cases were CMV infection. The immune recovery and the effect of CMV on it were evaluated.
1.2) preprocessing scheme
The children were given a myeloablative preconditioning program, including cyclosphosphamide (Cy) 100-120mg/kg, D-10 to D-9 two days intravenous drip, fludarabine, Flu, 200mg/m2, -6 to -2, 5 days intravenous drip, and cystopapidin (thiotepa, TT), two times 1 days. 3-4 days of intravenous injection (-8 to -6 days). All children were given cyclosporine A (cyclosporine A), mycophenolate mofetil (mycophenolate mofetil, MMF) and ATG (-3 - -1 days) to prevent short course of a course of methotrexate. Clusive disease, VOD).
1.3) detect and prevent cytomegalovirus infection before and after transplantation.
Ganciclovir (10mg/kg) was routinely treated for 8-10 days. The number of CMV-DNA replicas was monitored every week after allo-HSCT neutrophils, and the number of CMV-DNA copies was monitored every week until the 100 day. After the transplant January, the routine 7 days per month or 10 days of prophylactic.CMV infection per February were defined as the monitoring of the virus in peripheral blood. The number of replicas, the dose of 500/ul. ganciclovir or sodium phosphate, will be adjusted according to the renal function and peripheral neutrophils of the child, for example, the number of neutrophils in peripheral blood is less than 0.5 * 109/L for 2 days to stop ganciclovir.
1.4) the standard of implantation: 28 days, second months after transplantation, and the evidence for implantation is appropriate or even longer.
Indirect index: after 21 days of transplantation, the hematopoiesis returned to normal, including erythroid, granulocyte, lymphoid, megakaryocyte, neutrophils in 0.5 x 109/L, and the number of platelets maintained at 20 x 109/L; the liver and spleen were retracted to normal size in GVHD., and lactate dehydrogenase decreased to normal level.
Direct index: fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in the donor sex, and the donor sex was similar to the short tandem repeat (variable number of tandem repeat, VNTR) (HLA antigen turned into donor type, or donor gene proportion).
1.5) statistical analysis
SPSS13.0 software was used to analyze the effects of the infection of cytomegalovirus (CMV) after transplantation and the recovery of T cell subgroup in half a year. A single sample T test was used to compare the number of T cell subsets with the set reference value, and then the children were grouped with or without CMV infection, and the independent two-samples t Test test method was compared. The classification variables between the basic indexes of the group were compared with the chi square comparison, the level data could be used by the independent sample nonparametric test (Mann-Whitney), the quantitative variable was compared with the t test of 2 samples. The correlation analysis of the T cell subgroup CD8+ cells was less than 0.05 by linear correlation analysis. The second section after the transplantation was 1. Research objects and methods of T cell subsets in the year
For the subjects of the T cell subgroup within 1 years, the criteria for the study were the target of non related peripheral blood stem cell transplantation in children with thalassemia in my center from March 2009 to January 2011. No death or rejection occurred during the follow-up period of more than 1 years after transplantation, and there were still no disease after transplantation in January 2012. 1 years after transplantation, 4 subsets of T cell subsets (at least four items including CD3+, CD4+, CD8+, NK) and blood routine were evaluated. The patient had 38 cases. The detection of the virus was the same as the implantation test. The SPSS13.0 software was used for statistical analysis, and the number of each T cell subgroup was compared with the set reference value by a single sample t test. The.P value is less than 0.05. Second part of the research object and method of the immunization of the body fluid after transplantation and the first part of the study of humoral immunity in the second half of the transplantation
For the subjects of humoral immunity for half a year, the criteria for the study were the target of peripheral blood stem cell transplantation in all thalassemia children in my center from March 2010 to June 2011. No death or exclusion was taken during the half year follow-up period, and the immune evaluation of humoral immunity was completed at half a year. A total of 35 cases still had no disease surviving in January 2012. There were 35 patients. The detection of the virus was the same as that of the implant evidence. A single sample t test was used to compare the reference values of the humoral immunity and setting of each age group, the age of the children, the age of the donor and the half year after the transplant. The effect of the recovery of humoral immunity, using the bivariate Pearson correlation analysis, the.P value is less than 0.05, and the study objects and methods of humoral immunity for 1 years after the second node transplantation are statistically significant.
For the subjects of humoral immunity within 1 years, the criteria for the study were the target of peripheral blood stem cell transplantation in all children with thalassemia in my center from March 2009 to January 2011. No death or rejection occurred during the 1 year follow-up period after transplantation. Humoral immune evaluation was completed 1 years after transplantation. A total of 35 cases were still free from disease in January 2012. There were 35 cases. The detection of the virus was the same as that of the implant evidence. The SPSS13.0 software was used for statistical analysis, and a single sample t test was used to compare the reference values of the humoral immunity to the set reference values of each age group. The.P value was less than 0.05.
