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。
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 vitro
A 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 differentiation
Megakaryocytes 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+ cells
Lentivirus 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 megakaryopoiesis
MKL1 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.
引文
[1]张之南,杨天楹,郝玉书主编.血液病学(上册) [M].北京:人民卫生出版社, 2003.
[2]黄晓军主编.血液病[M].北京:中国医药科技出版社, 2003.
[3]林果为,余润泉主编.造血系统疾病的诊断与鉴别诊断[M].天津:天津科学技术出版社, 2004.
[4] A. Jemal, A. Thomas, T. Murray, et al. Cancer statistics, 2002 [J]. CA Cancer J Clin, 2002, 52: 23-47.
[5] J. Bennett, D. Catovsky, M. Daniel, et al. Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group [J]. Br J Haematol, 1976, 33: 451-458.
[6]雷琦,刘英,唐锁勤.儿童急性巨核细胞白血病[J].中国实验血液学杂志, 2007, 15: 528-532.
[7] K. Yumura-Yagi, J. Hara, A. Tawa, et al. Phenotypic characteristics of acute megakaryocytic leukemia and transient abnormal myelopoiesis [J]. Leuk Lymphoma, 1994, 13: 393-400.
[8] K. Kaushansky. Historical review: megakaryopoiesis and thrombopoiesis [J]. Blood, 2008, 111: 981-986.
[9] J. Italiano, S. Jr., Patel-Hett, J. Hartwig. Mechanics of proplatelet elaboration [J]. J Thromb Haemost, 2007, 5 Suppl 1: 18-23.
[10] R. Levine. Isolation and characterization of normal human megakaryocytes [J]. Br J Haematol, 1980, 45: 487-497.
[11] I. Branehog, B. Ridell, B. Swolin, et al. Megakaryocyte quantifications in relation to thrombokinetics in primary thrombocythaemia and allied diseases [J]. Scand. J. Haematol. 1975, 15: 321–332.
[12] D. Barnard, T. Alonzo, R. Gerbing, et al. Comparison of childhood myelodysplastic syndrome, AML FAB M6 or M7,CCG2891: report from the Children’s Oncology Group [J]. Pediatr Blood Cancer, 2007, 49: 17-22.
[13]阮长耿,吴德沛,李建勇等主编.现代血液病诊断治疗学[M].安徽:安徽科学技术出版社, 2007.
[14] S. Lewis, L. Szur. Malignant myelosclerosis [J]. Br Med J, 1963, 2:472-477.
[15] H. Hassan, M.Freund. Characteristic biological features of human megakaryoblastic leukaemia cell lines [J]. Leuk Res, 1995, 19: 589-594.
[16] L. Pagano, A. Pulsoni. Acute megakaryoblastic leukemia: experience of GIMEMA trials [J].Leukemia, 2002, 16: 1622-1626.
[17] Y. Ravindranath, E. Abella, J. Krischer, et al. Acute myeloid leukemia (AML) in Down's syndrome is highly responsive to chemotherapy: experience on Pediatric Oncology Group AML Study 8498 [J]. Blood, 1992, 80: 2210-2214.
[18] A. Gamis, W. Woods, T. Alonzo, et al. Increased age at diagnosis has a significantly negative effect on outcome in children with Down syndrome and acute myeloid leukemia: a report from the Children's Cancer Group Study 2891 [J]. J Clin Oncol, 2003, 21: 3415-3422.
[19] U. Creutzig, D. Reinhardt, S. Diekamp, et al. AML patients with Down syndrome have a high cure rate with AML-BFM therapy with reduced dose intensity [J]. Leukemia, 2005, 19: 1355-1360.
[20] B. Zeller, G. Gustafsson, E. Forestier, et al. Acute leukaemia in children with Down syndrome: a population-based Nordic study [J]. Br J Haematol, 2005, 128: 797-804.
[21] A. Rao, R. Hills, C. Stiller, et al. Treatment for myeloid leukaemia of Down syndrome: population-based experience in the UK and results from the Medical Research Council AML 10 and AML 12 trials [J]. Br J Haematol, 2006, 132: 576-583.
