环磷酰胺在移植治疗再生障碍性贫血中的应用及对造血功能的影响

发布时间：2018-01-01 11:23

  本文关键词：环磷酰胺在移植治疗再生障碍性贫血中的应用及对造血功能的影响　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 环磷酰胺 造血干细胞移植 移植物抗宿主病 动物模型 造血功能

【摘要】：研究背景及目的背景再生障碍性贫血(aplastic anemia,AA)是一组由理化因素、生物因素及不明原因引起的骨髓造血衰竭性疾病,具有致死率高、预后差等特点,造血干细胞移植成为根治AA的最有效方法。然而由于计划生育政策,同胞兄妹供者较少,对于单倍型造血干细胞移植来说,患者父母及兄弟姐妹均可作为其供者来源,使得单倍型造血干细胞移植成为主流力量。但严重的移植物抗宿主病(graft versus host disease,GVHD)和移植物排斥反应的发生成为主要障碍,直接影响移植后的长期生存率。环磷酰胺(CTX)具有强效的免疫抑制抗肿瘤作用,移植后高剂量CTX诱导免疫耐受在单倍型移植中的应用引起关注,但是应用时机和剂量很重要。CTX诱导耐受新方法在单倍型移植中占有重要作用,但对于CTX诱导免疫耐受的作用机制及特点目前尚未完全清楚,特别担心对造血重建的影响,本研究通过小鼠实体实验观察CTX对造血干细胞的影响,为移植工作提供理论依据。目的1.评价单倍型移植后应用高剂量CTX诱导免疫耐受治疗重型再生障碍性贫血(SAA)的临床疗效;2.通过动物实验观察不同剂量CTX对小鼠造血功能的影响。研究方法1.2014年1月至2015年8月期间单中心中国人民解放军陆军总医院血液科移植后使用高剂量CTX诱导免疫耐受单倍型造血干细胞移植新方法治疗的16例SAA患者,男7例,女9例,患者中位年龄14.5(4~30)岁。所有均为亲缘单倍型供者,其中父亲供者9例,母亲供者5例,同胞兄妹供者2例,供者中位年龄31(20~45)岁。观察全部患者移植后植入情况、造血重建(包括中性粒细胞及血小板植活时间)、移植相关并发症(移植物抗宿主病、感染及出血)、存活情况及随访时间等指标。2.选取雌性纯系BALB/c小鼠,按照常规方法制成再生障碍性贫血小鼠模型,所有造模成功后的小鼠随机分组以供实验需要。将环磷酰胺药物以无菌生理盐水溶解,按CTX处理剂量(380mg/kg、200mg/kg、100mg/kg)作用于实验组小鼠,动态观察实验组(HD-CTX组、MD-CTX组、LD-CTX组)、AA模型对照组、正常对照组小鼠一般状况、外周血血常规变化、双侧股骨有核细胞数(NC)及股骨骨髓病理切片、半固体甲基纤维素培养法测定红系集落形成单位(BFU-E)、粒单系集落形成单位(CFU-GM)及流式细胞术检测外周血及骨髓CD34+细胞变化。结果1.全部16例患者除1例原发排斥,余15例完全植入,其中有2例患者第1次未植入,行二次移植达到完全供者植入状态。粒细胞植活中位时间15.5(14~26)天、血小板植活中位时间22.0(17~32)天。随访至2016年8月,中位随访时间17.4(9~28)月,存活患者随访时间均在6个月以上,最长随访时间达28个月。移植后共有8例发生急性GVHD,其中IV度急性GVHD 2例,III度急性GVHD 3例,I~II度急性GVHD 3例,按部位分为肠道3例、肝脏3例、皮肤2例;2例发生慢性GVHD,均发生在皮肤。肺部感染7例;巨细胞病毒感染3例,其中2例发展为巨细胞肺炎,予更昔洛韦、磷甲酸钠治疗后好转,无肝静脉综合征及移植后淋巴细胞增殖发生;出血性膀胱炎4例。所有患者1例因未植入死亡,1例因严重肺部感染死亡,1例因急性GVHD死亡,其余13例仍存活。2.1)全部受鼠基于AA再障发病机制建造AA小鼠模型成功。2)正常对照组白细胞及血小板数目波动在正常范围;AA模型组白细胞及血小板变化大致相似,均进行性下降,第5天开始明显下降,分别为(0.46±0.32)×109/L、(33.4±14.67)×109/L,差异均有统计学意义(P0.05);LD-CTX及MD-CTX组白细胞数目在第9天开始回升。HD-CTX组白细胞数目变化与模型组大致相同。AA模型组骨髓及外周血CD34+细胞进行性下降,第5天降至较低水平,(1.76±0.28)×109/L,第7天开始回升,但仍低于正常,为(2.86±0.45)×109/L,差异有统计学意义(P0.05)。LD-CTX及MD-CTX组第3天骨髓及外周血CD34+细胞降至最低,分别(0.66±0.30)×109/L、(0.72±0.40)×109/L,差异有统计学意义(P0.01),第7天细胞数开始回升。HD-CTX组骨髓及外周血CD34+细胞第3天最低,为(0.49±0.38)×109/L,随后一直处于低水平,差异有统计学意义(P0.05)。模型AA组CFU-GM、CFU-E集落形成数明显减少,48h、96h分别形成的CFU-GM、CFU-E集落数为(10.00±5.00)个、(5.00±3.00)个、(12.67±3.79)个、(5.33±3.21)个,差异均有统计学意义(P0.05)。LD-CTX、MD-CTX、HD-CTX组与模型组比较无明显差异。结论1.移植后高剂量CTX诱导免疫耐受单倍型异基因造血干细胞移植治疗重型再生障碍性贫血,造血重建稳定,有较好的临床疗效。2.单倍型造血干细胞移植后应用高剂量CTX诱导免疫耐受可减少移植相关并发症,是预防单倍型移植GVHD发生的新方法。3.将受鼠BALB/C小鼠经Co60γ放射线亚致死量(5.5Gy)全身照射,将DBA/2小鼠的淋巴细胞悬液在4小时内经尾静脉输注受鼠(BALB/C鼠)体内,该过程大致模拟AA发病过程,制造AA小鼠模型,观察AA小鼠外周血血常规变化、骨髓有核细胞数及骨髓病理变化均符合AA的病理变化,说明小鼠AA造模成功。4.动物实验证实CTX对骨髓造血功能无明显损伤作用,为临床移植工作应用高剂量CTX诱导免疫耐受提供理论依据。
