骨髓增生异常综合征Evi1和MDS1-Evi1基因表达的研究

1、    骨髓增生异常综合征Evi1和MDS1-Evi1基因表达的研究        摘要目的:探讨骨髓增生异常综合征(MDS)患者Evi1、MDS1-Evi1基因表达及其在 MDS发病机制中的作用。方法:用半定量逆转录-聚合酶链反应技术检测了31例MDS、11例MDS转化的急性髓系白血病(post MDS AML)和34例原发性AML(de novo AML)患者的Evi1及MDS1-Evi1基因表达。结果:8名正常人骨髓单个核细胞中未测到Evi1转录本,其中3名有MDS1-

2、Evi1表达, 但表达量较低(MDS1-Evi1/GAPDH<0.1)。8例RA中1例、13例RAEB中8例、9例RAEB-t中6例有Evi1表达,RAEB和RAEB-t患者Evi1表达率高于RA(P<0.05)。8例RA中5例、13例RAEB中9例和9例RAEB-t患者中5例有MDS1-Evi1表达, 表达量较高(MDS1-Evi1/GAPDH>0.1)。MDS转化为 AML5例, 其中4例随病情进展Evi1表达量增加。post MDS AML患者Evi1、MDS1-Evi1表达率显著高于de novo AML (P值均<0.05)。Evi1 和 MDS1-Evi1

3、表达阳性的 MDS 患者祖细胞集落形成数低于Evi1和MDS1-Evi1 阴性者。结论:MDS患者Evi1的异常表达和MDS1-Evi1 过度表达在 MDS和post MDS AML的发病或病程进展中可能有一定的作用。关键词骨髓增生异常综合征白血病基因表达Study on expression of Evi1 and MDS1-Evi1 genes in myelodysplastic syndromesXu Kailin,Wang Li,Hao Yushu, et al. Institute of Hematology,CAMS and PUMC,Tianjin300020AbstractO

4、bjective: To investigate expression of Evi1 and MDS1-Evi1 genes in myelodysplastic syndromes (MDS) and its role in pathogenesis of MDS. Methods: Expression of Evi1 and MDS1-Evi1 genes was examined in 31 MDS,11 post MDS acute myeloid leukemia (post MDS AML) and 34 de novo AML patients by a semi-quant

5、itative RT-PCR. Results: Evi1 expression was not detected in 8 normal controls, but low MDS1-Evi1 expression levels (MDS1-Evi1/GAPDH<0.1) detected in 3 of the 8 controls. Evi1 mRNA was expressed in 1 of 8 RA, 8 of 13 RAEB and 6 of 9 RAEB-t patients, and the percentage of Evi1 expression in RAEB(t

6、) patients was higher than that in RA(P<0.05). MDS1-Evi1 expression was detected in 5 of 8 RA,9 of 13 RAEB and 5 of 9 RAEB-t patients, and MDS1-Evi1 expression levels (MDS1-Evi1/GAPDH>0.1) in the patients were markedly higher than those in the controls.Evi1 expression was gradually increased i

7、n 4 of 5 RAEB-t patients with transformation from MDS to AML. The percentages of Evi-1 and MDS1-Evi1 expression in post MDS AML patients were higher than those in de novo AML (P<0.01 and P<0.05, respectively). The numbers of colony formation of progenitor cells in Evi1 and MDS1-Evi1-positive M

8、DS patients were decreased as compared with Evi1 and MDS1-Evi1-negative patients.Conclusion: Abnormal expression of Evi1 and overexpression of MDS1-Evi1 might play a certain role in the pathogenesis or progression of MDS and post MDS AML.Key wordsMyelodysplastic syndromesLeukemiaGene expressionEvi1(

9、ecotropic virus integration site-1)基因定位于染色体3q26。在小鼠逆转录病毒诱发的急性髓系白血病(AML) 模型中,Evi1是病毒常见的插入位点, 与白血病的发生密切相关1，2。MDS1基因最初在骨髓增生异常综合征(MDS) 中发现, 又称MDS相关基因,定位于Evi1上游170400kb处, 通过可变性剪接, MDS1和Evi1可形成MDS1-Evi1融合转录本3，4。我们报告31例MDS患者Evi1和MDS1-Evi1的表达,探讨这两个基因在MDS发病中的作用。病例和方法1病例所有病例均为我院门诊和住院患者。MDS患者31例,其中难治性贫血(RA) 8例