[22] A. Hama, H. Yagasaki, Y. Takahashi, et al. Acute megakaryoblastic leukaemia (AMKL) in children: a comparison of AMKL with and without Down syndrome [J]. Br J Haematol, 2008, 140: 552-561.
[23] D. Reinhardt, S. Diekamp, C. Langebrake, et al. Acute megakaryoblastic leukemia in children and adolescents, excluding Down’s syndrome: improved outcome with intensified induction treatment [J]. 2005, Leukemia, 19: 1495–1496.
[24] J. Hitzler. Acute megakaryoblastic leukemia in Down syndrome [J]. Pediatr Blood Cancer, 2007, 49: 1066-1069.
[25] S. Gurbuxani, P. Vyas, J. Crispino. Recent insights into the mechanisms of myeloid leukemogenesis in Down syndrome [J]. Blood, 2004, 103: 399-406 .
[26] S. Malinge, S. Izraeli, J.Crispino. Insight into the nanifestations, outcomes, and mechanisms of leukemogesis in Down syndrome [J]. Blood, 2009, 113: 2619-2628.
[27] A. Carroll, C. Civin, N. Schneider, et al. The t (1;22) (p13;q13) is nonrandom and restricted to infants with acute megakaryoblastic leukemia: a Pediatric Oncology Group Study [J]. Blood, 1991, 78: 748-752.
[28] T. Lion, O. Haas. Acute megakaryocytic leukemia with the t (1;22)(p13;q13) [J]. Leuk Lymphoma, 1993, 11: 15-20.
[29] Z. Ma, S. Morris, V. Valentine, et al. Fusion of two novel genes, RBM15 and MKL1, in the t (1;22)(p13;q13) of acute megakaryoblastic leukemia [J]. Nat Genet, 2001, 28: 220-221.
[30] T. Hassold, S. Sherman. Down syndrome: genetic recombination and the origin of the extra chromosome 21 [J]. Clin Genet, 2000, 57: 95-100.
[31] M. Zwaan, D. Reinhardt, J. HItzler, et al. Acute leukemias in children with down syndrome [J]. Pediatr Clin North Am, 2008, 55: 53-70.
[32] J. Taub, Y. Ge. Down syndrome, drug metabolism and chromosome 21 [J]. Pediatr Blood Cancer, 2005, 44: 33-39.
[33] F. Wiseman, K. Alford, V. Tybulewicz, et al. Down syndrome-recent progress and future prospects [J]. Hum Mol Genet, 2009, 18: 75-83.
[34] A. Creavin, R. Brown. Ophthalmic abnormalities in children with Down syndrome [J]. J Pediatr Ophthalmol Strabismus, 2009, 46: 76-82.
[35] Y. Hawli, M. Nasrallah, G. EI-Hajj Fuleihan. Endocrine and musculoskeletal abnormalities in patients with Down syndrome [J]. Nat Rev Endocrinol, 2009, 5: 327-334.
[36] B. Lange, N. Kobrinsky, D. Barnard, et al. Distinctive demography, biology, and outcome of acute myeloid leukemia and myelodysplastic syndrome in children with Down syndrome: Children's Cancer Group Studies 2861 and 2891 [J]. Blood, 1998, 91:608-615.
[37] J. Bernstein, N. Dastugue, O. Haas, et al. Nineteen cases of the t (1;22)(p13;q13) acute megakaryblastic leukaemia of infants/children and a review of 39 cases: report from a t (1;22) study group [J]. Leukemia, 2000, 14: 216-218.
[38] H. Hsiao, M. Yang, Y. Liu, et al. RBM15-MKL1 (OTT-MAL) fusion transcript in an adult acute myeloid leukemia patient [J]. Am J Hematol. 2005, 79: 43-45.
[39] T. Lion, O. Haas, J. Harbott, et al. The translocation t (1;22)(p13;q13) is a nonrandom marker specifically associated with acute megakaryocytic leukemia in young children [J]. Blood, 1992, 79: 3325-3330.
