造血细胞分化相关基因的鉴别以及ASB-8和mir-223的功能研究

【作者】 袁金云；

【导师】 张俊武；

【作者基本信息】 中国协和医科大学 ， 生化与分子生物学， 2007， 博士

【摘要】 人类造血涉及多能干细胞的自我更新、向多个造血细胞链系的分化决定及进一步分化发育成熟。这个过程涉及关键的转录因子和造血生长因子的协同调控。为了研究造血干细胞链系分化的分子机制,我们首先用免疫磁珠（MACS）从正常成人骨髓中分离CD34+细胞,用特异的表面标志进一步分离获得造血干细胞（HSC;CD34+/CD38-/CD33-）和多能髓系祖细胞（MMP;CD34+/CD38-/CD33+）。同时用特异的细胞因子分别诱导CD34+细胞向红系、巨核系和粒系分化,收集红系祖细胞衍生的集落（EC）、巨核系祖细胞衍生的集落（MC）和粒系祖细胞衍生的集落（GC）。从这些细胞制备RNA并进行逆转录差异显示PCR（DDRT-PCR）分析,共获得差异表达条带（ESTs）近500条。挑选差异表达显著的条带进行PCR再扩增、克隆到T载体、DNA测序并在NCBI进行BLAST比对,发现这些ESTs所代表的基因为转录因子、信号转导分子、细胞代谢酶类、细胞周期调控因子、细胞凋亡相关分子、肿瘤相关分子、核糖体蛋白和未知功能的基因。我们发现特异的基因表达上调和特异的基因表达下调都在HSC链系特异分化中发挥重要作用。这些结果为揭示造血细胞分化的分子机制提供了线索。粒系形成包括HSC向粒系祖细胞的分化决定和进一步分化发育成具有功能活性的成熟粒细胞。这个过程不仅涉及粒系特异转录因子的调控,还涉及细胞因子及其受体、其它转录因子的调控。我们研究了DDRT-PCR分析中一个在GC中特异高表达的基因ASB-8在粒系分化中的作用。用real-time PCR分析验证了ASB-8在GC中高表达,在HSC、MMC、EC和MC中表达很低。在全反式视黄酸（ATRA）诱导NB4和HL-60向粒系分化的过程中,ASB-8 mRNA表达呈现上调趋势。pEGFP-N1-ASB-8融合蛋白定位于NIH-3T3的细胞质,并集中在核周围。我们构建了ASB-8的过表达质粒pcDNA3.1-ASB-8和RNAi质粒pAVU6+27-ASB-8i,分别转染NB4获得ASB-8的过表达稳定转化子pcDNA3.1-ASB-8/NB4和RNAi稳定转化子pAVU6+27-ASB-8i/NB4。与NB4相比,在ATRA诱导之前,在pcDNA3.1-ASB-8/NB4中CD11b mRNA的表达升高,而MPO mRNA的表达无明显变化。在ATRA诱导向粒系分化中,CD11b mRNA在pcDNA3.1-ASB-8/NB4的表达均要高出在相应诱导时间的NB4细胞中的表达,而MPO mRNA的表达无明显差别,但在96h降低为1/3。在ATRA诱导之前,在pAVU6+27-ASB-8i/NB4中CD11bmRNA和MPO mRNA的表达分别只有NB4中的1/10和1/2。在ATRA诱导向粒系分化过程中,在pAVU6+27-ASB-8i/NB4中CD11b mRNA一直处于极低的表达水平,而MPO mRNA的表达基本保持不变。