10、,原始细胞增多的RA(RAEB)13例, 转化中RAEB(RAEB-t)9例,慢性粒-单核细胞白血病(CMML)1例；MDS后急性髓系白血病(post MDS AML)11例；原发性AML(de novo AML)34例。正常对照者8名;贫血对照为5例再生障碍性贫血(再障)患者。2试剂Taq酶, 脱氧核糖核苷三磷酸(dNTPs) 和随机引物(上海生工公司), M-MLV(Gibco公司),焦碳酸二乙酯(DEPC,Promega 公司),异硫氰酸胍(Fluke公司)。聚合酶链反应(PCR)引物: Evi1基因的PCR引物由美国国立卫生研究院(NIH)王建祥博士和亚利桑那癌症研究中心Taetle教

11、授惠赠, MDS1-Evi1基因和内对照GAPDH的PCR引物由上海生工公司合成。Evi1引物序列:5AGCAACGTCGAATCAAGACCTGCTTCAGAT3ACTGACTGTAAGAGCTCACTGGCCTCAGGTMDS1-Evi1引物序列:5TGGGAGAGCAGAGGTCAAACC3TTTCATGGGGATAGTCTTCGCGAPDH引物序列见文献5。3逆转录-聚合酶链反应(RT-PCR)采集患者骨髓液,ACD抗凝,分离单个核细胞。用异硫氰酸胍一步法提取细胞总RNA。反应体系45l 内含RNA5g, M-MLV300U, 2.5mmol/L dNTPs 6l,随机引物60pmol

12、,0.1mol/L DTT 4.5l,5×第1链缓冲液 9l,37反应1小时。Evi1、MDS1-Evi1反应体系均为100l,cDNA均为来自同一逆转录体系的产物10l(1gRNA合成的cDNA)。Evi1的循环参数为94 30秒, 64 60秒,7260秒, 35个循环;MDS1-Evi1的循环参数为94 30秒, 60 60秒,72 60秒,30个循环。取20l扩增产物在1.5%的琼脂糖凝胶电泳, 70V 80分钟后在紫外灯下照相。胶片在岛津 SC-9000型双波长色谱扫描仪上扫描, 计算Evi1和MDS1-Evi1对GAPDH的比值。4染色体核型分析按常规R显带法。5祖细胞集

13、落培养CFU-E, BFU-E和CFU-GM均采用我所常规的甲基纤维素半固体培养法6。6统计学处理率的比较用2检验,两组之间均数的比较用t检验。结果1对照组Evi1、MDS1-Evi1基因的表达8名正常人骨髓单个核细胞中无 Evi1 mRNA表达(阴性);其中3名有 MDS1-Evi1表达(阳性), 但表达量较低, MDS1-Evi1/GAPDH分别为0.067,0.039和0.054。5例再障患者Evi1和MDS1-Evi1表达均阴性。2MDS患者Evi1、MDS1-Evi1基因表达结果见表1。31例MDS患者15例Evi1表达阳性，表达率为 48.4%。经2检验, RA患者Evi1表达率明

14、显低于RAEB(t)(2=4.261,P<0.05)。31例患者19例MDS1-Evi1表达阳性，表达率为61.3%。各亚型之间MDS1-Evi1表达率无明显差异(P>0.05)。仅有MDS1-Evi1表达者主要见于RA阶段, 仅有Evi1或Evi1与MDS1-Evi1 同时表达主要见于RAEB和RAEB-t。部分阳性病例的电泳结果见1和2。1例CMML患者Evi1和MDS1-Evi1均为阴性。3Evi1、MDS1-Evi1基因表达与 MDS病情转化的关系随访中, 2例患者由RA分别转化为RAEB和RAEB-t, 5例患者由RAEB-t转化为AML。除1例RA转化为RAEBt者Ev

15、i1表达由阴性转化为阳性外, 其余6例均未发现Evi1、MDS1-Evi1 由阴性转化为阳性或阳性转为阴性。但随病情的进展,5例(例1,6,22,26,29)转化为AML的患者中4例Evi1基因表达量有增加的趋势,MDS1-Evi1表达水平无明显变化。4post MDS AML 与 de novo AML患者Evi1和MDS1-Evi1的表达post MDS AML患者Evi1和MDS1-Evi1表达率分别为54.5%和63.6%,34例de novo AML患者Evi1和MDS1-Evi1表达率分别为14.7%和23.5%。经统计学处理2值分别为5.148和4.347,P值均<0.05

