【作者】 周健；

【导师】 宋永平；

【作者基本信息】 郑州大学 ， 内科血液学， 2004， 硕士

【摘要】 背景与目的：异基因造血干细胞移植(allo-HSCT)成为近年来治疗恶性血液病、再生障碍性贫血、自身免疫性疾病、某些遗传性疾病及实体肿瘤的一种重要的有效手段。人类巨细胞病毒(human cytomegalovirus，hCMV)感染是造血干细胞移植患者早期的主要感染并发症及死亡原因，既往因CMV感染导致的间质性肺炎死亡率高达80％以上。CMV属于人类疱疹病毒乙组DNA病毒，在人群中感染非常普遍(80％～100％)，正常人感染CMV后，由于机体免疫功能正常，CMV在感染细胞内复制水平低下，以一种潜伏整合状态存在，通常无临床症状。当机体免疫功能尤其是细胞免疫功能受到抑制，潜伏的CMV可被重新激活，从而持续高水平的复制，引起CMV活动性感染，产生有严重临床症状的CMV感染性疾病。移植物抗宿主病(GVHD)是异基因造血干细胞移植的主要障碍，CMV感染可以诱导GVHD出现：CMV感染还可影响造血干细胞的植入，使造血重建延迟；引起间质性肺炎；是继发曲霉菌感染的高危因素，易并发细菌、真菌、原虫等混合感染，从而造成移植失败。近年来，随着大量新型免疫抑制剂的出现及多种免疫抑制剂联合应用，非亲缘关系造血干细胞移植、单倍体造血干细胞移植、非清髓造血干细胞移植广泛开展，造血干细胞移植得到快速发展。同时巨细胞病毒感染率也不断增高，已引起医学界高度重视，CMV感染引起严重疾病再次成为医学领域的研究热点。虽然目前更昔洛韦的使用使CMV疾病的治疗有较大转机，但取得抗CMV显著疗效的切入点还在于早期诊断和及时治疗。在CMV病发生之前，及时应用抗病毒治疗，可以预防和减少CMV病的发生，郑州大学2004届硕士研究生毕业论文异基因造血干细胞移植中巨细胞病毒的检测及临床意义降低移植受者的死亡率。巨细胞病毒活动性感染早期所致疾病临床表现多为非特异性的，早期诊断主要依靠实验室检测。尽管目前诊断CMV手段日臻完善，但诊断方法多种多样，哪种方法更能简便快捷、灵敏可靠地早期诊断，从而进行早期干预性治疗争议很大。本研究分别应用ELISA、流式细胞仪、荧光定量PCR检测巨细胞病毒IgG、lgM抗体、pp65抗原、DNA负荷定量，旨在探讨HcMv的早期有效诊断方法。 材料与方法:本研究分别采用酶联免疫吸附法(ELISA)检测血清巨细胞病毒抗体IgG、IgM;流式细胞仪检测巨细胞病毒抗原pp65;荧光聚合酶链反应(FQ一PCR)核酸扩增技术检测血浆巨细胞病毒DNA负荷量。选择造血干细胞移植术受者(移植组)预处理前常规检测lgG、IgM抗体，排除IgM抗体阳性移植患者。于回输当天、回输后每10天左右;正常造血干细胞移植健康供者(正常组)，于粒细胞集落刺激因子(G一CSF)动员之前;长期化疗的白血病患者(化疗组)于化疗后白细胞升至2.0xl夕/L时，同时检测巨细胞病毒血清IgG、IgM抗体、粒细胞pp65抗原、血浆DNA负荷量。ELISA采用间接法，待测抗原包被微孔、洗板、加抗原一酶结合复合物、孵育后洗板，加底物液后置室温暗处显色，用酶联免疫检测仪(波长45Onln)测定OD值。流式细胞仪检测采用单色间标法，取标本分离单个核细胞、固定、破膜、分别加入pp“抗体和小鼠IgGI抗原(对照)，再加FITC标记的兔抗鼠IgGI抗体(二抗)，用流式细胞仪检测阳性细胞数比例。荧光定量PCR检测采用外标法，用DNA提取液提取血浆DNA后和阴、阳性对照一起加入荧光引物、Taq酶等反应液，置于荧光PCR扩增仪测定DNA含量。统计学分析:所有资料采用SPSS 10.0软件处理，a=0.05为显著性检验水准。 结果: (l)巨细胞病毒IgG抗体阳性检出率在正常组、化疗组、移植组之间分别为82.24%，81 .48%和86.91%，三者之间的差别无统计学意义(尸>0.05)。 (2)在化疗组中IgM抗体、pp65抗原、DNA定量三者阳性检出率差别无统计学意义(P>0.05)o (3)正常组中巨细胞病毒lgM抗体和DNA荧光定量、pp65抗原流式细胞仪检测结果基本相同。郑州大学2004届硕士研究生毕业论文异基因造血干细胞移植中巨细胞病毒的检测及临床意义 (4)移植组中巨细胞病毒IgM抗体阳性检测率明显低于DNA荧光定量和pp65抗原流式细胞仪检出率，其差别有统计学意义(p<0.05)。两种检测方法有关系，但关系不密切。 (5)无论在移植组还是化疗组中巨细胞病毒DNA荧光定量检测与pp65抗原流式细胞仪阳性检出率的差别都无统计学意义(尸>0.05)，两种检测方法有明显的相关性。 (6)lgM抗体、I〕NA荧光定量和pp65抗原流式细胞仪检测阳性率各自在化疗组和移植组之间的差异具有显著性(P<0.05)。 (7)移植组治疗中应用免疫抑制剂三种及三种以上与三种以下在DNA荧光定量和pp65抗原流式细胞仪检测阳性率的差异有统计学意义(尸<0.05)。 结论: (1) ELIsA法检测血清cMV IgG和IgM抗体相结合可以用于造血干细胞移植术供者的选择。IgM抗体不适用于移植术后早期诊断CMV激活感染，但可用于化疗后CMV活动性感染的诊断，还可以用于筛选阳性受者及时采取措施。 (2)流式细胞仪检测CMV PP65抗原快捷、客观、易于定量，适用于造血干细胞移植术后造血恢复以后CMV感染的诊断。 (3)荧光定量PCR检测血浆CMV DNA病毒方法简单快速、更多还原