[40] T. Mercher, M. Coniat, F. Khac, et al. Involvement of a human gene related to the Drosophila spen gene in the recurrent t (1; 22) translocation of acute megakaryocytic leukemia [J]. Proc Natl Acad Sci USA, 2001, 98: 5776-5779.
[41] R. Paredes-Aguilera, L. Romero-Guzman, N. Lopez-Santiago, et al. Biology, clinical, and hematologic features of acute megakaryoblastic leukemia in children [J]. Am J Hematol. 2003, 73: 71-80.
[42] A. Gamis. Acute myeloid keukemia and Down syndrome evolution of modern therapy—state of the art review [J]. 2005, 44: 13-20.
[43] T. Sawada, C. Nishiyama, T. Kishi, et al. Fusion of OTT to BSAC results in aberrant up-regulation of transcriptional activity [J]. J Biol Chem, 2008, 283: 26820-26828.
[44] G. Raffel, T. Mercher, H. Shigematsu, et al. Ott1 (Rbm15) has pleiotropic roles inhematopoietic development [J]. Proc Natl Acad Sci U S A, 2007, 104: 6001-6006.
[45] X. Ma, M. Renda, L. Wang, et al. Rbm15 modulates Notch-induced transcriptional activation and affects myeloid differentiation [J]. Mol Cell Biol, 2007, 27: 3056-3064.
[46] K. Du, M. Chen, J. Li, et al. Megakaryoblastic leukemia factor-1 transduces cytoskeletal signals and induces smooth muscle cell differentiation from undifferentiated embryonic stem cells [J]. J Biol Chem, 2004, 279: 17578-17586.
[47] A. Selvaraj, R. Prywes. Megakaryoblastic leukemia-1/2, a transcriptional co-activator of serum response factor, is required for skeletal myogenic differentiation [J]. J Biol Chem, 2003, 278: 41977-41987.
[48] E. Cheng, Q. Luo, M. Emanuela, et al. Role for MKL1 in megakaryocytic maturation [J]. Blood, 2009, 113: 2826-2834.
[49]姚泰主编.生理学(第六版) [M].北京:人民卫生出版社, 2007.
[50] I. Weissman, J. Shizuru. The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmue diseases [J]. Blood, 2008, 112: 3543-3553.
[51] H. Kawamoto, Y. Katsura. A new paradigm for hematopoietic cell lineages: revision of the classical concept of the myeloid-lymphoid dichotomy [J]. Trends Immunol, 2009, 30: 193-200.
[52]裴雪涛主编.干细胞生物学[M].北京:科学出版社, 2003.
[53]王琦如,谭孟群,程腊梅主编.造血生理学[M].长沙:中南大学出版社, 2005.
[54]达万明,裴雪涛主编.现代血液病学[M].北京:人民军医出版社2003.
[55] E. Bruno, R. Hoffman. Human megakaryocyte progenitor cells [J]. Semin Hematol, 1998, 35: 183-191.
[56] M. Long. Megakaryocyte differentiation events [J]. Semin Hematol, 1998, 35: 192-199.
[57] A. Wickrema, J. Crispino. Erythroid and megakaryocytic transformation [J]. Oncogene, 2007, 26: 6803-6815.
[58] I. Matsumura, Y. Kanakura. Molecular control of megakaryopoiesis and thrombopoiesis [J]. Inter J Hematol, 2002, 75: 473-483.
[59] D. Bluteau, L. Lordier, A. DI Stefano, et al. Regulation of megakaryocyte maturation and platelet formation [J]. J Thromb Haemost, 2009, 7 (Suppl 1): 227-234).
[60] M. Long. Thrombopietin stimulation of hematopoietic stem/progenitor cells [J]. Curr Opin Hematol, 1999, 6: 159-163.
[61] S. Bord, E. Frith, D. Ireland, et al. Estrogen stimulates differentiation of megakaryocytes and modulates their expression of estrogen receptors alpha and beta [J]. J Cell Biochem, 2004, 92:249-257.
