移植前输注供体细胞诱导单倍体相合造血干细胞植入的实验研究

发布时间：2018-03-18 01:05

本文选题：非清髓性造血干细胞移植　切入点：供者细胞输注　出处：《第四军医大学》2011年硕士论文　论文类型：学位论文

【摘要】：背景造血干细胞移植(hematopoietic stem cell transplantation, HSCT)已经成为治愈血液系统恶性疾病最有效的手段之一。但由于生物种系及个体间的主要组织相容性复合体(major histocompability complex, MHC)存在差异,同种异体移植时将产生双向排斥反应,即移植物抗宿主(graft versus host, GVH)和宿主抗移植物(host versus graft, HVG)反应。由于单倍体相合供受者间人类白细胞分化抗原(Human leukocyte antigen, HLA)差异显著,移植排斥反应重,移植前往往需要大剂量的放化疗清除宿主免疫系统及肿瘤细胞,预防排斥、促进植入,同时需去除供者干细胞中的T细胞以减轻移植物抗宿主病(graft-versus-host disease, GVHD),并且加大输注的供者造血干细胞数量以促进植入。非清髓性造血干细胞移植(nonmyeloablative hematopoietic stem cell transplantation, NST)由于移植前放化疗强度降低,移植相关毒性减轻,越来越符合现代治疗学的要求。然而非清髓性单倍体或不相合造血干细胞移植(mismatched hematopoieticstem cell transplantation, mis-HSCT)一方面预处理强度降低,另一面供受者主要组织相容性抗原不合,常导致移植物排斥,移植后供者细胞植入率低,甚至植入失败。如何促进植入一直是非清髓性单倍体相合造血干细胞移植的重点研究课题之一。如何在非清髓或减低毒性的(reduced intensity conditioning, RIC)前提下进一步降低预处理强度,实现单倍体相合造血干细胞的植入,对于临床移植具有重要意义。因其不仅保留了非清髓性单倍体相合造血干细胞移植的长处,进一步降低了移植相关毒性,解决了相合供者来源不足的问题,同时弊弃了其短处,即移植排斥反应大。目前已知,宿主体内的T细胞是介导同种异体排斥反应的主要免疫细胞,其清除或者诱导免疫无能对于促进植入具有重要意义。去除或抑制供者同种异体反应性T细胞可以明显降低GVHD的发生率,表明同种异体反应性细胞的去除对于减低异体排斥反应确实可以减轻异体排斥。因而我们设想,通过去除受者体内同种异体反应性T的方法减轻宿主对供者细胞的排斥,促进植入。阿糖胞苷为抗嘧啶类抗代谢药,主要作用于S期的细胞,对于分裂活跃的细胞尤为敏感。体外实验证实免疫细胞接触抗原64h时细胞毒作用达到峰值,我们通过移植前输注供鼠细胞诱导宿主T细胞产生针对供鼠的同种异体反应,并在细胞免疫达到峰值之前利用阿糖胞苷将宿主体内这种同种反应性T细胞清除,这样宿主体内淋巴细胞池针对同一抗原的免疫能力相对耗竭,短时间内接触同一抗原,将产生免疫耐受,从而促进供体细胞植入。第一部分非清髓性单倍体相合骨髓移植小鼠模型的建立目的建立小鼠非清髓性单倍体相合骨髓移植模型,为研究移植前输注供体细胞诱导免疫耐受提供研究平台。方法实验分为实验组和对照组2组。以CB6F1雌性小鼠为受鼠,移植-1d予450cGy全身照射(TBI)后,随机分为2组,实验组移植0d输注C57BL/6雄性小鼠骨髓有核细胞5×107/只,对照组不予移植。监测受鼠造血恢复、检测供鼠Y染色体上性别决定基因(SRY),以及外周血供体细胞尤其CD3+细胞嵌合状态,同时观察小鼠急性移植物抗宿主病(acute graft-versus-host disease, aGVHD)的发生情况。结果对照组及实验组小鼠均未见小鼠死亡,各组小鼠均未见明显aGVHD表现,移植后30d内白细胞基本恢复正常水平。实验组小鼠SRY基因在移植后14d、30d、60d时经聚合酶链式反应(polymerase chain reaction, PCR)检测,结果均阳性,供鼠外周血淋巴细胞、单核细胞、粒细胞嵌合率移植后14d、30d、60d时分别为23.8%±4.6%、36.9%%±13.7%、19.4%±7.5% vs 49.9%±3.6%、53.2%±7.6%、54.4%±4.8% vs 67.6%±3.1%、51.6%±4.3%、56.9%±2.9%,其中CD3+细胞嵌合率分别为4.4%±4.6%、21.2%±6.4%、54.4%±4.1%。结论450cGyTBI的非清髓性预处理方案,可以诱导受鼠免疫耐受、供体骨髓细胞植入,嵌合率处于中低水平混合嵌合状态。第二部分移植前输注供体细胞诱导单倍体相合造血干细胞植入的实验研究目的观察移植前输注供体细胞,能否诱导宿主免疫耐受、促进供体骨髓细胞植入。方法实验分为Ara-C+TBI+BMT, SC+Ara-C+TBI+BMT, BMC+ Ara-C+ TBI+BMT 3组,以CB6F1雌性小鼠为受鼠,C57BL/6雄性小鼠为供鼠,移植前-3d经尾静脉输注供鼠脾细胞或骨髓细胞3×107,输注细胞后24h和48h分别通过腹腔注射阿糖胞苷0.015g/d,-1d时予450cGy全身照射(TBI),移植当天输注供鼠骨髓有核细胞5×107/只。监测受鼠白细胞恢复、外周血供鼠CD3+细胞嵌合状态及小鼠存活、GVHD的发生。结果各组分别有两只小鼠死亡,并表现不同程度GVHD。移植前输注脾细胞组和对照组白细胞恢复中位时间相当,为17d(12d~37d),输注骨髓细胞组恢复时间稍慢,中位时间为22d(22d~22d)。移植前输注供体脾细胞组及对照组供鼠CD3+细胞嵌合率相当,均达到完全供者型嵌合,+30d时移植前输注脾细胞组供鼠源CD3+细胞比例达93.5%±4.8%,明显高于输注骨髓细胞组(P0.05),+60d时,输注骨髓细胞组供体细胞嵌合率上升为87.2%±7.4%,与输注脾细胞组嵌合水平相当(91.7%±4.0%,P0.05),各组小鼠均出现不同程度的GVHD。结论移植前输注脾细胞可在一定程度上促进供体造血干细胞的早期植入。
[Abstract]:The background of hematopoietic stem cell transplantation (hematopoietic stem cell transplantation, HSCT) has become one of the most effective cure of malignant hematological diseases means. But due to the biological tissue and major histocompatibility complex between individuals (major histocompability, complex, MHC) differences in allograft rejection will have a two-way, or graft the host (graft versus host, GVH) and graft versus host (host