骨髓增生異常綜合征Evi1和MDS1-Evi1基因表達(dá)的研究

1、    骨髓增生異常綜合征Evi1和MDS1-Evi1基因表達(dá)的研究        摘要目的:探討骨髓增生異常綜合征(MDS)患者Evi1、MDS1-Evi1基因表達(dá)及其在 MDS發(fā)病機(jī)制中的作用。方法:用半定量逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)技術(shù)檢測了31例MDS、11例MDS轉(zhuǎn)化的急性髓系白血病(post MDS AML)和34例原發(fā)性AML(de novo AML)患者的Evi1及MDS1-Evi1基因表達(dá)。結(jié)果:8名正常人骨髓單個核細(xì)胞中未測到Evi1轉(zhuǎn)錄本,其中3名有MDS1-

2、Evi1表達(dá), 但表達(dá)量較低(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表達(dá),RAEB和RAEB-t患者Evi1表達(dá)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表達(dá), 表達(dá)量較高(MDS1-Evi1/GAPDH>0.1)。MDS轉(zhuǎn)化為 AML5例, 其中4例隨病情進(jìn)展Evi1表達(dá)量增加。post MDS AML患者Evi1、MDS1-Evi1表達(dá)率顯著高于de novo AML (P值均<0.05)。Evi1 和 MDS1-Evi1

3、表達(dá)陽性的 MDS 患者祖細(xì)胞集落形成數(shù)低于Evi1和MDS1-Evi1 陰性者。結(jié)論:MDS患者Evi1的異常表達(dá)和MDS1-Evi1 過度表達(dá)在 MDS和post MDS AML的發(fā)病或病程進(jìn)展中可能有一定的作用。關(guān)鍵詞骨髓增生異常綜合征白血病基因表達(dá)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)基因定位于染色體3q26。在小鼠逆轉(zhuǎn)錄病毒誘發(fā)的急性髓系白血病(AML) 模型中,Evi1是病毒常見的插入位點(diǎn), 與白血病的發(fā)生密切相關(guān)1，2。MDS1基因最初在骨髓增生異常綜合征(MDS) 中發(fā)現(xiàn), 又稱MDS相關(guān)基因,定位于Evi1上游170400kb處, 通過可變性剪接, MDS1和Evi1可形成MDS1-Evi1融合轉(zhuǎn)錄本3，4。我們報告31例MDS患者Evi1和MDS1-Evi1的表達(dá),探討這兩個基因在MDS發(fā)病中的作用。病例和方法1病例所有病例均為我院門診和住院患者。MDS患者31例,其中難治性貧血(RA) 8例

10、,原始細(xì)胞增多的RA(RAEB)13例, 轉(zhuǎn)化中RAEB(RAEB-t)9例,慢性粒-單核細(xì)胞白血病(CMML)1例；MDS后急性髓系白血病(post MDS AML)11例；原發(fā)性AML(de novo AML)34例。正常對照者8名;貧血對照為5例再生障礙性貧血(再障)患者。2試劑Taq酶, 脫氧核糖核苷三磷酸(dNTPs) 和隨機(jī)引物(上海生工公司), M-MLV(Gibco公司),焦碳酸二乙酯(DEPC,Promega 公司),異硫氰酸胍(Fluke公司)。聚合酶鏈反應(yīng)(PCR)引物: Evi1基因的PCR引物由美國國立衛(wèi)生研究院(NIH)王建祥博士和亞利桑那癌癥研究中心Taetle教

11、授惠贈, MDS1-Evi1基因和內(nèi)對照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引物序列見文獻(xiàn)5。3逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)采集患者骨髓液,ACD抗凝,分離單個核細(xì)胞。用異硫氰酸胍一步法提取細(xì)胞總RNA。反應(yīng)體系45l 內(nèi)含RNA5g, M-MLV300U, 2.5mmol/L dNTPs 6l,隨機(jī)引物60pmol

12、,0.1mol/L DTT 4.5l,5×第1鏈緩沖液 9l,37反應(yīng)1小時。Evi1、MDS1-Evi1反應(yīng)體系均為100l,cDNA均為來自同一逆轉(zhuǎn)錄體系的產(chǎn)物10l(1gRNA合成的cDNA)。Evi1的循環(huán)參數(shù)為94 30秒, 64 60秒,7260秒, 35個循環(huán);MDS1-Evi1的循環(huán)參數(shù)為94 30秒, 60 60秒,72 60秒,30個循環(huán)。取20l擴(kuò)增產(chǎn)物在1.5%的瓊脂糖凝膠電泳, 70V 80分鐘后在紫外燈下照相。膠片在島津 SC-9000型雙波長色譜掃描儀上掃描, 計算Evi1和MDS1-Evi1對GAPDH的比值。4染色體核型分析按常規(guī)R顯帶法。5祖細(xì)胞集