The first part of the.T cell subgroup
The recovery of T cell subsets in the first half of the first half of the year
(1) the mean value of CD3+ cells in the T cell subgroup for half a year was 1.46 x 109/L, the mean value of NK cells was 0.33 x 109/L, the mean value of CD4+ cells was 0.25 x 109/L, the mean value of CD8+ cells was 1.14 x 109/L, and CD4+ cells were lower than the set reference value 0.5 x 109/L level (t=-6.874, wasting).
(2) the number of CD3+, CD8+ and CD8+CD28- cells in CMV infection group was 1.87 + 1.47 x 109/L, 1.51 + 1.27 x 109/L and 1.20 + 1.13 x 109/L, which was significantly higher than that of the uninfected cytomegalovirus. The number of cells was 1.05 + 0.60 * 109/L, 0.78 + 0.50 x 109/L, 0.51 + 0.41 * 109/L respectively. The CD8+CD28- ratios of the two groups were 46.17% + 19.68%, 33.32% + 12.50% (t=-2.465, P=0.018), respectively.
(3) in the CMV infection group, CD8+ cells were linearly and positively correlated with CD8+CD28- cells (r=0.980, P0.001), but negatively correlated with the number of CD8+CD28+T cells, but there was no linear linear correlation (r=0.585, P=0.007).
Recovery of T cell subsets over 1 years after second transplantation
The mean value of CD3+ cells in T cell subsets for more than 1 years after hemopoietic stem cell transplantation in children with thalassemia is 2.22 x 109/L, the mean value of NK cells is 0.27 x 109/L, and the mean value of CD4+ cells is 0.63 x 109/L.
The second part. The situation of humoral immunity
Humoral immune recovery in the first half of the first half of the year
(1) the average and standard deviation of IgA, IgM and IgG of 3-6 year old children were 0.40 + 0.22g/L, 0.71 + 0.35g/L and 7.17 + 2.30g/L, respectively, and the recovery of IgA and IgM was lower than that of normal children (t=-8.467, P0.001; t=-2.305, P=0.042), and the average and standard difference of 7 years old. It is 0.81 + 0.71g/L, 1.02 + 0.40g/L, 8.13 + 3.28g/L, all lower than the normal children of the same age (t=-2.305, P=0.042:t=-6.161, P0.001, t=-2.733, P=0.019, respectively).
(2) the antibody level after transplantation was not related to the age of donor, but the age of the children was positively correlated with the level of IgA, C3 and C4, the correlation coefficient was 0.667,0.511,0.341, and the P value was 0.001,0.002,0.045. respectively.
(3) the average value of IgA, IgM, IgG in 2 children after splenectomy after allogeneic hematopoietic stem cell transplantation is 1.58g/L, 0.76g/L, 14.25g/L second, respectively, for more than 1 years of humoral immune recovery.
The mean and standard deviation of IgA, IgG and IgM of children aged 3-6 years after 1 years were 0.83 + 0.40g/L, 9.81 + 2.92g/L and 0.86 + 0.27g/L respectively. The mean and standard deviation of IgA, IgG, IgM were 1.35 + 0.74g/L, 11.45 + 4.85g/L, 1.05 + 0.53g/L. two age groups were lower than those of normal age children.
(1) the ratio of NK cells and the number and ratio of CD4+ cells and CD4+ cells in the latter half of the year of TM were not affected by CMV infection or not. Except the number of CD4+ cells was lower than 0.5 x 109/L, the rest of the cells recovered to normal, but the subgroups of T cells in the children after one year were at the normal reference level.
(2) the cytomegalovirus infection after allo-HSCT in TM children can lead to the monoclonal amplification of CD8+CD28- cells in the high CD8+ cells, thereby changing the conversion rate between the T cell subsets, and the ratio of CD8+ cells to the CD8+CD28- ratio is linearly related to the ratio of CD8+CD28-.
(3) the level of immunoglobulin at the latter half of allo-HSCT in TM children was lower than that of normal children, except for the level of IgG at the age of 3-6, and the age of children was positively correlated with the recovery of IgA, C3, and C4.
(4) the immunoglobulin level of TM children returned to normal children in 1 years after allo-HSCT, except IgM level was lower than that of normal children of the same age.
(5) spleen
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.5
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本文编号：1923984