【作者】 罗庆；

【导师】 宋关斌； Diane S. Krause；

【作者基本信息】 重庆大学 ， 生物医学工程， 2009， 博士

【摘要】 急性巨核细胞白血病(Acute Megakaryoblastic Leukemia, AMKL),在急性髓系白血病(acute myeloid leukemia, AML)分型中也称为M7,至少同唐氏综合症(Down’s syndrome, DS)和t (1;22) (p13;q13)染色体易位两类染色体异常有关,伴有骨髓中原始巨核细胞(megakaryoblast)增加、骨髓纤维化和血小板减少等症状。t (1;22) (p13;q13)是AMKL特有的遗传特征,在1号和22号染色体上形成了RBM15 (RNA-binding motif protein 15, RBM15)和MKL1 (megakaryoblastic leukemia-1, MKL1)的融合基因。目前人们对于RBM15-MKL1融合蛋白在AMKL致病中发挥的作用所知有限,充分认识RBM15和MKL1正常情况下在造血系统中的功能特点有助于了解RBM15-MKL1融合蛋白在AMKL形成、发展中的作用。目前,关于MKL1在巨核细胞发育中的功能尚未见报道。本实验组之前的研究发现在人红白细胞白血病细胞(human erythroleukemia cells,HEL)中表达外源MKL1显著增加了TPA (12-O-tetradecanoylphorbol-13-acetate)刺激下巨核细胞的分化以及多倍体化。为了进一步研究MKL1在造血系统巨核细胞生成(megakaryopoiesis)中的作用,本文以动员人外周血CD34+细胞(mobilized human peripheral blood CD34+ cells, PB CD34+ cells)巨核细胞分化为研究对象,利用实时定量PCR考察MKL1基因在巨核细胞分化过程中的表达情况;通过慢病毒载体在PB CD34+细胞中表达MKL1,考察外源MKL1对PB CD34+细胞来源巨核细胞分化、成熟的影响;最后通过构建MKL1基因敲除小鼠模型,研究MKL1功能缺陷对小鼠外周血血小板数量和骨髓中巨核细胞生成的影响。主要研究工作和结果如下:①动员人外周血CD34+细胞来源巨核细胞体外两阶段分化模型的建立建立并优化了动员人外周血CD34+细胞体外分化为巨核细胞的两阶段培养条件:采用自制的Cocktail培养液和Stem Cell Technologies公司购买的CC100培养液刺激细胞增殖3天、4天、5天或6天,然后转入含有TPO (thrombopoietin)和SCF (stem cell factor)的分化培养液培养7天、8天或9天,培养结束后通过比较CD41+细胞和多倍体细胞相对初始CD34+细胞的倍增情况,优化培养条件。实验结果表明在本研究范围内,PB CD34+细胞在Cocktail培养液中扩增3天后转入分化培养液培养7天,细胞能获得16倍数量的CD41+细胞和3倍数量的多倍体巨核细胞,优于其它实验组。同时,该实验结果在CD41+细胞和多倍体细胞产量上优于目前常用的单阶段法体外PB CD34+细胞来源巨核细胞分化条件,可以为巨核细胞相关的体外研究提供大量的实验细胞来源。②MKL1基因在巨核细胞分化中的表达利用流式细胞仪和BSA非连续梯度法分离了不同成熟阶段的巨核细胞,实时定量RT-PCR研究MKL1在不同成熟阶段巨核细胞中的表达情况:1.5% BSA组(含67.2% CD41+细胞,1.3%多倍体巨核细胞)、3% BSA组(含74.4% CD41+细胞,39.3%多倍体巨核细胞)和流式分选细胞组(含98.0% CD41+细胞,21.5%多倍体巨核细胞)相对于对照组PB CD34+组(CD41+细胞和多倍体细胞含量均为0%)MKL1基因表达量的平均变化分别为3.0、6.8和5.0倍,均显著高于PB CD34+组(p<0.01)。此外,MKL1表达量流式分选组显著高于1.5% BSA组(p<0.01),3% BSA组显著高于流式分选组,提示MKL1基因在CD41+细胞中的表达量高于未分化的PB CD34+细胞,在成熟多倍体巨核细胞中的表达量高于单核巨核细胞,即随着CD34+细胞来源巨核细胞的分化和成熟,MKL1表达逐渐升高。③MKL1外源表达对PB CD34+细胞来源巨核细胞生成的影响利用慢病毒载体在PB CD34+细胞中表达外源MKL1,MKL1过表达的pCCL-MKL1组经两阶段法诱导分化后CD41+巨核细胞的比例为61.49%,显著高于pCCL对照组36.26%的分化比例(p<0.05)。在DNA分布方面,pCCL-MKL1组相对pCCL组,2N倍体细胞比例由35.46%降低为23.86% (p=0.013),4N倍体细胞比例由30.52%降低为21.52% (p=0.001),16N倍体细胞比例由9.15%增加为19.44% (p=0.014),32N倍体细胞比例由2.39%增加为7.77% (p=0.010),多倍体细胞(8N及以上倍体)总比例由31.10%增加为50.81% (p=0.001)。因此,MKL1外源表达促进了PB CD34+细胞来源巨核细胞分化,增加了巨核细胞中多倍体细胞比例,促进了巨核细胞的成熟。④MKL1功能缺陷对小鼠巨核细胞生成的影响构建MKL1基因敲除小鼠,通过外周血和骨髓细胞的研究发现,MKL1功能缺陷小鼠外周血中的红细胞、白细胞数量同野生型小鼠没有差异,血小板数量为5.2×105/μL显著低于野生型对照组8.2×105/μL (p<0.001),表现为血小板生成障碍;MKL1基因敲除小鼠骨髓细胞总数、LSK细胞(Lin-Sca-1+c-Kit+ cells, LSK cells)比例、pre-MegE (erythro-megakaryocytic progenitor cells, pre-MegE)细胞比例和巨核细胞集落形成能力与野生型小鼠没有显著差异;CD41+细胞比例显著高于对照组(p<0.0001),CD41+c-kit+巨核系祖细胞比例也显著高于野生型对照组(p<0.01);在DNA分布方面,MKL1基因敲除小鼠的骨髓CD41+细胞中,2N倍体细胞比例为32.15%显著高于野生型小鼠14.97%的比例(p<0.001);同时8N和16N倍体细胞比例MKL1 KO组为18.20%和19.67%,显著低于野生型对照组25.90% (p<0.01)和30.65% (p<0.05)的比例。因此,MKL1功能缺陷并未影响造血干/祖细胞到巨核细胞分化的早期过程,但阻滞了巨核系祖细胞或早期巨核细胞向多倍体巨核细胞的发育成熟过程,导致大量的未成熟的巨核细胞在骨髓中的堆积;而巨核细胞的成熟功能缺陷也直接导致了在外周血中血小板数量的减少。综合以上结论我们推测,MKL1直接或通过与其它蛋白共同作用参与了巨核细胞成熟过程的调控,RBM15-MKL1融合蛋白可能导致了MKL1指导巨核细胞多倍体化功能的丧失,这可能是t (1;22) (p13;q13)AMKL形成的原因。更多还原

