高剂量MHC不相合供体细胞输注诱导供体细胞植入及避免GVHD的动物实验研究

发布时间：2018-04-08 23:16

本文选题：造血干细胞移植　切入点：预处理　出处：《中国人民解放军军事医学科学院》2011年博士论文

【摘要】：研究目的:异基因造血干细胞移植(allo-HSCT)是目前治疗恶性血液系统疾病、新陈代谢疾病、免疫系统缺陷、自身免疫系统疾病以及实体肿瘤等多种疾病的有效方法。长期以来一致认为:足够强度的预处理(髓系和淋巴系的彻底清除-清髓性移植;淋巴系的彻底清除及一定程度的髓系清除-非清髓移植)及骨髓腾空是供体细胞稳定植入及诱导移植物抗白血病效应(GVL)的基础和必要条件。然而预处理相关的毒性反应及移植后GVHD、排斥、感染等并发症严重限制了allo-HSCT的疗效。与传统的清髓方案相比,非清髓等减毒方案虽然显著减轻了预处理相关的并发症,但预处理本身所包含的放化疗、移植后免疫抑制剂的应用,以及免疫功能低下所致的细菌、真菌及病毒等严重感染并发症仍严重威胁着病人的生命和健康。因此,如何从根本上减轻移植相关并发症及病死率,进一步提高移植疗效是临床亟需解决的问题。本课题旨在既往清髓和非清髓移植和HLA半相合移植研究的基础上,试图通过最大限度的减小或去除预处理,但调整供体细胞数量等条件,达到供体细胞稳定植入及预防GVHD。本课题拟从另一角度研究异基因供体细胞植入及免疫耐受和GVHD相关科学问题及机制研究,同时探讨无预处理条件下进行allo- HSCT的可行性及安全性,为非恶性疾病、无预处理条件下的allo-HSCT的临床应用提供实验依据。研究内容:首先建立FCM检测小鼠供体大嵌合和Real-time PCR检测小鼠供体微嵌合的方法,并评价方法的有效性,其次研究不同TBI强度(8GY~1GY)对H-2半相合移植小鼠供体植入和GVHD的影响规律,进一步研究不同供体细胞输注数量(1×10~7～9×10~7)对H-2半相合移植小鼠供体植入和GVHD的影响规律。然后研究在无预处理条件下,输注不同数量的H-2半相合供体细胞组小鼠供体植入情况与GVHD情况,探讨无预处理条件下H-2半相合移植的可行性及安全性。最后通过无预处理条件下供体细胞分布、淋巴细胞亚群变化及细胞因子变化等研究探讨无预处理条件下H-2半相合供体细胞输注诱导供体细胞稳定植入的可能机制。研究方法:通过检测移植后受鼠H-2表型变化建立FCM检测供体大嵌合的方法;建立Real-time PCR法检测SRY基因表达率即供体微嵌合的方法:首先根据小鼠β-actin基因及sry基因序列设计引物探针、进行PCR扩增,再将电泳产物回收构建质粒,测序正确后建立标准曲线,然后进行实时荧光定量PCR扩增并优化扩增条件,最后以雄性小鼠DNA标本为阳性对照,雌性小鼠为阴性对照,检验Real-time PCR法的敏感性、特异性以及可重复性。以TBI 6GY与2GY条件下H-2半相合骨髓移植小鼠模型为例,通过检测两组小鼠移植后DC验证FCM法联合Real-time PCR法作为DC定量检测方法的有效性; 分别以亲代和子代小鼠作为供鼠与受鼠,TBI作为预处理,进行H-2半相合造血干细胞移植研究。实验组根据不同TBI强度(8GY、6GY、4GY、2GY、1GY)分为5组,分别输注动员后的供鼠脾单个核细胞1×10~7/只,对照组在TBI后输注等量生理盐水。比较不同TBI强度组移植后小鼠供体植入情况、GVHD发生率与病死率、生存情况的差异;实验组在相同TBI条件下,再根据不同供体细胞输注数量(1×10~7、3×10~7、6×10~7、9×10~7)分为A组～D组。比较不同细胞数量组小鼠移植后供体植入及GVHD情况的差异;在无预处理条件下根据不同供体细胞输注数量(1×10~7、3×10~7、6×10~7、9×10~7、12×10~7、15×10~7)分为6组,比较各组小鼠输注H-2半相合供体细胞后供体植入及GVHD的差异;检测受鼠在无预处理条件下输注H-2半相合供体细胞后淋巴细胞亚群变化、细胞因子变化、供体细胞组织分布以及细胞系中DC情况。研究结果:成功建立了小鼠供体嵌合率定量检测方法;6GY组小鼠在移植2w后DC90%,利用FCM法检测精确度较高,Real-time PCR法检测误差较大;2GY组小鼠在移植1月后DC1% ,利用Real-time PCR法检测良好,FCM法灵敏度较低无法检测。