versus, graft, HVG) reaction. Because haploidentical donor human leukocyte differentiation antigen (Human leukocyte, antigen, HLA) had significant difference, transplant rejection, often require high dose chemotherapy before transplantation of the host immune system and tumor removal cells, preventing rejection, promoting implantation, and removal of donor stem cells in T cells to reduce graft-versus-host disease (graft-versus-host disease, GVHD), and Increase the infusion of donor hematopoietic stem cell implantation. In order to promote the number of non myeloablative hematopoietic stem cell transplantation (nonmyeloablative hematopoietic stem cell transplantation, NST) before transplantation due to chemotherapy intensity decrease, reduce transplant related toxicity, more in line with the requirements of modern therapeutics. However non myeloablative hematopoietic stem haploid or not cell transplantation (mismatched hematopoieticstem cell transplantation, mis-HSCT) pretreatment reduces the intensity of the one hand, on the other side of the main organization for recipients of MHC incompatibility, often leads to graft rejection, low rate of donor cell engraftment after transplantation, or implant failure. How to promote implantation has been the focus of study of nonmyeloablative haploidentical hematopoietic stem cell transplantation. In non myeloablative or reduced toxicity (reduced intensity, conditioning, RIC) under the premise of further reducing pretreatment The strength of haploidentical hematopoietic stem cell implantation, has important significance for clinical transplantation. Because it not only retains the non myeloablative allogeneic hematopoietic stem cell transplantation strengths, further reducing transplant related toxicity, solve the matched donor shortage problem at the same time, abandoned the disadvantages of its weaknesses, namely the transplant rejection the reaction. It is now known that host T cells are the main immune cell mediated allograft rejection, the scavenging or induce immune anergy and plays an important role in promoting or inhibiting implantation. Removal of donor alloreactive T cells can significantly reduce the incidence of GVHD, suggesting that removal of alloreactive cells for reduce allograft rejection can indeed reduce rejection. So we assume that, by removing the body by the method of alloreactive T donor cells to reduce the host The rejection, promote implantation. Cytarabine for anti pyrimidine antimetabolites, a major role in the S phase of the cell, particularly sensitive to mitotically active cells. In vitro immune cells with the antigen 64H cytotoxic effect reached the peak, we through the pretransplant infusion of donor cells induced by host T cells for the same allogeneic response of donor rats, and reached the peak in cellular immunity before using cytarabine will host the alloreactive T cell depletion, immunity against the same antigen that host lymphocyte pool is depleted, contact time will produce the same antigen, immune tolerance, thereby promoting engraftment.