【Abstract】 Background and Objective: Allogeneic hematopoietic stem cell transplantation, allo-HSCT has been an effective way to treat malignant hematologic disease, aplastic anemia, autoimmune diseases and some solid tumors. Human cytomegalo-virus (hCMV) infection is the early main infection and death-cause to the recipients of allo-HSCT. The prevenient mortality of interstitial pneumonia (I?) caused by CMV infection was above 80 precent. CMV belongs to DNA virus of human herpersviruses, and CMV is very common among people. When infecting normal people, because of the normal immune function of the host, CMV reproduces slow, stays in a latent and concordant state and usually causes no clinical syndrome. When the host immune function especially the cellar immunofunction is suppressed, the latent CMV can be reactivated, and CMV reproduce in a high level constantly, the causes CMV active infection and CMV infectious disease with serious clinical syndromes. At the same time the infectious rate of CMV has been increasing constantly and drawing much attention to by the medical field, then the serious disease caused by CMV infection became a focal point of medical research again. Graft-verse-host disease, GVHD is a main obstacle to the allo-HSCT, and CMV can induce GVHD; affluence the survival of HSCT, delaying the hematopoiesis rebuilding; causing IP. It is a high-dangerous factors of secondary aspergillosis infection, also can be subject to bacteria, fungi andprotozoon infections ,thus lead to to the failure of HSCT. As the occurrence and combined application of many new immune inhibitors, unrelated-HSCT, haploid-HSCT, non-myeloablative-HSCT developed extensively, and HSCT has been developed quickly in recent years. Though the treatments of CMV using ganciclovirus have turned for better, the key of antivirus therapy depends on early-stage diagnose and timely treatment. Using antivirus treatment in time before the occurrence of CMV diseases can prevent and decrease the occurrence of the disease and lower the mortality of HSCT recipients . The early clinical signs and symptoms of the disease by CMV active infection are mostly non-specific, and its early diagnosis mainly depends on laboratorial examination. At present methods for CMV diagnosis are various. Inspite of the more intact diagnostic methods, there is much controversy on which method is simpler, more sensitive and reliable to diagnose CMV early, and then early intervening treatment is in great dilemma. In this paper, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry (FCM), fluorescence quantitative PCR (FQ-PCR/real-time PCR) to examine IgG antibodies and IgM antibodies of CMV, pp65 antigen, quantity of DNA for exploring an effective way to diagnose early CMV infection.Methods and Material: In our research, we used ELISA to detect IgG antibodies, IgM antibodies of CMV; used FCM to detect pp65 antigen; used FQ-PCR to examine HCMV DNA load of blood plasma. Before conditioning we examined CMV IgG, IgM and excluded IgM positive HSCT recipients from our research. We simultaneously examined CMV IgG, IgM, pp65 antigen and DNA load of recipients (the transplantation group) at the day of transfusion of hematopoietic stem cell, and every 10 days after the transfusion of hematopoietic stem cells, of normal HSCT donors (the normal group) before G-CSF mobilization; of leukemic patients (the chemotherapy group)with long term chemotherapy when their WBC to 2.0x109/L after chemo- therapy. We used indirect ELISA: the antigen to examine was in lamellan aperture, washing plate, added Ag-enzyme complex, washing plate again after incubation added substratum, put in darkness to colorate in room temperature,then examined OD valves by enzyme immunoassay (X=450nm); we used indirect simple color method by FCM selecting single-nuclear cells, then fixing, effecting cell membrance, adding pp65 antibodies and mouse IgG1 antigen (the control) then adding rat-anti-mouse IgGl (antibody), then detecting the proportion of positive cells. The FQ-PCR took the outer-marking method: abstrac更多还原