本文選題：重型地中海貧血 + 兒童��； 參考：《南方醫(yī)科大學》2012年碩士論文

【摘要】：異基因造血干細胞移植(allogenic hematopoietic stem cell transplantation, allo-HSCT)仍然是治愈遺傳性血紅蛋白病如重型β地中海貧血(Thalassemia Major,TM)患兒的唯一手段,但其臨床應(yīng)用受到以下因素的限制,如預處理的相關(guān)毒性,供者細胞對受者細胞抗原的免疫反應(yīng)引起的移植物抗宿主病(graft versus host disease,GVHD)及延遲的免疫重建或免疫缺陷導致感染,復發(fā)及偶爾的排斥等等。造血干細胞移植后的免疫重建在成人已有較為深入的研究,但對于兒童研究很少。經(jīng)過大劑量的化療后,受者免疫細胞的大量減少,但受者漿細胞往往可能持續(xù)存在至移植后數(shù)月到數(shù)年。移植前許多因素也可影響移植后的免疫狀態(tài)及其恢復,如年齡,干細胞來源,組織相容性抗原(histocompatibility antigen,HLA)相合的程度等等。移植后的急性GVHD,巨細胞病毒感染(cytomegalovirus,CMV),免疫抑制劑應(yīng)用、抗菌素,免疫球蛋白的應(yīng)用及供者的淋巴細胞輸注(donor lymphocyte infusions,DLI)等均可影響移植后免疫的恢復。提高移植后的免疫重建是決定其臨床治療效果的重要因素。我們研究在TM的兒童在清髓性的allo-HSCT后2年內(nèi)的T細胞亞群及體液免疫的恢復及其相關(guān)影響因素。 本課題旨在探討TM兒童在異基因外周血造血干細胞移植后2年內(nèi)的免疫恢復情況,評估CMV感染對T細胞亞群的影響,評估不同的供者年齡和受者年齡對體液免疫的恢復的影響,從而評估免疫細胞在造血干細胞移植中可能的作用,期望通過該研究為移植后免疫重建的快速恢復,減少移植后感染死亡提供指導意義。 我們回顧性的分析在南方醫(yī)科大學南方醫(yī)院兒科層流病房進行外周血造血干細胞移植的地貧患兒免疫恢復情況。第一部分移植后T細胞亞群的研究對象和方法第一節(jié)：移植后半年的T細胞亞群的研究對象和方法 1.1)對于半年內(nèi)的T細胞亞群的研究對象,入選研究范圍的標準是：2009年3月至2011年6月內(nèi)所有在我中心的地中海貧血患兒行非親緣外周血造血干細胞移植的對象,在移植后半年的隨訪期間內(nèi)未發(fā)生死亡或排斥,半年時完成T細胞亞群8項(包括CD3+、CD4+、CD8+、NK、CD4+CD45RA+、CD4+CD29+、 CD8+CD28+、CD8+CD28-這八項)和血常規(guī)的評估,移植后截止于2012年1月仍無病存活的病例。共有40例患兒,其中CMV感染有20例。評估其免疫恢復情況和CMV對其的影響。 1.2)預處理方案 患兒均給予清髓性的預處理方案,包括環(huán)磷酰胺(cyclosphosphamide,Cy)100-120mg/kg,d-10至d-9分兩天靜脈滴注,氟達拉濱(fludarabine,Flu),200mg/m2,-6至-2天分5天靜脈滴注,塞替哌(thiotepa,TT),10mg/kg,-5天1天分兩次靜脈滴注,白消安(buslfan, BU),9.6-17.6mg/kg,分3-4天靜脈注射(-8至-6天)。所有患兒給予環(huán)孢素A(cyclosporine A),霉酚酸酯(mycophenolate mofetil, MMF)和ATG(-3—-1天)預防GVHD。短療程的甲氨蝶呤(methotrexate、MTX)在移植后第1,3,6天分別給予15,10and10mg/m2治療。肝素鈉預防肝靜脈阻塞綜合癥(hepatic veno-occlusive disease,VOD)。 1.3)移植前及移植后檢測及預防巨細胞病毒定量 移植前常規(guī)進行更昔洛韋(10mg/kg)治療,療程8-10天。在allo-HSCT后中性粒細胞數(shù)0.5×109/L開始每周監(jiān)測CMV-DNA復制數(shù),直到100天,移植后1月開始,常規(guī)進行每月7天的或每2月10天的預防性更昔洛韋治療。CMV感染定義為在外周血中可監(jiān)測到病毒的復制數(shù),即500/ul。