两种方法结合运用,可适于移植后不同供体嵌合状态的检测。在相同的供体细胞数量1×10~7的移植条件下,TBI 8GY组小鼠移植后2w即获得完全供体植入(CC),并出现典型的GVHD体征,GVHD发生率100%,在移植后18d～25d全部死亡;6GY组小鼠在移植后6w也获得了CC,3/8小鼠出现了轻度的GVHD,全部小鼠存活时间大于3个月;4GY组小鼠移植后仅获得了MC,1/8小鼠出现轻度GVHD,小鼠均长期存活;2GY组与1GY组小鼠移植后1w获得了低比例的MC,然后转为微嵌合状态,供体细胞分别在10w与8w内完全消失,无GVHD的发生。在TBI-6GY条件下,供体细胞数量3×10~7组、6×10~7组及9×10~7组小鼠移植后MC转为CC的时间分别是3w、2.5w、2w,且均出现了典型的GVHD体征,GVHD发病率100%,病死率分别为75%～100%;在TBI-4GY条件下供体细胞数量3×10~7组、6×10~7组及9×10~7组小鼠移植后均获得了CC,GVHD死亡率分别为25%、50%、100%;在TBI-2GY/1GY条件下供体细胞数量3×10~7组小鼠移植后均获得了MC及短暂的供体微嵌合状态,未发生GVHD;TBI-2GY条件下6×10~7组及9×10~7组小鼠移植后均获得CC,出现典型了GVHD症状,GVHD发生率为62.5%与100%,GVHD病死率分别为25%与50%;TBI-1GY条件下6×10~7组及9×10~7组小鼠移植后均获得CC,出现轻度GVHD症状,GVHD发病率分别为25%与62.5%,小鼠均长期存活。无预处理条件下,1×10~7组、3×10~7组以及6×10~7组小鼠在输注H-2半相合供体细胞后获得一过性的供体细胞微嵌合,供体细胞在受鼠体内存在的中位时间分别是5w、7w及9w,未发生GVHD。9×10~7组5/8小鼠在输注细胞后8w(5w～12w)获得CC,且未见明显的GVHD体征。12×10~7组及15×10~7组小鼠在移植后全部获得了CC,MC转为CC的中位时间分别是6w与5w,GVHD发生率分别为0和25%,小鼠存活时间均大于6个月。无预处理条件下输注半相合供体细胞后受鼠造血及淋巴细胞亚群短暂抑制但可逐渐恢复正常;无预处理组受鼠血清中TH1细胞因子(IFN-γ、IL-2)及炎性因子TNF-α表达较GVHD组降低,TH2细胞因子(IL-4、IL-10、IL-6)较对照组明显升高;MLR实验提示6×10~7组和12×10~7组小鼠在移植后对供体细胞的反应性较正常小鼠下降,而对第三方供体细胞的反应性与正常鼠无差异;组织中及亚群中供体嵌合率检测结果表明无预处理条件下12×10~7组小鼠移植后获得的供体嵌合体为多系嵌合体,供体细胞植入特点及在各免疫器官的分布与预处理组类似。研究结论:本研究成功建立了FCM联合Real-time PCR法定量检测小鼠供体嵌合率的方法;阐明了在H-2半相合移植模型中TBI强度与供体细胞输注数量均是影响小鼠供体植入与GVHD的重要因素;成功建立了在无预处理条件下的小鼠H-2半相合移植模型;初步确定了无预处理方案中保障移植成功而无GVHD的适宜供体细胞数量;揭示无预处理方案可能通过形成组织中混合嵌合体(MC)、调节TH1/TH2平衡、调节性T淋巴细胞等机制诱导供体细胞稳定植入和免疫耐受。此模型的建立及相关机制研究为无预处理的半相合移植方案在临床的应用提供动物实验基础和理论依据。
[Abstract]:Research purposes : Allogeneic hematopoietic stem cell transplantation ( allogeneic hematopoietic stem cell transplantation ) is an effective method for the treatment of malignant blood system diseases , metabolic diseases , immune system defects , autoimmune diseases and solid tumors .