[62] Z. Han, L. Sensebe, J. Abgrall, et al. Platelet factor 4 inhibits human megakaryocyto-poiesis in vitro [J]. Blood,1990, 75: 1234-1239.
[63] A. Gewirtz, B. Calabretta, B. Rucinski, et al. Inhibition of human megakaryocyto-poiesis in vitro by platelet factor4 (PF4) and a synthetic COOH-terminal PF4 peptide [J]. J Clin Invest, 1989, 83: 1477-1486.
[64] M. Long, V. Dixit. Thrombospondin functions as a cytoadhesion molecule for human hematopoietic progenitor cells [J]. Blood, 1990, 75: 2311-2318.
[65] H. Sasaki, M. Matsuda, Y. Lu, et al. A fraction unresponsive to growth inhibition by TGF-beta among the high proliferative jprtential progenitor cells in bone marrow of p532 deficient mice [J]. Leukemia, 1997, 11: 239-244.
[66] J. Abgrall, Z. Han, L. Sensebe, et al. Inhibitory effect of highly purified human platelet beta-thromboglobulin on in vitro human megakaryocyte colony formation [J]. Exp Hematol, 1991, 19: 202-205.
[67] B. Kuang, S. Wu, Y. Shin, et al. split ends encodes large nuclear protein that regulate neuronal cell fate and axon extension in the Drosophila embryo [J]. Development, 2000, 127: 1517-1529.
[68] L. Sanchez-Pulido, A. Rojas, A. Valencia, et al. ACRATA: a novel electron transfer domain associated to apoptosis and cancer [J]. BMC Cancer, 2004, 29: 98.
[69] G. Raffel, G. Chu, J. Jesneck, et al. Ott1 (Rbm15) is essential for placental vascular branching morphogenesis and embryonic development of the heart and spleen. Mol Cell Biol, 2009, 29: 333-341.
[70] L. Firth, J. Manchester, J. Lorenzen, et al. Identification of genomic regions that interact with a viable allele of the Drosophila protein tyrosine phosphatase corkscrew [J]. Genetics, 2000, 156: 733-748.
[71] I. Rebay, F. Chen, F. Hsiao, et al. A genetic screen for novel components of the Ras/Mitogen-activated protein kinase signaling pathway that interact with the yan gene of Drosophila identifies split ends, a new RNA recognition motif-containing protein [J]. Genetics, 2000, 154: 695-712.
[72] E. Wiellette, K. Harding, K. Mace, et al. spen encodes an RNP motif protein that interacts with Hox pathways to repress the development of head-like sclerites in the Drosophila trunk [J]. Development, 1999, 126: 5373-5385.
[73] K. Mace, A. Tugores. The product of the split ends gene is required for the maintenance of positional information during Drosophila development [J]. BMC Dev Biol, 2004, 13: 15.
[74] J. Chang, H. Lin, T. Blauwkamp, et al. Spenito and Split ends act redundantly to promote Wingless signaling [J]. Sev Biol, 2008, 314: 100-111.
[75] Y. Feng, G. Bommer, Y. Zhai, et al. Drosophila split ends homologue SHARP functions as a positive regulator of Wnt/beta-catenin/T-cell factor signaling in neoplastic transformation [J]. Cancer Res, 2007, 67: 482-491.
[76] K. Kurodak, H. Han, S. Tani, et al. Regulation of marginal zone B cell development by MINT, a suppressor of Notch/RBP-J signaling pathway [J]. Immunity, 2003, 301-312.
[77] F. Chen, I. Rebay. split ends, a new component of the Drosophila EGF receptor pathway, regulates development of midline glial cells [J]. Curr Biol, 2000, 10: 943-946.
[78] C. Niu, J. Zhang, P. Breslin, et al. c-Myc is a target of RNA-binding motif protein 15 in the regulation of adult hematopoietic stem cell and megakaryocyte development [J]. Blood, 2009, 114: 2087-2096.
[79] B. Cen, A. Selvaraj, R. Prywe. Myocardin/MKL family of SRF coactivators: key regulators of immediate early and muscle specific gene expression [J]. J Cell Biochem, 2004, 93: 74-82.