【Abstract】 Acut megakaryoblastic leukemia (AMKL), also known as M7 in AML (acute myeloid leukemia), is associated with at least two distinct chromosome abnormalities: Down’s syndrome (DS) and t (1;22)(p13;q13) chromosomal translocation which are characterized by an expansion of megakaryoblasts in bone marrow, myelofibrosis and thrombocytopenia. t (1;22) (p13;q13) translocation is the unique character of AML and results in the fusion of RBM15 and MKL1 genes on chromosomes 1 and 22, respectively. So far, the role of RBM15-MKL1 fusion protein remains poor investigated. To understand the role of RBM15-MKL1 fusion protein in AMKL, we must understand the normal functions of RBM15 and MKL1 in hematopoiesis. However no role for MKL1 has been defined in hematopoietic differentiation.Our previous data indicated that the ectopic MKL1 expression promoted the differentiation and polyploidization of megakaryocytes induced from human erythroleukiea (HEL) cells with TPA. To further investigate the role of MKL1 in megakaryopoiesis, a two-phase megakaryocyte differentiation system of mobilized human peripheral blood CD34+ cells was developed; real-time quantitative PCR was used to detect relative MKL1 gene expression during megakaryocyte differentiation; lentivirus was constructed to study the effect of ectopic MKL1 expression on megakaryocyte differentiation and maturation; MKL1 gene knockout mice were produced to investigate the effect of MKL1 deficiency on platelet volume in peripheral blood and megakaryopoiesis in bone marrow.The main experiments and results are as follows:1. The development of two-phase megakaryocyte differentiation model of PB CD34+ cells in vitroA two-phase megakaryocyte differentiation model of PB CD34+ cells was developed and optimized. The homemade Cocktail medium and CC100 medium purchased from Stem Cell Technologies were used to induce expansion of PB CD34+ cells for 3, 4 or 5 days, respectively. And then the cells were tranfered into differentiation medium containing TPO and SCF for 7, 8 or 9 days. Culture conditions were optimized by comparing the relative fold expansion of CD41+ cells and polyploid cells to the initial CD34+ cells. After cultured in Cocktail medium for 3 days and in differentiation medium for additional 7 days, PB CD34+ cells obtained 16-fold expansion of CD41+ cells and 3-fold increasing of polyploid cells, which was the optimal condition we studied. With the optimized two-phase condition, PB CD34+ cells can be expanded and differentiate into more CD41+ and polyploid cells than only with one-phase culture protocol, which provides a new higher efficiency megakaryocyte differentiation model for our further researches.2. MKL1 expression during megakaryocyte differentiationMegakaryocytes at different mature stage of differentiation were selected by flow cytometry and BSA gradient and real time quantitative PCR was used to investigate the expression of MKL1. Comparing to PB CD34+ control group (with 0% CD41+ cell and polyploid cell), MKL1 expression of 1.5% BSA group (with 67.2% CD41+ cells and 1.3% polyploid cells), 3% BSA group (with 74.4% CD41+ cells and 39.3% polyploid cells) and FACS sorted group (with 98.0% CD41+ cells and 21.5% polyploid cells) was 3.0, 6.8 and 5.0 folds higher (p<0.01), respectively. Moreover, the expression of MKL1 in FACS sorted group was higher than 1.5% BSA group (p<0.01), and in 3% BSA