16、。5Evi1、MDS1-Evi1表达与染色体核型的关系7号染色体异常 (-7)的8例患者中,Evi1和MDS1-Evi1表达阳性者各有7例;无-7的21例患者中分别有7例表达Evi1和12例表达MDS1-Evi1,有-7的MDS患者两个基因的表达率均较高, 但仅Evi1有统计学意义(2值>3.84,P<0.05)。8号染色体异常(+8,-8)的7例MDS中，Evi1和MDS1-Evi1表达阳性者分别为5例和4例,表达率亦较高,但无显著性差异。在Evi1和MDS1-Evi1表达阳性的病例中,均未检出3号染色体异常。6Evi1、MDS1-Evi1基因表达与祖细胞集落形成的关系与Evi1

17、 和MDS1-Evi1阴性组比较,这两个基因阳性的病例祖细胞集落有不同程度的减少(表2)。表131例MDS患者Evi1、MDS1-Evi1基因的表达例号性别年龄亚型Evi1MDS1-Evi1Evi1/GAPDHMDS1-Evi1/GAPDH染色体核型1男72RAEB-t0.1840.58045,XY,-21;47,XY,+82男57RAEB0.9120.20247,XY,+8,t(2;5);45,XY,-7,-16,+mar3男25RA-00.31746,XY4男26RAEB-0.346046,XY;47,XY,+85女46RAEB-t-0046,XX;45,XX,-206女43RAEB-t-

18、1.718046,XX;45,XX,-14;45,XX,-7;45,XX,-137男51RAEB-0.10708男68RA-0046,XY9女30RAEB-01.06446,XX10女63RAEB-+00.36846,XX;46,XX,5q-11男66RAEB3.8971.79345,XY,-7;45,XY,-2244,XY,-7,-812男48RAEB-t-0046,XY13男55RAEB-0045,XY,+8,-11,-15;42,X,-Y,-8,-13,-1514女36CMML-0046,XX15男37RA-0045，XY,-18;47,XY,+816男58RAEB-t0.2720.23

19、046,XY,5q-17男48RA-0046,XY18男64RAEB1.9300.40044,XY,-5,-7,-15,+2119男46RAEB-00.26746,XY;45,XY,-1820男35RAEB1.0510.31546,XY;45,XY,-721男21RA0.5422.46645,XY,-7,-8,+1622女40RAEB-t1.1341.51146,XX,-17,+mar;46,XX,11q+23男43RAEB-0046,XY24女27RA-00.78446,XX25男42RAEB-t-0046,XY26男40RAEB-t2.1441.53746,XY,13p+27女25RAEB

20、2.5920.30146,XX28女39RAEB1.7730.29446,XX29男62RAEB-t0.5060.23746,XY;45,XY,-730男39RA-00.33844,XY,-5,-7,6p+31男45RA-00.24046,XY         M：pBR322/Msp；H：HEL细胞系；17：MDS患者1部分MDS患者Evi1 PCR扩增产物电泳    M：pBR322/Hinf标志；N：正常对照；17：MDS患者2部分MDS患者MDS1-Evi1 PCR扩

21、增产物电泳表2部分MDS患者骨髓体外培养的祖细胞集落(<"01 (866 bytes)" src="/med/cano/201003/20100317190939365" 9 12>±s)组别例数CFU-E (集落/2×104细胞)BFU-E(集落/2×104细胞)CFU-GM(集落/1×105细胞)Evi1阳性组1119.7±23.76.5±6.61.1±2.1阴性组1343.4±58.013.9±21.44.5±4.6P值>0.05&

22、gt;0.05<0.05MDS1-Evi1阳性组1320.1±23.06.6±6.60.9±1.9阴性组1149.4±62.016.2±22.85.5±4.8P值>0.05>0.05<0.005     讨论资料表明, Evi1基因在小鼠脏器的发育中起重要作用。小鼠心脏的发育过程中, Evi1表达的高峰在心脏形成的早期阶段, 较晚期不再有Evi1的表达;在呼吸系统和肾脏组织, Evi1表达的时相与发育的心脏相似, 提示Evi1 基因表达与组织细胞的“幼稚化”密切相关7。E