[80] G. Pipes, E. Creemers, E. Olson. The myocardin family of transcriptional coactivators: versatile regulator of cell growth, migration, and myogenesis [J]. Genes Dev, 2006, 20: 2545-1546.
[81] C. Mack, J. Hinson. Regulation of smooth muscle differentiation by the myocardin family of serum response factor co-factors. J Thromb Haemost, 2005, 1976-1984.
[82] Z. Wang, D. Wang, G. Pipes, et al. Mycardin is a master regulator of smooth muscle gene expression [J]. Proc Natl Acad Sci U S A, 2003, 100: 7129-7134.
[83] T. Yoshida, S. Sinha, F. Dandre, et al. Myocardin is a key regulator of CArG-dependent transcription of multiple smooth muscle marker genes [J]. Circ Res, 2003, 92: 856-864.
[84] F. Miralle, G. Posern, A. Zaromytidou, et al. Actin dynamics control SRF activity by regulation of its coactivator MAL [J]. Cell, 2003, 113: 329-342.
[85] A. Selvaraj, R. Prywes. Expression profiling of serum inducible genes identifies a subset of SRF target genes that are MKL dependent [J]. BMC Mol Biol, 2004, 5: 13.
[86] T. Sasazuki, T. Sawada, S. Sakon, et al. Identification of a novel transcriptional activator, BSAC, by a functional cloning to inhibit tumor necrosis factor-induced cell death [J]. J Biol Chem, 2002, 277: 28853-28860.
[87] T. Morita, T. Mayanagi, K. Sobue. Dual roles of myocardin-related transcription factors in epithelial mesenchymal transition via slug induction and actin remodeling [J]. J Cell Biol, 2007, 179:1027-1042.
[88] Y. Sun, K. Boyd, W. Xu, et al. Acute myeloid leukemia-associated MKL1 (Mrtf-a) is a keyregulator of mammary gland fuction [J]. Mol. Cell Biol, 2006, 26: 5809-5826.
[89] S. Li, S. Chang, X. Qi, et al. Requirement of a myocardin-related transcription factor for development of mammary myoepithelial cells [J]. Mol Cell Biol, 2006, 26: 5797-5808.
[90] K. Hinohara, T. Nakajima, M. Yasunami, et al. Megakaryoblastic leukemia factor-1 gene in the susceptibility to coronary artery disease [J]. Hum Genet, 2009, E-pub.
[91] B. Cen, A. selvaraj, R. Burgess, et al. Megakaryoblastic leukemia 1, a potent transcriptional coactivator for serum response factor (SRF), is required for serum induction of SRF target genes [J]. Mol Cell Biol, 2003, 23: 6597-6608.
[92] A. Descot, M. Rex-Haffner, G. Courtois, et al. OTT-MAL is a deregulated activator of serum response factor–dependent gene expression [J]. Mol Cell Biol, 2008, 28: 6171-6181.
[93] T. Mercher, G. Raffel, S. Moore, et al. The OTT-MAL fusion oncogene activates RBPJ-mediated transcription and induces acute megakaryoblastic leukemia in a knockin mouse model. J Clin Invest, 2009, 119: 852-864.
[94] KIRTO K. Kirto and K. Kaushanskey. Transcriptional regulation of megakaryopoiesis: thrombopoietin signaling and nuclear factors [J]. Curr Opin Hematol, 2006, 13: 151-156.
[95] K. Kaushanskey. Thrombopoietin: the primary regulator of platelet production [J]. Blood, 1995, 86: 419-431.
[96] M. Majka, M. Baj-Krzyworzeka, J. Kijowski, et al. In vitro expansion of human megakaryocytes as a tool for studying megakaryocytic development and function. Platelets, 2001, 12: 325-332.
[97] G. Mattia, F. Vulcano, L. Milazzo, et al. Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release [J]. Blood, 2002, 99: 888-897.