The purpose of this study was to study the feasibility and safety of allogeneic donor cell implantation and immune tolerance and GVHD , and to explore the feasibility and safety of allogeneic donor cell implantation and immune tolerance and GVHD .

In this paper , the effects of donor implantation and GVHD in mice with H - 2 half - phase transplantation were studied by means of FCM . The effects of different donor - donor infusion numbers ( 8GY - 1GY ) on donor implantation and GVHD in H - 2 half - phase grafting mice were studied . The possible mechanism of donor cell distribution , lymphocyte subsets and cytokine changes was studied under the condition of no pretreatment .

Methods : To establish a method for the detection of donor microchimerism by detecting H - 2 phenotype changes in mice after transplantation . A method for detecting SRY gene expression rate , i.e . donor microchimerism , was established by establishing a real - time PCR method . The PCR amplification was carried out according to the mouse 尾 - actin gene and the sry gene sequence . The sensitivity , specificity and repeatability of the real - time PCR method were determined .

The experimental group was divided into 6 groups according to different donor cell infusion numbers ( 1 脳 10 ~ 7 , 3 脳 10 ~ 7 , 6 脳 10 ~ 7 , 9 脳 10 ~ 7 , 12 脳 10 ~ 7 , 15 脳 10 ~ 7 ) . The experimental groups were divided into 6 groups according to different donor cell infusion numbers ( 1 脳 10 ~ 7 , 3 脳 10 ~ 7 , 6 脳 10 ~ 7 , 9 脳 10 ~ 7 , 12 脳 10 ~ 7 , 15 脳 10 ~ 7 ) . The experimental groups were divided into 6 groups according to the number of different donor cells ( 1 脳 10 ~ 7 , 3 脳 10 ~ 7 , 6 脳 10 ~ 7 , 9 脳 10 ~ 7 , 12 脳 10 ~ 7 , 15 脳 10 ~ 7 ) .

Results : The method of quantitative determination of mouse donor chimerism was successfully established ; 6GY group of mice were DC90 % after 2w transplantation , the accuracy was high with FCM method , the detection accuracy was high with the method of real - time PCR , and the sensitivity of the 2GY group was less than that of the method of real - time PCR , and the sensitivity of FCM method was low . The two methods were combined and applied , which could be suitable for the detection of the chimeric state of different donor after transplantation .

At the same donor cell number of 1 脳 10 ~ 7 , the total donor implant ( CC ) was obtained after transplantation of 8GY group of mice . The incidence of GVHD was 100 % . After transplantation , the mice showed mild GVHD . All the mice survived longer than 3 months . After transplantation , only MC and 1 / 8 mice received low proportion of MC , then the mice survived . The donor cells disappeared completely within 10w and 8w , and no GVHD occurred .

There were 3 脳 10 ~ 7 groups of donor cells , 6 脳 10 ~ 7 groups and 9 脳 10 ~ 7 groups of mice after transplantation . The rates of GVHD and GVHD were 25 % , 50 % and 100 % , respectively .

There were no obvious GVHD signs . The median time of CC and MC to CC was 0 and 25 % in 12 脳 10 ~ 7 groups and 15 脳 10 ~ 7 groups , respectively , and the survival time of mice was more than 6 months .

The results showed that the donor chimeras obtained after transplantation of 12 脳 10 ~ 7 groups and 12 脳 10 ~ 7 groups was similar to that of normal mice after transplantation . The donor chimeras obtained after transplantation of mice with no pre - treatment was 6 脳 10 ~ 7 and 12 脳 10 ~ 7 groups .

Conclusion : This study successfully established a method for detecting the donor chimerism rate in mice by means of FCM combined with Real - time PCR . It has been shown that both the intensity and the number of donor cells in the H - 2 half - phase grafting model are important factors affecting donor implantation and GVHD in mice . The establishment of a pretreatment protocol and the mechanism of non - pretreatment can induce the stable implantation and immune tolerance of donor cells . The establishment of this model and the related mechanism are used to provide animal experimental basis and theoretical basis for the clinical application of the non - pretreatment half - phase grafting scheme .

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1

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