[Abstract]:Background and objective background of aplastic anemia (aplastic anemia AA) is a group of physical and chemical factors, biological factors and unexplained bone marrow failure disease, with high mortality and poor prognosis, hematopoietic stem cell transplantation has become the most effective method to cure AA. However, because of the one-child policy sibling donor, less for haploidentical hematopoietic stem cell transplantation for patients with parents and siblings can be used as the donor, the haploidentical hematopoietic stem cell transplantation has become main force. But severe graft versus host disease (graft versus host disease, GVHD) and graft rejection has become main obstacle and directly influence the long-term survival rate after transplantation. Cyclophosphamide (CTX) immune suppression has potent antitumor effect, application of high dose of CTX after transplantation induced immune tolerance in transplantation induced by haplotype Now, but the timing and dose of.CTX is very important to a new method for inducing tolerance in haplotype transplantation plays an important role, but the mechanism and characteristics of immune tolerance induced by CTX is not fully understood, particularly worried about the impact on hematopoietic reconstruction, the effects on hematopoietic stem cell research by small rat experimental observations of CTX entity, provide the theoretical basis for the transplantation. Objective 1. to evaluate the haplotype after transplantation immune tolerance induced by high dose CTX in treatment of severe aplastic anemia (SAA) clinical curative effect; 2. through animal experiments to observe the effects of different doses of CTX on hematopoietic function in mice. Methods 16 cases of SAA patients 1.2014 years from January to August 2015 during the use of a single center China people the PLA General Hospital Department of Hematology after transplantation immune tolerance induced by high dose CTX haploidentical hematopoietic stem cell transplantation therapy, 7 cases were male, 9 were female, Patients with a median age of 14.5 (4~30) years old. All were haploidentical donor, the father for 9 cases, 5 cases of maternal donors, 2 cases of sibling donor donor, the median age was 31 years (20~45). The implantation was observed in all patients after transplantation, hematopoietic reconstruction (including neutrophils and platelet engraftment time), transplantation related complications (graft-versus-host disease, infection and bleeding), survival rate and follow-up time index.2. female inbred BALB/c mice, according to the conventional method made the model of AA mice, all afterinjecting mice were randomly divided to experiment. The cyclophosphamide drugs with sterile saline solution, treatment dose according to CTX (380mg/kg, 200mg/kg, 100mg/kg) in experimental mice, dynamic observation of the experimental group (HD-CTX group, MD-CTX group, LD-CTX group), AA model group, normal control group of Mice Peripheral Blood 甯歌�勫彉鍖�,鍙屼晶鑲￠�ㄦ湁鏍哥粏鑳炴暎�

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