[98] S. Van Den Oudenrijn, M. De Haas, J. Calafat, et al. A combination of megakaryocyte growth and development factor and interleukin-1 is sufficient to culture large numbers of megakaryocytic progenitors and megakaryocytes for transfusion purposes [J]. Br J Haematol, 1999, 106: 553-563.
[99] L. Giammona, P. Fuhrken, E. Papoutsakis, et al. Nicotinamide (vitamin B3) increases the polyploidisation and proplatelet formation of cultured primary human megakaryocytes [J]. Br J Haematol, 2006, 135: 554-566.
[100] L. Boyer, A. Robert, C. Proulex, et al. Increased production of megakaryocytes near purity from cord blood CD34+ cells using a short two-phase culture system [J]. J Immunol Methods, 2008, 332: 82-91.
[101] P. Lefebvre, J. Winter, L. Kahn, et al. Megakaryocyte ex vivo expansion potential of threehematopoietic sources in serum and serum-free medium [J]. J Hematother, 1999, 8: 199-208.
[102] C. De Bruyn, A. Delforge, P. Martiat, et al. Ex vivo expansion of megakaryocyte progenitor cells: cord blood versus mobilized peripheral blood [J]. Stem Cell Dev, 2005, 14: 415-424.
[103] M. Gaur, T. Kamata, S. Wang, et al. Megakaryocytes derived from human embryonic stem cells: a genetically tractable system to study megakaryocytopoiesis and integrin function [J]. J Thromb Haemost, 2006, 4: 436-442.
[104] J. Unkeless. Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors [J]. J Exp Med. 1979, 150:580-596.
[105] L. Robb. Cytokine receptors and hematopoietic differentiation [J]. Oncogene, 2007, 26:6715-6723.
[106] S. Baker, S. Rane, E. Reddy. Hematopoietic cytokine receptor signaling [J]. Oncogene, 2007, 26: 6724-6737.
[107] D. Metcalf Hematopoietic cytokines [J]. Blood, 2008, 111: 485-491.
[108] K. Payne, G. Crooks. Human hematopoietic lineage commitment [J]. Immunol Rev, 2002, 187: 48-64.
[109] A. Tomer, L. Harker, S. Burstein SA. Purification of human megakaryocytes by fluorescence-activated cell sorting [J]. Blood, 1987, 70: 1735-1742.
[110] A. Tomer, L. Harker, S. Burstein SA. Flow cytometric analysis of normal human megakaryocytes [J]. Blood, 1988, 71: 1244-1252.
[111] J. Kostyak, U. Naik. Megakaryopoiesis: transcriptional insight into megakaryocyte maturation [J]. Front Biosci, 2007, 12: 2050-2062.
[112] J Drachman. , D. Sabath, N. Fox, et al. Thrombopoietin signal transduction in purified murine megakaryocytes [J]. Blood, 1997, 89:483-492.
[113] C. Rubin, D. French, G. Atweh. Statehmin expression and megakaryocyte differentiation: a potential role in polyploidy [J]. Exp Hematol, 2003, 31: 389-397.
[114] A. Lovatt. Applications of quantitative PCR in the biosafety and genetic stability assessment of biotechnology products [J]. J Biotechnol, 2002, 82: 279-300.
[115] D. Ginzinger. Gene quantification using real-time quantitative PCR: an emerging technology hits the mainstream [J]. Exp Hematol, 2002, 30: 503-512.
[116] C. Ding, C. Cantor. Quantitative analysis of the nucleic acids—the last few years of progress [J]. J biochem Mol Biol, 2004, 37: 1-10.
[117] V. Deutsch, A. Tomer. Megakaryocyte development and platelet production [J]. Br J Haematol, 2006, 134: 453-466.
[118] K. Ravid , J. Lu, J. Zimmet, et al. Roads to polyploidy: the megakaryocyte example [J]. J CellPhysiol. 2002, 190: 7-20.
[119] D. Kute, W. Sheridan, D. Zucker-Franklin. Thrombopoiesis and Thrombopoietins: Molecular, Cellular, Preclinical, and Clinical Biology [M]. 1997, Totowa, NJ: Springer-Verlag.