13、落培養(yǎng)CFU-E, BFU-E和CFU-GM均采用我所常規(guī)的甲基纖維素半固體培養(yǎng)法6。6統(tǒng)計學(xué)處理率的比較用2檢驗,兩組之間均數(shù)的比較用t檢驗。結(jié)果1對照組Evi1、MDS1-Evi1基因的表達(dá)8名正常人骨髓單個核細(xì)胞中無 Evi1 mRNA表達(dá)(陰性);其中3名有 MDS1-Evi1表達(dá)(陽性), 但表達(dá)量較低, MDS1-Evi1/GAPDH分別為0.067,0.039和0.054。5例再障患者Evi1和MDS1-Evi1表達(dá)均陰性。2MDS患者Evi1、MDS1-Evi1基因表達(dá)結(jié)果見表1。31例MDS患者15例Evi1表達(dá)陽性，表達(dá)率為 48.4%。經(jīng)2檢驗, RA患者Evi1表達(dá)率明

14、顯低于RAEB(t)(2=4.261,P<0.05)。31例患者19例MDS1-Evi1表達(dá)陽性，表達(dá)率為61.3%。各亞型之間MDS1-Evi1表達(dá)率無明顯差異(P>0.05)。僅有MDS1-Evi1表達(dá)者主要見于RA階段, 僅有Evi1或Evi1與MDS1-Evi1 同時表達(dá)主要見于RAEB和RAEB-t。部分陽性病例的電泳結(jié)果見1和2。1例CMML患者Evi1和MDS1-Evi1均為陰性。3Evi1、MDS1-Evi1基因表達(dá)與 MDS病情轉(zhuǎn)化的關(guān)系隨訪中, 2例患者由RA分別轉(zhuǎn)化為RAEB和RAEB-t, 5例患者由RAEB-t轉(zhuǎn)化為AML。除1例RA轉(zhuǎn)化為RAEBt者Ev

15、i1表達(dá)由陰性轉(zhuǎn)化為陽性外, 其余6例均未發(fā)現(xiàn)Evi1、MDS1-Evi1 由陰性轉(zhuǎn)化為陽性或陽性轉(zhuǎn)為陰性。但隨病情的進(jìn)展,5例(例1,6,22,26,29)轉(zhuǎn)化為AML的患者中4例Evi1基因表達(dá)量有增加的趨勢,MDS1-Evi1表達(dá)水平無明顯變化。4post MDS AML 與 de novo AML患者Evi1和MDS1-Evi1的表達(dá)post MDS AML患者Evi1和MDS1-Evi1表達(dá)率分別為54.5%和63.6%,34例de novo AML患者Evi1和MDS1-Evi1表達(dá)率分別為14.7%和23.5%。經(jīng)統(tǒng)計學(xué)處理2值分別為5.148和4.347,P值均<0.05

16、。5Evi1、MDS1-Evi1表達(dá)與染色體核型的關(guān)系7號染色體異常 (-7)的8例患者中,Evi1和MDS1-Evi1表達(dá)陽性者各有7例;無-7的21例患者中分別有7例表達(dá)Evi1和12例表達(dá)MDS1-Evi1,有-7的MDS患者兩個基因的表達(dá)率均較高, 但僅Evi1有統(tǒng)計學(xué)意義(2值>3.84,P<0.05)。8號染色體異常(+8,-8)的7例MDS中，Evi1和MDS1-Evi1表達(dá)陽性者分別為5例和4例,表達(dá)率亦較高,但無顯著性差異。在Evi1和MDS1-Evi1表達(dá)陽性的病例中,均未檢出3號染色體異常。6Evi1、MDS1-Evi1基因表達(dá)與祖細(xì)胞集落形成的關(guān)系與Evi1

17、 和MDS1-Evi1陰性組比較,這兩個基因陽性的病例祖細(xì)胞集落有不同程度的減少(表2)。表131例MDS患者Evi1、MDS1-Evi1基因的表達(dá)例號性別年齡亞型Evi1MDS1-Evi1Evi1/GAPDHMDS1-Evi1/GAPDH染色體核型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細(xì)胞系；17：MDS患者1部分MDS患者Evi1 PCR擴(kuò)增產(chǎn)物電泳    M：pBR322/Hinf標(biāo)志；N：正常對照；17：MDS患者2部分MDS患者M(jìn)DS1-Evi1 PCR擴(kuò)

