骨髓增生异常综合征论文：MDS、AA和AL患者骨髓细胞周期及增殖特征的研究

骨髓增生异常综合征论文：MDS、AA和AL患者骨髓细胞周期及增殖特征的研究【中文摘要】检测骨髓增生异常综合征(myelodysplastic syndrome, MDS)、再生障碍性贫血(aplastic anemia, AA)和急性白血病(acute leukemia, AL)患者骨髓单个核细胞(bone marrow mononuclear cells, BMMNC)的细胞周期分布及CD34、Ki67抗原的表达。

方法选取2009年6月至2010年6月在山东大学附属省立医院就诊的住院及门诊患者68例,MDS组30例,其中难治性贫血(refractory anemia, RA)组18例,难治性贫血伴原始细胞增多(refractory anemia with excess of blasts, RAEB)组12例；AA组22例,其中急性重型再障组7例,慢性非重型再障组15例；AL组16例,其中急性髓系白血病(acute myeloid leukemia, AML)组12例,急性淋巴细胞白血病(acute lymphoblastic leukemia, ALL)组4例。

另外选取正常对照组18例(均为非血液病患者)。

采用常规骨髓穿刺术,抽取骨髓各2ml；应用流式细胞仪(flow cytometry, FCM),通过碘化丙啶(propidium iodide, PI)细胞核染色及免疫荧光双标法,对其BMMNC 的细胞周期分布及CD34、Ki67的表达进行检测。

结果1.MDS和AL患者与正常对照组患者骨髓单个核细胞中细胞周期分布及增殖特征表达的比较与正常对照组相比,MDS组和AL组患者其BMMNC中细胞周期G0/G1期、造血干/祖细胞(haemopoietic stem cell, HSC)CD34细胞、Ki67+细胞、CD34+Ki67+细胞、CD34+细胞中Ki67+细胞和Ki67+细胞中CD34+细胞各自的比例均显著升高(P0.05),而CD34+细胞中Ki67+细胞比例显著降低(P<0.05),且CD34+细胞、CD34+Ki67+细胞和Ki67+细胞中CD34+细胞比例均显著降低(P<0.01)。

4.AA患者和RA患者骨髓单个核细胞中细胞周期分布及增殖特征表达的比较与AA组相比,RA组BMMNC中G0/G1期、CD34+细胞、Ki67+细胞、CD34+Ki67+细胞、CD34+细胞中Ki67+细胞和Ki67+细胞中CD34+细胞比例均显著升高(P<0.01),而RA组的S和S+G2/M期细胞比例均显著降低(P<0.01)。

结论1.MDS患者骨髓细胞G1期阻滞且伴有造血干/祖细胞的高增殖特性,可解释MDS患者骨髓细胞分化成熟障碍,在细胞形态学上可表现为病态造血,在临床上可表现为骨髓增生活跃而外周血细胞减少,支持MDS的本质是造血干/祖细胞的恶性克隆性疾病。

2.MDS与AL患者骨髓细胞存在G1期阻滞的同时,都伴有造血干/祖细胞的高增殖特性。

由RA进展到RAEB,再由RAEB进展到AL,BMMNC中CD34+细胞、CD34+细胞中Ki67+细胞比例均升高,G0/G1期细胞比例、Ki67+细胞比例亦升高,提示来源于造血干/祖细胞的恶性克隆性扩增逐渐获得优势而积聚,和/或其细胞本身质的缺陷越来越严重；MDS的不同发展阶段及其最终转化为急性白血病,从细胞周期理论及其增殖特征分析,它们之间并无质的差异,可能是同一疾病的不同发展阶段,支持MDS和AL 具有相似的发病机制。

3.与正常对照组相比,AA患者骨髓细胞的细胞周期分布差异无统计学意义,不支持AA是造血干/祖细胞恶性克隆性疾病,同时AA患者的造血干/祖细胞不仅数量减少,而且增殖活性减低,不同于MDS和AL患者。

4.RA和AA患者相比,其骨髓细胞周期分布及增殖特性的表达差异均具有显著性意义,可用于二者的诊断及鉴别诊断,尤其是BMMNC中CD34+Ki67+细胞比例两者差异更大,用作本研究RA与AA的鉴别诊断指标准确率达100%。