更昔洛韋的劑量或改用磷甲酸鈉會依據(jù)患兒的腎功能和外周血中性粒細胞數(shù)進行調(diào)整,如外周血中性粒細胞數(shù)連續(xù)2天小于0.5×109/L停止更昔洛韋。 1.4)植入的標準：移植后28天,第二個月,適當時甚至更長時間的檢測植入證據(jù)。 間接指標：移植后臨床存活21天；各系統(tǒng)造血恢復正常,包括紅系,粒系,淋巴系,巨核系等,中性粒細胞數(shù)在0.5×109/L,血小板數(shù)維持在20×109/L；臨床上出現(xiàn)GVHD。腫大的肝脾回縮至正常大��；乳酸脫氫酶降至正常水平。 直接指標：供受者性別不同采用免疫熒光原位雜交法(fluorescence in situ hybridization,FISH)檢測性染色體的轉(zhuǎn)變,供受者性別相同采用短串聯(lián)重復序列(variable number of tandem repeat, VNTR)(HLA抗原轉(zhuǎn)變成供者型,或供者基因占受者的比例)。 1.5)統(tǒng)計分析 應(yīng)用SPSS13.0軟件進行統(tǒng)計分析,移植后巨細胞病毒感染與否與半年內(nèi)的T細胞亞群的恢復的影響。先采用單個樣本T檢驗對各個T細胞亞群數(shù)量與設(shè)定的參考值進行比較,然后將患兒按有無CMV感染分組,采用獨立two-samples t Test檢驗方法比較。組間的基礎(chǔ)指標的分類變量采用卡方比較,等級資料可采用獨立樣本的非參數(shù)檢驗(Mann-Whitney),定量變量采用2個樣本的t檢驗比較。采用線性相關(guān)分析分析T細胞亞群CD8+細胞之間的相關(guān)性。P值小于0.05為有統(tǒng)計學意義。第二節(jié)移植后1年的T細胞亞群的研究對象和方法 對于1年內(nèi)的T細胞亞群的研究對象,入選研究范圍的標準是：2009年3月至2011年1月內(nèi)在我中心的地中海貧血患兒行非親緣外周血造血干細胞移植的對象,在移植后1年以上的隨訪期間內(nèi)未發(fā)生死亡或排斥,移植后截止于2012年1月仍無病存活的病例,移植后1年完成T細胞亞群4項(至少包括CD3+、CD4+、CD8+、NK這四項)及血常規(guī)的評估。病人共有38例。對其病毒的檢測與植入檢測同上。應(yīng)用SPSS13.0軟件進行統(tǒng)計分析,采用單個樣本t檢驗對各個T細胞亞群數(shù)量與設(shè)定的參考值進行比較。P值小于0.05為有統(tǒng)計學意義。第二部分移植后體液免疫的研究對象和方法第一節(jié)移植后半年的體液免疫的研究對象和方和 對于半年內(nèi)的體液免疫的研究對象,入選研究范圍的標準是：2010年3月至2011年6月內(nèi)所有在我中心的地中海貧血患兒行外周血造血干細胞移植的對象,在移植后半年的隨訪期間內(nèi)未發(fā)生死亡或排斥,半年時完成體液免疫的免疫評估,移植后截止于2012年1月仍無病存活的病例,病人共有35例。對其病毒的檢測與植入證據(jù)檢測同上。應(yīng)用SPSS13.0軟件進行統(tǒng)計分析,先采用單個樣本t檢驗對各個年齡段的體液免疫與設(shè)定的參考值進行比較,患兒年齡,供者年齡與移植后半年內(nèi)體液免疫的恢復的影響,采用雙變量的Pearson相關(guān)分別分析。P值小于0.05為有統(tǒng)計學意義第二節(jié)移植后1年的體液免疫的研究對象和方法 對于1年內(nèi)的體液免疫的研究對象,入選研究范圍的標準是：2009年3月至2011年1月內(nèi)所有在我中心的地中海貧血患兒行外周血造血干細胞移植的對象,在移植后1年的隨訪期間內(nèi)未發(fā)生死亡或排斥,移植后1年完成體液免疫評估,移植后截止于2012年1月仍無病存活的病例。病人共有35例。對其病毒的檢測與植入證據(jù)檢測同上。應(yīng)用SPSS13.0軟件進行統(tǒng)計分析,采用單個樣本t檢驗對各個年齡段的體液免疫與設(shè)定的參考值進行比較。P值小于0.05為有統(tǒng)計學意義。 