The first part of a mouse model of non myeloablative haploid allograft bone marrow transplantation
Objective to establish a model of non myelohaploid allograft bone marrow transplantation in mice, and to provide a research platform for the study of immune tolerance induced by donor cells before transplantation.
Methods the experiments were divided into experimental group and control group. 2 groups of female CB6F1 mice as the recipients in transplantation -1d to 450cGy irradiation (TBI), were randomly divided into 2 groups, experimental group was transplanted with 0d infusion of C57BL/6 male mice bone marrow nucleated cells of 5 * 107/, the control group was not monitored by murine hematopoietic transplantation. Recovery, detection for the mouse Y chromosome sex determining gene (SRY), and peripheral blood donor cells especially CD3+ cell chimerism, and observe the mice of acute graft-versus-host disease (acute graft-versus-host, disease, aGVHD) were observed.
The results of the control group and the experimental group mice showed no death of the mice, the mice showed no obvious aGVHD expression, white blood cell 30d returned to normal levels after transplantation. The experimental group of mice after transplantation of SRY gene in 14d, 30d, 60d by polymerase chain reaction (polymerase chain reaction, PCR) detection, the results were positive for rats peripheral blood lymphocytes, monocytes, myeloid chimerism after transplantation of 14d, 30d, 60d were 23.8% + 4.6%, 36.9%% + 13.7%, 19.4% + 7.5% vs 49.9% + 3.6%, 53.2% + 7.6%, 54.4% + 4.8% vs 67.6% + 3.1%, 51.6% + 4.3%, 56.9% + 2.9%, including CD3+ cell chimerism were 4.4% + 4.6%, 21.2% + 6.4%, 54.4% + 4.1%.
Conclusion the non myeloablative preconditioning scheme of 450cGyTBI can induce immune tolerance, bone marrow cells implantation and chimerism in low and middle level.
Experimental study of haploid allogeneic stem cell implantation induced by donor cells in second parts before transplantation
Objective To observe whether donor cells were transfused before transplantation to induce host immune tolerance and to promote donor bone marrow cells implantation.
Methods the experiment was divided into Ara-C+TBI+BMT, SC+Ara-C+TBI+BMT, BMC+ Ara-C+, TBI+BMT 3 groups, CB6F1 female mice as recipients were male C57BL/6 mice as donors and -3d before transplantation via tail vein infusion of donor spleen cells or bone marrow cells 3 * 107 cells after infusion of 24h and 48h respectively through abdominal cavity injection of cytarabine 0.015g/d -1d, with 450cGy total body irradiation (TBI), on the day of transplantation by infusion of bone marrow nucleated cells of 5 * 107/. Recipients were monitored the recovery of leukocytes, peripheral blood donor mice CD3+ cell chimerism and survival, the occurrence of GVHD.
The results of each group were two mice died, and showed different degrees of GVHD. before transplantation group and control group injection of spleen cells of white blood cell recovery in time is equivalent to 17D (12d~37d), infusion of bone marrow cells group the recovery time is slower, the median time to 22d (22d~22d) before transplantation. Infusion of donor spleen cells group and the control group of donor CD3+ cell chimerism, achieved complete donor chimerism, +30d infusion of spleen cells group before transplantation for murine CD3+ cell ratio was 93.5% + 4.8%, was significantly higher than that of infusion of bone marrow cell group (P0.05), +60d, infusion of bone marrow cells for somatic cell chimerism rate rise 87.2% + 7.4%, and lose chimeric horizontal injection spleen cells group (91.7% + 4%, P0.05), the mice showed varying degrees of GVHD.
Conclusion the injection of splenocytes before transplantation can promote the early implantation of donor hematopoietic stem cells to a certain extent.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【共引文献】