21、增產(chǎn)物電泳表2部分MDS患者骨髓體外培養(yǎng)的祖細(xì)胞集落(<"01 (866 bytes)" src="/med/cano/201003/20100317190939365" 9 12>±s)組別例數(shù)CFU-E (集落/2×104細(xì)胞)BFU-E(集落/2×104細(xì)胞)CFU-GM(集落/1×105細(xì)胞)Evi1陽性組1119.7±23.76.5±6.61.1±2.1陰性組1343.4±58.013.9±21.44.5±4.6P值>0.05&

22、gt;0.05<0.05MDS1-Evi1陽性組1320.1±23.06.6±6.60.9±1.9陰性組1149.4±62.016.2±22.85.5±4.8P值>0.05>0.05<0.005     討論資料表明, Evi1基因在小鼠臟器的發(fā)育中起重要作用。小鼠心臟的發(fā)育過程中, Evi1表達(dá)的高峰在心臟形成的早期階段, 較晚期不再有Evi1的表達(dá);在呼吸系統(tǒng)和腎臟組織, Evi1表達(dá)的時相與發(fā)育的心臟相似, 提示Evi1 基因表達(dá)與組織細(xì)胞的“幼稚化”密切相關(guān)7。E

23、vi1基因可以抑制粒系細(xì)胞對集落刺激因子 (G-CSF)的反應(yīng), 阻礙粒細(xì)胞分化, 使其成熟障礙8。Evi1的表達(dá)還能抑制GATA-1誘導(dǎo)的基因轉(zhuǎn)錄, 從而阻礙紅細(xì)胞生成素誘導(dǎo)的紅系祖細(xì)胞的分化9。MDS 是一克隆性疾病,大部分是白血病前期, 發(fā)病的本質(zhì)是惡性克隆的形成, 異常幼稚白細(xì)胞的產(chǎn)生和分化障礙。我們觀察了31例MDS患者Evi1基因的表達(dá),其中近半數(shù)為陽性, 而8名正常人均陰性, 顯示MDS患者存在 Evi1基因異常表達(dá)。8名正常對照中, 雖3名有MDS1-Evi1融合基因表達(dá), 但MDS1-Evi1/GAPDH均<0.1, 而 MDS中 MDS1-Evi1陽性者,其比值均&g

24、t;0.1,表明該融合基因在MDS有過度表達(dá)。對AML的發(fā)病機(jī)制已有較為廣泛而深入的研究, 雖然目前還未能完全闡明, 但許多學(xué)者注意到de novo AML和post MDS AML在血液學(xué)和生物學(xué)特征等許多方面存在著差異。例如post MDS AML 骨髓造血細(xì)胞常有較明顯的發(fā)育異常的形態(tài)學(xué)改變, 預(yù)后較差, 常見 7號染色體異常等。本組post MDS AML患者Evi1、MDS1-Evi1 基因表達(dá)率明顯高于de novo AML,這是兩組AML生物學(xué)特征不同的又一個例證,并提示兩組AML的發(fā)病機(jī)制也不完全相同。在較早期的報道中,Evi1 mRNA的表達(dá)見于3號染色體異常,例如t(3;3

25、)(q21;q26)或 inv(3)(q21;q26)10。后來的資料表明, 無3號染色體異常的白血病中也有Evi1的異常表達(dá)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還可與AML1形成AML1-Evi1和AML1-MDS1-Evi1融合基因12，13。我們對MDS 患者進(jìn)行了核型分析, 均未發(fā)現(xiàn) 3號染色體異常, 而 7號染色體異常者 Evi1表達(dá)率較高。7號染色體異常見于治療相關(guān)MDS,但這些患者均未發(fā)現(xiàn)烷化劑等用藥史和放射線接觸史。因此,這些患者的Evi1表達(dá)率高的原因尚待進(jìn)一步闡明。本組MDS中, 向

26、AML轉(zhuǎn)化患者隨著病情向AML進(jìn)展，其Evi1表達(dá)量有遞增趨勢,雖然動態(tài)觀察的病例數(shù)較少, 不足以斷定Evi1在 AML轉(zhuǎn)化中的作用, 故有必要進(jìn)行更多病例Evi1表達(dá)的隨訪。更重要的是RAEB和RAEB-t患者的Evi1表達(dá)率顯著高于RA, 這進(jìn)一步支持Evi1與MDS病情進(jìn)展有關(guān)。此外,Evi1和MDS1-Evi1陽性的病例CFU-GM集落數(shù)顯著少于這兩個基因陰性的病例,陽性病例的CFU-E和BFU-E數(shù)亦較少(未達(dá)到統(tǒng)計學(xué)顯著性), 這也許為解釋MDS無效造血的發(fā)生機(jī)制提供了一個線索。參 考 文 獻(xiàn)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劉薏玲，湯曉培，郭亞東，等. 再生障礙性貧血患者粒、紅祖細(xì)胞的觀察. 中華血液學(xué)雜志，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