group was higher than FACS sorted group (p<0.05). These results indicated that the expression of MKL1 was higher in CD41+ cells than in PB CD34+ cells and higher in more matured polyploid cells than mononuclear megakaryocytes, which suggested MKL1 was upregulated within the process of megakaryocyte differentiation and maturation.3. Effect of ectopic MKL1 expression on megakaryopoiesis of PB CD34+ cellsLentivirus was constructed to express MKL1 in PB CD34+ cells and megakaryocyte differentiation and polyploidization were studied. After cultured in optimized two-phase condition, pCCL-MKL1 group with enhanced MKL1 expression obtained 61.49% of CD41+ cells, which was significantly higher than 36.26% of pCCL control group (p<0.05). Comparing the DNA distribution, ectopic MKL1 expression decreased the percentage of cells with 2N ploidy from 35.46% to 23.86% (p=0.013), 4N ploidy from 30.52% to 21.52% (p=0.001), and increased the 16N ploidy from 9.15% to 19.44% (p=0.014), 32N ploidy from 2.39% to 7.77% (p=0.010). The total percentage of cells with polyploidy (8N and above) was increased from 31.10% to 50.81% (p=0.001). Thus, the ectopic MKL1 promoted the differentiation of PB CD34+ cells and increased the percentage of polyploid cells, which suggested that ectopic MKL1 expression promoted the maturation of megakaryocytes.4. Effect of MKL1 deficiency on mouse megakaryopoiesisMKL1 gene knockout mice were produced to investigate the effect of MKL1 deficiency on mouse megakaryopoiesis in vivo. MKL1 deficiency didn’t affect the number of red blood cells and white blood cells in peripheral blood, however the platelet number of MKL1 knockout mice was 5.2×105/μL, significantly lower than 8.2×105/μL of wild-type mice (p<0.001), suggesting a thrombocytopenia in MKL1 deficiency mice. There was no significant difference between MKL1 knockout mice and wild-type mice in bone marrow cell number, LSK cell percentage, pre-MegE cell percentage and the ability to form CFU-MK. The percentages of CD41+ cells (p<0.0001) and CD41+c-kit+ cells (p<0.01) in the bone marrow of MKL1 knockout mice were higher than wild-type. Comparing the DNA distribution of CD41+ cells in bone marrow, in MKL1 knockout mice, the cells with 2N ploidy was 32.15%, higher than 14.97% in wild-type (p<0.001). Meanwhile, the percentages of cells with 8N and 16N ploidy were 18.20% and 19.67% in MKL1 knockout mice, which were lower than 25.90% (p<0.01) and 30.65% (p<0.05) in wild-type mice. The initial differentiation from hematopoietic stem/progenitor cells to megakaryocyte progenitor cells was not affected by the MKL1 deficiency, however the maturation of megakaryocyte progenitor cells or early stage megakaryocytes was blocked and resulted in the accumulation of immature megakryoblasts in the bone marrow. The deficiency of megakaryocyte maturation may be responsible for the decreased platelet volume in peripheral blood.In summary, our results suggested that MKL1 acts an important role in megakaryocyte maturation. For this reason, RBM15-MKL1 fusion protein may affect the function of endogenous MKL1, inhibit the polyploidization of megakaryocyte differentiation and results in t (1;22) (p13;q13) AMKL.更多还原