[120]冯义,韩忠朝.巨核细胞生成中的细胞因子和转录因子调节[J].国外医学输血及血液学分册, 2004, 1: 4-7.
[121] A. Goldfarb. Megakaryocytic programming by a transcriptional regulatory loop: A circle connecting RUNX-1, GATA-1 and P-TEFb [J]. J Cell Biochem, 2009, 107: 377-382.
[122] H. Raslova, L. Roy, C. Vourch, et al. Megakaryocyte ployploidization is associated with a functional gene amplification [J]. Blood, 2003, 15: 541-544.
[123] R. Shivdasani. The role of transcription factor NF-E2 in megakaryocyte maturation and platelet production [J]. Stem Cell, 1996, 14 (Suppl 1): 112-115.
[124] P. Romeo, M. Prandini, V. Joulin, et al. Megakaryocytic and erythrocytic lineages share specific transcription factors [J]. Nature, 1990, 344: 447-449.
[125] N. Andrews, H. Erdjument-Bromage, M. Davidson, et al. Erythroid transcription factor NF-E2 is a haematopoietic-specific basic-leucine zipper protein [J]. Nature, 1993, 362: 722-728.
[126] R. shivdasani, M. Rosenblatt, D. Zucker-Franklin, et al. Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development [J]. Cell, 1995, 81: 695-704.
[127] P. Lecine, J. Villeval, P. Vyas, et al. Mice lacking transcription factor NF-E2 provide in vivo validation of the proplatelet model of thrombocytopoiesis and show a platelet production defect that is intrinsic to megakaryocytes [J]. Blood, 1998, 92: 1608-1616.
[128] A. Radonic, S. Thulke, I. Mackay, et al. Guideline to reference gene selection for quantitative real-time PCR [J]. Biochem Biophys Res Commun, 2004, 313: 856-862.
[129] B. Small, C. Murdock, A. Bilodeau-Bourgeois, et al. Stability of reference genes for real-time PCR analyses in channel catfish (lctalurus punctatus) tissues under varying physiological conditions [J]. Comp Biochem Physiol B Biochem Mol Biol, 2008, 151: 296-304.
[130] O. Thellin, B. Elmoualij, E. Heinen, et al. A decade of improvements in quantification of gene expression and internal standard selection [J]. Biotechnol Adv, 2009, 27: 323-333.
[131] S. Guenin, M. Mauriat, J. Pelloux, et al. Normalization of qRT-PCR data: the necessity of adopting a systematic, experimental conditions-specific, validation of references [J]. J Exp Bot, 2009, 60: 487-493.
[132] J. Jang, K, Shaw, X. Yu, et al. Specific and stable gene transfer to human embryonic stem cells using pseudotyped lentiviral vectors [J]. Stem Cells Dev, 2006, 15: 109-117.
[133] L. Naldini , U. Blomer, P. Gallay, et al . In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector [J]. Science, 1996, 272: 263-267.
[134] T. Kafri, U. Blomer, D. Peterson, et al. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors [J]. Nat Genet, 1997, 17: 314-317.
[135]徐晓明,杨慧,王晓萍.慢病毒载体的构建及优化[J].中国临床康复,2006 ,10 (9) :147-149.
[136]尹春光,杜立新.慢病毒载体构建策略及生物安全性研究进展[J].动物医学进展, 2008, 29: 72-75.
[137] A. Follenzi A , L. Santambrogio, A. Annoni. Immune responses to lentiviral vectors [J]. Curr Gene Ther, 2007 , 7: 306-315.
[138] G. Buchschacher, F. Wong-Staal. Development of lentiviral vectors for gene therapy for human disease [J]. Blood, 2000, 95: 2499-2504.
[139] C. Thomas, A. Ehrhardt, M. Kay. Progress and problems with the use of viral vectors for gene therapy [J]. Nat Rev Genet, 2003, 4: 346-358.
[140] T. Hopp, K. Prickett, V. Price, et al. A short polypeptide marker sequence useful for recombinant protein identification and purification [J]. Bio/Technology, 1988, 6: 1204-1210.