23、vi1基因可以抑制粒系细胞对集落刺激因子 (G-CSF)的反应, 阻碍粒细胞分化, 使其成熟障碍8。Evi1的表达还能抑制GATA-1诱导的基因转录, 从而阻碍红细胞生成素诱导的红系祖细胞的分化9。MDS 是一克隆性疾病,大部分是白血病前期, 发病的本质是恶性克隆的形成, 异常幼稚白细胞的产生和分化障碍。我们观察了31例MDS患者Evi1基因的表达,其中近半数为阳性, 而8名正常人均阴性, 显示MDS患者存在 Evi1基因异常表达。8名正常对照中, 虽3名有MDS1-Evi1融合基因表达, 但MDS1-Evi1/GAPDH均<0.1, 而 MDS中 MDS1-Evi1阳性者,其比值均&g

24、t;0.1,表明该融合基因在MDS有过度表达。对AML的发病机制已有较为广泛而深入的研究, 虽然目前还未能完全阐明, 但许多学者注意到de novo AML和post MDS AML在血液学和生物学特征等许多方面存在着差异。例如post MDS AML 骨髓造血细胞常有较明显的发育异常的形态学改变, 预后较差, 常见 7号染色体异常等。本组post MDS AML患者Evi1、MDS1-Evi1 基因表达率明显高于de novo AML,这是两组AML生物学特征不同的又一个例证,并提示两组AML的发病机制也不完全相同。在较早期的报道中,Evi1 mRNA的表达见于3号染色体异常,例如t(3;3

25、)(q21;q26)或 inv(3)(q21;q26)10。后来的资料表明, 无3号染色体异常的白血病中也有Evi1的异常表达6,7，10，11。在有t(3;21)(q26;q22)的 AML 和慢性髓系白血病 (CML), Evi1和MDS1-Evi1还可与AML1形成AML1-Evi1和AML1-MDS1-Evi1融合基因12，13。我们对MDS 患者进行了核型分析, 均未发现 3号染色体异常, 而 7号染色体异常者 Evi1表达率较高。7号染色体异常见于治疗相关MDS,但这些患者均未发现烷化剂等用药史和放射线接触史。因此,这些患者的Evi1表达率高的原因尚待进一步阐明。本组MDS中, 向

26、AML转化患者随着病情向AML进展，其Evi1表达量有递增趋势,虽然动态观察的病例数较少, 不足以断定Evi1在 AML转化中的作用, 故有必要进行更多病例Evi1表达的随访。更重要的是RAEB和RAEB-t患者的Evi1表达率显著高于RA, 这进一步支持Evi1与MDS病情进展有关。此外,Evi1和MDS1-Evi1阳性的病例CFU-GM集落数显著少于这两个基因阴性的病例,阳性病例的CFU-E和BFU-E数亦较少(未达到统计学显著性), 这也许为解释MDS无效造血的发生机制提供了一个线索。参 考 文 献1Mucenski ML,Taylor BA,Ihle JN,et al.Identifi

27、cation of a common ecotropic viral integration site, Evi-1, in the DNA of AKXD murine myeloid tumors. Mol Cell Biol,1988,8:301-308.2Perkins AS,Fishel R,Jenkins NA,et al. Evi-1, a murine zinc finger proto-oncogene,encodes a sequence-specific DNA-binding protein. Mol Cell Biol, 1991,11:2665-2674.3Nuci

28、fora G,Begy CR,Kobayashi H,et al. Consistent intergenic splicing and production of multiple transcripts between AML1 at 21q22 and unrelated genes at 3q26 in (3;21)(q26;q22) translocations. Proc Natl Acad Sci USA, 1994,91:4004-4008.4Fears S,Mathieu C,Zeleznik-Le N, et al. Intergenic splicing of MDS1

29、and Evi-1 occurs in normal tissues as well as in myeloid leukemia and produces a new member of the PR domain family. Proc Natl Acad Sci USA, 1996,93:1642-1647.5Lipman ML, Stevens AC, Strom TB, et al. Heightened Intragraft CTL gene expression in acutely rejecting renal allografts.J Immunol,1994,152:5

30、120-5127.6刘薏玲，汤晓培，郭亚东，等. 再生障碍性贫血患者粒、红祖细胞的观察. 中华血液学杂志，1987，8：602-605.7Lopingco MC, Perkins AS. Molecular analysis of Evi-1, a zinc finger oncogene involved in myeloid leukemia. Curr Top Microbiol Immunol, 1996, 211:211-222.8Morishita K,Parganas E,Matsugi T, et al. Expression of the Evi-1 zinc finger gene in 32D13