【英文摘要】To explore the cell cycle distributions and the expressions of CD34 and Ki67, the cell proliferation-related protein in bone marrow mononuclear cells among myelodysplastic syndromes, aplastic anemia, and acute leukemiapatients.METHODS Bone marrow aspirates were collected from 68 patients between June 2009 and June 2010 from the Department of Hematology, Provincial Hospital affiliated to Shandong University. The 68 cases consist of 30 MDS, including 18 refractory anemia and 12 refractory anemia with excess blasts,22 AA, including 7 severe aplastic anemia and 15 none severe aplastic anemia, and 18 AL, including 12 acute myeloid leukemia and 4 acute lymphoblastic leukemia.18 healthy individuals (non-hematologic patients) were used as normal control. Take 2 ml bone marrow from the patients. Propidium iodide and immunofluorescent double staining through flow cytometry were utilized to explore cell cycle distributions and the expression of CD34 and Ki67, the cell proliferation-related protein in bone marrow mononuclear cells.RESULTS1. Compared with the control group, the percentages of G0/G1 phase of cellcycle, haemopoietic stem/progenitor cells CD34+ cells, Ki67+ cells, CD34+Ki67+ cells, Ki67+ cells in CD34+ cells, and CD34+ cells in Ki67+ cells of BMMNC were all significantly increased in the MDS and AL group (P0.05). The percentages of Ki67+ cells in CD34+ cells was significantly lower (P<0.05), as well as the percentages of CD34+ cells, CD34+Ki67+ cells, and CD34+ cells in Ki67+ cells of BMMNC were all significantly lower in the AA group than those in the control group (P<0.01).4. The percentages of G0/G1 phase, CD34+, Ki67+, CD34+Ki67+, Ki67+ cells in CD34+, and CD34+ cells in Ki67+ cells were all significantly increased (P<0.01), although the proportions of S and S+G2/M phases were both significantly decreased in the RA group than the AA group (P<0.01).CONCLUSIONS1. Most of the cells are kept at the G1 phase as well as the high proliferation of the haemopoietic stem/progenitor cells in MDS patients that can explain the dysdifferentiation and dysmaturity, which is the impaired peripheral blood cell production (cytopenias) as well as most commonly a hypercellular, dysplastic-appearing bone marrow. This greatly proves that the disorder of MDS might occur when the malignant damage of the haemopoieticstem/progenitor cells occurs.2. There are higher percentages of G1 phase as well as the high proliferations of the CD34+ cellsin both MDS and AL patients. In our study, the ratios of G0/G1 phase, CD34+, Ki67+, and Ki67+ cells in CD34+cells were all significantly higher as RAEB progressed to AL as well as RA progressed to RAEB. This indicates that the disorder of MDS evolves the different stages and may eventually have the transformation to acute leukemia because of the expansion of malignant clonal cells from the haemopoietic stem/progenitor cells or because of more and more seriously qualitative defections in cells. Both of the two disorders may have the similar pathogenesis and may be the different stage of a common disease according to the theory of cell cycle and cell proliferation.3. There was no significant difference on the cell cycle between patients with AA and normal controls. It proves that AA is an illness without the malignant clonal mutation of haemopoietic stem/progenitor cells. We also found that besides the haemopoietic stem/progenitor cells were lower, the expressions of proliferations of BMMNC were all significantly lower in the AA group. It is different from patients with MDS and AL.4. The disturbance of cell cycle and the expression of cell proliferation were all different in patients with RA from AA, which can be used for the diagnosis and differential diagnosis of them. It is most different on theratio of CD34+Ki67+ cells that may be 100% accuracy for the diagnosis and differential diagnosis of RA and AA.【关键词】骨髓增生异常综合征贫血,再生障碍性急性白血病细胞周期细胞增殖【英文关键词】Myelodysplastic syndromes Anemia, aplastic Acute leukemia Cell cycle Cell proliferation【目录】MDS、AA和AL患者骨髓细胞周期及增殖特征的研究中文摘要6-9ABSTRACT9-11符号说明12-17前言17-20对象和方法20-23结果23-25讨论25-32结论32-33附图和附表33-35参考文献35-39综述39-46参考文献44-46英文文章46-60致谢60-61攻读硕士研究生期间发表的论文目录61-62学位论文评阅及答辩情况表62。