[141] S. Zielske, S. Braun. Cytokines: value-added products in hematopoietic stem cell gene therapy [J]. Mol Ther, 2004, 10: 211-219).
[142] D. Haas, S. Case, G. Crooks, et al: Critical factors influencing stable transduction of human cd34+ cells with hiv-1-derived lentiviral vectors [J]. Mol Ther, 2000, 2: 71-80.
[143] A. Szyda, M. Paprocka, A. Krawczenko, et al. Optimization of a retroviral vector for transduction of humancd34 positive cells [J]. Acta Biochim Pol, 2006, 53: 815-823.
[144] S. Zielske, S. Gerson: Lentiviral transduction of p140k mgmt into human cd34+ hematopoietic progenitors at low multiplicity of infection confers significant resistance to BG/BCNU and allows selection in vitro [J]. Mol Ther, 2002, 5: 381-387.
[145] S. Jeanpierre, F. Nicolini, B. Kaniewski, et al. BMP4 regulation of human megakaryocytic differentiation is involved in thrombopoietin signaling [J]. Blood, 2008, 112: 3154-3164).
[146] L. Pang L, H. Xue, G. Szalai, et al. Maturation stage-specific regulation of megakaryopoiesis by pointed-domain Ets protein [J]. Blood, 2006, 108: 2198-2206.
[147] K. Elagib, F. Racke, M. Mogass, et al. RUNX1 and GATA-1 coexpression and cooperation in megakaryocytic differentiation [J]. Blood, 2003, 101: 4333-4341.
[148] L. Waltzer, G. Ferjoux, L. Bataille, et al. Cooperation between the GATA and RUNX factors Serpent and Lozenge during Drosophila hematopoiesis [J]. EMBO J, 2003, 22: 6516-6525.
[149] V. Hamelin, C. Letourneux, P. Romeo, et al. Thrombopoietin regulates IEX-1 gene expression 86through ERK-induced AML1 phosphorylation [J]. Blood, 2006, 107: 3106-3113.
[150] P. Heller, A. Glembotsky, M. Gandhi, et al. Low Mpl receptor expression in a pedigree with familial platelet disorder with predisposition to acute myelogenous leukemia and a novel AML1 mutation [J]. Blood, 2005, 105: 4664-4670.
[151] L. Gilles, R. Guieze, D. Bluteau, et al. P19INK4D links endomitotic arrest and megakaryocyte maturation and is regulated by AML-1 [J]. Blood, 2008, 111: 4081-4091.
[152] K. Ravid, J. Lu, M. Zimmet, et al. Roads to polyploidy: the megakaryocyte example [J]. J Cell Physiol, 2002, 190: 7-20.
[153] D. Mccrann, H. Nguyen, M. Jones, et al. Vascular smooth muscle cell polyploidy: an adaptive or maladaptive response [J]. J Cell Physiol, 2008, 215: 588-592.
[154]卢丽,陈系古,黄冰.基因敲除动物的研究和应用[J].中国实验动物学报, 2006, 14: 252-256.
[155]刘建忠,敖敬,田为宇,等.基因敲除技术研究进展[J].化学与生物工程, 2006, 23: 4-6.
[156]李湘萍,徐慰倬,李宁.动物基因敲除研究的现状与展望[J].遗传, 2003, 25: 81-88.
[157] M. Glotzer. The molecular requirements for cytokinesis [J]. Science, 2005, 307: 1735-1739.
[158] A. Piekny A, M. Werner, M. Glotzer M. Cytokinesis: welcome to the Rho zone [J]. Trends Cell Biol, 2005, 15: 651~658.
[159] A. Jaffe, A. Hall. Rho GTPase: biochemistry and biology [J]. Annu Rev Cell Deb Biol, 2005, 21: 247-269.
[160] L. Lordier, A. Jalil, F. Aurade, et al. Megakaryocyte endomitosis is a failure of late cytokinesis related to defects in the contractile ring and Rho/Rock signaling [J]. Blood, 2008, 112: 3164-3174.