在ATRA诱导向粒系分化48h后,NBT还原试验发现pcDNA3.1-ASB-8/NB4和pAVU6+27-ASB-8i/NB4中的A₅₇₀nm/10⁶（细胞）要比NB4分别增加13%和减少20%;Gimesa染色发现pcDNA3.1-ASB-8/NB4中的细胞核分叶比NB4中更多,而pAVU6+27-ASB-8i/NB4中的细胞核分叶则更少。这些结果说明过表达ASB-8促进ATRA诱导的NB4向粒系的分化,而ASB-8的表达阻遏则抑制ATRA诱导NB4向粒系的分化。近来的研究表明miRNAs可能参与造血细胞分化的调控。我们课题组先前发现mir-223在红系分化中可能具有一定的调节作用,本文进一步研究mir-223调节红系分化的功能和机制。通过分别转染寡核苷酸小RNA223前体（Pre-mir-223）和小RNA223抑制剂（Anti-mir-223）,分别观察到联苯胺染色阳性细胞百分比以及γ-珠蛋白mRNA表达的降低和升高,说明在K562中mir-223的过表达和表达阻遏分别抑制和促进hemin诱导的红系分化。我们构建了mir-223过表达质粒pcDNA3.1-pri-mir-223（小RNA223初级转录本）和pSilencer 2.1-U6-Neo-pre-mir-223,分别转染K562细胞,获得mir-223过表达稳定转化子pcDNA3.1-pri-mir-223/K562和pSilencer 2.1-U6-Neo-pre-mir-223/K562。在hemin诱导向红系分化中,在pcDNA3.1-pri-mir-223/K562和pSilencer 2.1-U6-Neo-pre-mir-223/K562中的联苯胺染色阳性细胞百分比和γ-珠蛋白mRNA表达均要低于K562,进一步说明在K562中过表达mir-223会抑制hemin诱导的红系分化。为了鉴别mir-223的靶基因,把LMO2和HLF的3’-UTR插入到pRL-TK和pEGFP-C1,构建了LMO2和HLF-双萤光和荧光报告基因质粒,与Pre-mir-223共转染K562和NIH-3T-3,发现LMO2和HLF-报告基因的表达受到抑制,说明LMO2和HLF可能是mir-223的靶基因。把LMO2-报告基因质粒分别转染到过表达稳定转化子pcDNA3.1-pri-mir-223/K562、pSilencer2.1-U6-Neo-pre-mir-223/K562和K562中,发现LMO2-报告基因在过表达稳定转化子中的表达受到抑制。Real-time PCR还发现LMO2 mRNA在mir-223过表达稳定转化子中的表达要低于K562,这些说明LMO2很可能就是mir-223的靶基因。这些结果提示在hemin诱导K562向红系分化过程中,mir-223表达的降低部分解除了对LMO2表达的抑制,进而促进了红系分化。在PMA诱导向巨核系分化过程中,Giemsa染色发现pcDNA3.1-pri-mir-223/K562的大核和多核细胞比K562多,PI染色后FACS分析发现在pcDNA3.1-pri-mir-223/K562的4倍体细胞数目多于K562。这些结果说明过表达mir-223可能会促进PMA诱导K562向巨核系的分化。更多还原