第一部分.T細胞亞群的情況 第一節(jié)移植后半年的T細胞亞群的恢復 (1)半年的T細胞亞群內(nèi)CD3+細胞的均值1.46×109/L,NK細胞均值0.33×109/L,CD4+細胞均值0.25×109/L,CD8+細胞均值1.14×109/L,其中CD4+細胞低于設(shè)定參考值0.5×109/L水平(t=-6.874,P0.001) (2)CMV感染組的半年CD3+、CD8+、CD8+CD28-的細胞數(shù)分別為1.87±1.47×109/L、1.51±1.27×109/L、1.20±1.13×109/L,明顯高于巨細胞病毒未感染組,其細胞數(shù)分別是1.05±0.60×109/L、0.78±0.50×109/L、0.51±0.41×109/L(分別為t=-2.315,P=0.029；t=-2.414,P=0.024；t=-2.540,P=0.018；),其兩組的CD8+CD28-比率分別是46.17%±19.68%,33.32%±12.50%(t=-2.465,P=0.018)。 (3)在CMV感染組內(nèi),CD8+細胞與CD8+CD28-細胞呈線性正相關(guān)(r=0.980,P0.001),而與CD8+CD28+T細胞數(shù)負相關(guān),但無直線線性相關(guān)(r=0.585,P=0.007)。 第二節(jié)移植后1年以上的T細胞亞群的恢復 地中海貧血患兒在造血干細胞移植后1年以上T細胞亞群內(nèi)CD3+細胞的均值2.22×109/L,NK細胞均值0.27×109/L,CD4+細胞均值0.63×109/L。 第二部分.體液免疫的情況 第一節(jié)移植后半年的體液免疫恢復 (1)地中海貧血患兒移植后半年的免疫球蛋白水平,3-6歲的IgA、IgM, IgG的平均值和標準差分別是0.40±0.22g/L、0.71±0.35g/L、7.17±2.30g/L,其中IgA、IgM恢復低于正常同齡兒童水平(分別t=-8.467,P0.001；t=-2.305,P=0.042),7歲的IgA、IgM,IgG的平均值和標準差分別是0.81±0.71g/L、1.02±0.40g/L、8.13±3.28g/L,均低于正常同齡兒童水平(分別t=-2.305,P=0.042：t=-6.161,P0.001；t=-2.733,P=0.019)。 (2)移植后的抗體水平和供者年齡無關(guān),但患兒年齡分別與IgA、C3,C4的水平成正相關(guān)關(guān)系,相關(guān)系數(shù)分別是0.667、0.511、0.341,P值分別0.001、0.002、0.045。 (3)2例脾切除后的患兒在造血干細胞移植后半年內(nèi)的IgA、IgM, IgG的平均值分別1.58g/L,0.76g/L,14.25g/L第二節(jié)移植后1年以上的體液免疫恢復 1年后地貧患兒的3-6歲的IgA、IgG, IgM的均值和標準差分別是0.83±0.40g/L、9.81±2.92g/L、0.86±0.27g/L,7歲的IgA, IgG, IgM的均值和標準差分別是1.35±0.74g/L、11.45±4.85g/L、1.05±0.53g/L。兩年齡段IgM低于正常同齡兒童(t分別為-8.982,-5.380,P均為0.001)。 (1)TM兒童allo-HSCT后半年時NK細胞數(shù)比率及CD4+細胞數(shù)和比率不受CMV感染與否的影響,除了CD4+細胞數(shù)低于0.5×109/L外,其余均恢復正常。但一年后的患兒的T細胞各亞群處于正常參考值水平。 (2)TM兒童allo-HSCT后的巨細胞病毒感染會導致高的CD8+細胞中的CD8+CD28-細胞的單克隆擴增,從而改變T細胞亞群的之間的轉(zhuǎn)化率,且CD8+細胞升高比率與CD8+CD28-比率呈直線線性相關(guān)。 (3)對于TM兒童allo-HSCT后半年時免疫球蛋白水平均低于正常同齡兒童,除了3-6歲的IgG水平外；同時患兒年齡與IgA,C3,C4的恢復成正相關(guān)。 (4)對于TM兒童allo-HSCT后1年時免疫球蛋白水平均恢復至正常同齡兒童,除了IgM水平低于正常同齡兒童的外。 (5)脾切除的TM患兒allo-HSCT后半年內(nèi)有低的IgM水平和更高的IgG和IgA水平的趨勢。
[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
【學位授予單位】：南方醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R725.5
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本文編號：1923984