【作者】 李辉；

【导师】 彭军；

【作者基本信息】 山东大学 ， 内科学（专业学位）， 2014， 硕士

【摘要】 研究背景与目的再生障碍性贫血(简称再障)(aplastic anemia,AA)是一种获得性人类骨髓衰竭综合征,以全血细胞减少为临床特征,其发病率约为为0.74/10万人口。再障的发病呈明显的异质性和重叠性特征,目前认为其发病机制为免疫介导的1类细胞毒性T淋巴细胞的激活。再障的治疗采用干细胞移植和免疫抑制药物为主的综合治疗措施。MicroRNA (miRNA)是一种小保守非编码RNA分子,主要在翻译后水平调节基因的表达,对于正常细胞生长、增殖、分化、凋亡等功能的维持和多种免疫过程的调节起着至关重要的作用,目前已发现多种miRNA在免疫性血小板减少症、类风湿性关节炎和系统性红斑狼疮等多种免疫相关性疾病有异常表达。miRNA芯片检测结果发现miR-34a在再障患者骨髓T淋巴细胞中有差异表达,其表达量高于正常对照。本研究的目的是探讨miR-34a在各型再障患者骨髓T淋巴细胞中表达状态的临床特点。研究方法我们选取了再障患者及健康对照者作为研究对象。2012年3月至2013年9月期间于山东大学齐鲁医院血液科病房和门诊收集初诊再障患者31例,皆符合国内获得性再障的诊断标准,其中非重型再障(NSAA)16例,重型再障(SAA)10例,极重型再障(VSAA)5例。14例健康者作为正常对照组(NC)。收集以上病例及健康对照组的临床资料：一般资料包括年龄和性别,实验室检查包括粒细胞计数、网织红细胞计数、血小板计数等。手工法分离31例再障患者及14例正常对照者骨髓T淋巴细胞,然后采用实时定量荧光PCR测定其中的miR-34a的表达水平。采用SPSS17.0分析性别、年龄、再障严重程度、中性粒细胞(NEU)计数、网织红细胞(RET)计数、血小板(PLT)计数与miR-34a水平的关系。研究结果1.不同性别(P=0.5109)的再障患者骨髓T淋巴细胞miR-34a的表达水平无显著性差异。年龄与再障患者骨髓T淋巴细胞miR-34a的表达水平无明显相关(r=-0.2608,P=0.1565)。2.再障患者与健康对照骨髓T淋巴细胞miR-34a的表达水平有显著性差异(P<0.0001),再障患者骨髓T淋巴细胞miR-34a的表达水平明显增高；且不同严重程度再障患者骨髓T淋巴细胞miR-34a的表达水平皆明显高于正常(VSAA vs.NC, P<0.0001; SAA vs. NC,p<0.0001; NSAA vs. NC, P<0.0001)。对于不同严重程度AA患者之间骨髓T淋巴细胞miR-34a的表达水平,VSAA与SAA (P=0.0324)、VSAA与NSAA (p=0.0192)有显著性差异,而SAA与NSAA无明显差异(P=0.9054)。3.再障患者骨髓T淋巴细胞miR-34a的表达水平与再障的严重程度有相关性,且呈正相关关系(rs=0.451,P=0.011)。4.再障患者NEU计数(r=-0.4279,P=0.0163)、RET计数(r=-0.3603,P=0.0465)与骨髓T淋巴细胞miR-34a的表达水平皆有相关性,且呈负相关关系,而PLT计数(r=-0.2005,P--0.2794)与骨髓T淋巴细胞miR-34a的表达水平无明显相关性。结论我们的结果表明,再障患者骨髓T淋巴细胞中miR-34a的表达水平是明显高于正常对照的,且与性别和年龄无关。再障患者骨髓T淋巴细胞miR-34a的表达水平与再障的严重程度呈正相关关系。再障患者骨髓T淋巴细胞miR-34a的表达水平与NEU计数、RET计数呈负相关关系,而与PLT计数无明显相关。由此可以得出结论,miR-34a的表达水平对于再障的临床诊断与分型、治疗和预后评估具有一定的指导意义。更多还原

【Abstract】 Backgrounds and ObjectivesAplastic anemia(AA), characterized by pancytopenia, is a paradigm of the human bone marrow failure syndromes. The pathophysiology of AA is mostly immune mediated, with activated type1cytotoxic T cells implicated. AA can now be cured or ameliorated by stem-cell transplantation or immunosuppressive drug therapy. MicroRNA (miRNA)s are a family of evolutionarily conserved small non-coding RNAs regulating gene expression by posttranscriptional gene repression or mRNA degradation, which have been found to control cell division, differentiation, death and normal immune function. To date, various miRNAs have been found abnormally expressed in immune-related diseases such as ITP(immune thrombocytopenia), RA(retinoid arthritis) and SLE(systemic lupus erythematosus). MiRNA chip result found that miR-34a was expressed significantly higher in AA patients bone marrow T cells than those in normal controls. This research is to detect the expression pattern of miR-34a in bone marrow T cells of different types of AA patients.MethodsBone marrow T cells were isolated from31AA patients and14healthy controls, whose miR-34a expressions were detected using quantitative real time RT-PCR. The relationship between sex, age, AA severity, NEU number, RET number, PLT number and miR-34a level was analyzed respectively using SPSS17.0.Results1. No significant differences of miR-34a level were detected in AA patients bone marrow T cells with different sexes(P=0.5109) or ages(P=0.1565).2. Significantly higher miR-34a level was detected in AA patients bone marrow T cells than in normal controls(p<0.0001), showing a positive correlation with AA severity(r=0.451,p=0.011).3. MiR-34a level in AA patients bone marrow T cells was in a negative correlation with NEU number(r=-0.4279,p=0.0163) and RET number(r=-0.3603,P=0.0465), while uncorrelated with PLT number(r=-0.2005,P=0.2794).ConclusionsOur results show that miR-34a level in AA patients bone marrow T cells was significantly higher than in normal controls, which was with a positive correlation with AA severity and a negative correlation with NEU number and RET number, but was uncorrelated with sex, age and PLT number. This is instructive for laboratory diagnosis, typing, clinical therapy choice and prognosis in AA.更多还原