【Abstract】 Haematopoiesis,the differentiation of haematopoietic stem cells and progenitors into various lineages,involves complex interactions of transcription factors that modulate the expression of downstream genes and mediate proliferation and differentiation signals and complex interplay of hematopoietic cytokines and their receptors.To better understand the transcriptional program that accompanies orderly lineage-specific hematopoietic differentiation,we analyzed expression changes during the lineage-specific differentiation of human hematopoietic stem cells（HSC）.HSC （CD34+/CD38-/CD33-） and multipotent myeloid progenitors（MMP; CD34+/CD38-/CD33+） were isolated from the bone marrow of healthy individuals by Magnetic Activated Cell Sorting（MACS）.CD34+ cells in semi-solid culture were stimulated with the cytokines erythropoietin（EPO）,interleukin 6（IL-6） and granulocyte colony-stimulating factor（G-CSF） to promote differentiation to committed erythroid, megakaryocytic,and granulocytic clones,respectively.Differential display reverse transcription polymerase chain reaction analysis was performed to compare the mRNA transcripts in HSC,MMP,and the clones derived from their committed lineage-specific progenitors.Expressed sequence tags（ESTs;n=256） that were differentially expressed were identified.194 were homologous to known genes,and some of which were related to genes involved in hematopoiesis.These known genes were classified as involved in transcription/translation,signal transduction,cell surface receptors/ligands,cell signaling, cell metabolism,cell cycle,cell apoptosis,and oncogenesis.We identified genes that were specifically up-or down-regulated in the lineage-committed clones compared with HSC or/and MMP,suggesting that both specific gene activation and repression might be necessary for specific lineage commitment and differentiation.Granulopoiesis is a complex process by which HSC differentiates into fully differentiated,functionally active granulocytes.Various transcription factors both specific to the granulocytic lineage as well as more widely expressed molecules control the differentiation,including growth factors and their receptors,other transcription factors as well as various molecules important for the function of mature cells.The role of ASB-8 in granulocytic differentiation was studied in the paper.ASB-8 mRNA was mainly expressed in GC from DDRT-PCR and confirmed this by real-time PCR.ASB-8 mRNA was increased during the granulocytic differentiation of NB4 and HL-60 treated by ATRA.pEGFP-N1-ASB-8 was located in cytoplasma of NIH-3T3 and around nucleus.Enforced plasmid of ASB-8 and RNAi plasmid of ASB-8 was constructed and plasmid was transfectted into NB4 and stable transfectants with enforced expression of ASB-8 and with RNA interfere of ASB-8 were created,pcDNA3.1-ASB-8/NB4, pAVU6+27-ASB-8i/NB4.CD11b mRNA in pcDNA3.1-ASB-8/NB4 was much more than that in NB4 and remained this following granulocytic differentiation treated by ATRA.MPO mRNA was comparative to NB4 and was one third time in NB4 at 96h treated by ATRA.CD11b and MPO mRNA in pAVU6+27-ASB-Si/NB4 was lower than that in NB4.CD11b mRNA was hardly undetectable and MPO mRNA remained almost no change following granulocytic differentiation treated by ATRA.A₅₇₀nm/10⁶ of pcDNA3.1-ASB-8/NB4 was more and pAVU6+27-ASB-8i/NB4 less than that in NB4 from NBT reduction.The morphological changes in cell smears of pcDNA3.1-ASB-8/NB4 were more and pAVU6+27-ASB-Si/NB4 less than that in NB4 from Giemsa staining.The results suggested that the enforced expression of ASB-8 in NB4 may accelerate the granulocytic differentiation treated by ATRA and RNA interfere of ASB-8 in NB4 may delay the granulocytic differentiation treated by ATRA.MiRNAs have been implicated in the differentiation of mammalian blood cell lineages.Our lab had found that mir-223 may be associated with and regulate erythroid differentiation.The fraction of benzidine positive cells andγ-globin mRNA of K562 with endogenous over expression and suppression of mature mir-223 via olio-nucleotide transfection were much less and more than that of K562.Stable transfectants with enforced expression of mir-223 in K562 was constructed and they are pcDNA3.1-pri-mir-223/K562 and pSilencer 2.1-U6-Neo-pre-mir-223/K562.The fraction of benzidine positive cells andγ-globin mRNA in pcDNA3.1-pri-mir-223/K562 and pSilencer 2.1-U6-Neo-pre-mir-223/K562 was much lower than that in K562 following erythroid differentiation treated by hemin.These results suggested that the enforced expression of mir-223 may suppress erythroid differentiation of K562 treated by hemin. The expression of LMO2 and HLF was suppressed in K562 and NIH-3T3 transfected with pre-mir-223 examined by reporter system of luciferase and fluorescence.The mRNA of LMO2 in pcDNA3.1-pri-mir-223/K562 and pSilencer 2.1-U6-Neo-pre-mir-223/K562 was much lower than that in K562 and reporter system of luciferase and fluorescence also confirmed this decrease.These results indicated that LMO2 may be one of target genes of mir-223.The mechanism may be that mir-223 may unblock LMO2 to promote eryhtroid differentiation of K562 treated by hemin.The increase in cell size and extensive multinuclearity in pcDNA3.1-pri-mir-223/K562 in May-Gru"nwald-Giemsa staining was much more than that in K562 following megakaryocytic differentiation treated by PMA.Ploidy analysis by FACS after PI staining showed that the peak corresponding to 4N cells in pcDNA3.1-pri-mir-223/K562 was much higher than that in K562 following megakaryocytic differentiation treated by PMA.These results suggested that the enforced expression of mir-223 may promote megakaryocytic differentiation of K562 treated by PMA.更多还原