骨髓增生異常綜合征淋巴細(xì)胞亞群及其激活狀態(tài)的分析

1、骨髓增生異常綜合征淋巴細(xì)胞亞群及其激活狀態(tài)的分析               作者：楊雋，王椿，謝匡成，顏式可，高彥榮，               蔡琦，秦尤文，萬理萍，蔡宇【摘要】  本研究探討骨髓增生異常綜合征（MDS）患者外周血T細(xì)胞亞群，B細(xì)胞，NK細(xì)胞數(shù)量及激活狀態(tài)的臨床意義。采用流式細(xì)胞術(shù)對(duì)30例MDS患

2、者外周血T細(xì)胞，B細(xì)胞，NK細(xì)胞及其表面活化分子CD28，CD45RA，CD45RO，CD69，HLA-DR的表達(dá)進(jìn)行檢測(cè)和分析。30例MDS患者中低危組（RA+RAS）22例，高危組(RAEB+RAEBT)8例。結(jié)果表明：MDS組T細(xì)胞（CD3+細(xì)胞）的百分率低于正常對(duì)照組，CD4+CD45RA+細(xì)胞（未致敏CD4+細(xì)胞）的數(shù)值低于正常對(duì)照組。MSD組早期活化T細(xì)胞（CD3+CD69+細(xì)胞）和晚期活化T細(xì)胞（CD3+HLA-DR+細(xì)胞）的數(shù)值均顯著高于正常對(duì)照組。低危組(RA和RAS)主要表現(xiàn)為T細(xì)胞活化功能的改變：早期活化T細(xì)胞（CD3+CD69+細(xì)胞）和晚期活化T細(xì)胞（CD3+HLA-D

3、R+細(xì)胞）比例均增高，以及B細(xì)胞數(shù)量的減少。高危組（RAEB和RAEBT）主要表現(xiàn)為T細(xì)胞亞群數(shù)量的改變：CD3+細(xì)胞，CD3+CD4+CD8-細(xì)胞(T輔助細(xì)胞)數(shù)量的減少，CD3+細(xì)胞HLA-DR和CD69的表達(dá)并不增高。NK細(xì)胞數(shù)量減少。結(jié)論：MDS病人存在有T細(xì)胞數(shù)量和功能的異常，且隨著病情的進(jìn)展而發(fā)生改變，所以T細(xì)胞亞群及活化功能的檢測(cè)對(duì)于判斷疾病的進(jìn)程和指導(dǎo)治療具有重要的意義。 【關(guān)鍵詞】  骨髓增生異常綜合征；T細(xì)胞亞群；B細(xì)胞；NK細(xì)胞；激活標(biāo)志Evaluation of the Subsets    of Lymphocytes 

4、; and Their Activated Status   in Patients with Myelodysplastic Syndrome Abstract    This study was purposed to investigate  the clinical significance of the amount and activated status of T cell subsets，B cells，NK cells in peripheral blood from patients with myelo

5、dysplastic syndrome (MDS).  The proportion of T cells，B cells，NK cells in peripheral blood from 30 patients with MDS and their surface activation markers of CD28，CD45RA，CD45RO，CD69，HLA-DR were analyzed by flow cytometry. Twenty-two patients were in the low risk group (RA+RAS) while eight patien

6、ts were in the high risk group (RAEB+RAEBT). The result showed  that the amounts of T cells (CD3+cells) in peripheral blood from patients with MDS were lower than those in control group .The amounts of naived CD4+ cells (CD4+CD45RA+ cells) in MDS patients were lower than those in control. The e

7、xpression rates of early activation marker (CD69) and late activation marker (HLA-DR) on CD3+ cells in MDS patients were significantly higher than those in control. The abnormalities of the immunologically competent cells were mainly observed in the low risk group (RA+RAS)，and were characterized by

8、the high expression rates of CD69+ and HLA-DR+ on CD3+cells，the decrease of  B cell amounts. The amount abnormalities of T cell subsets were mainly observed in high risk group (RAEB+RAEBT)，and were characterized by the decrease of CD3+ cells and CD3+CD4+CD8-cells (Th cells)    am

9、ounts without  high expression of the CD69 and HLA-DR，the decrease of NK cells amounts. It is concluded that there are the abnormalities of T cell subsets and function in the patients with MDS and may change with  disease progression，so the measurement of amonnt and activated status of T c

10、ell subsets in peripherat blood from MDS patients can have predictive role for diagnosis of disease progression and guide of therapy.Key words    myelodysplastic syndrome; T cell subset;  B cell; NK cell; activation marker    骨髓增生異常綜合征（MDS）是一組由于造血干細(xì)胞突變而引起的惡性克隆增殖性疾病

11、，該病具有異質(zhì)性，以骨髓無效造血，三系細(xì)胞減少和形態(tài)學(xué)異常為特征?，F(xiàn)有的臨床資料表明，部分MDS病人用免疫抑制劑（如ATG，環(huán)孢菌素） 治療有效，且MDS和其它各種腫瘤一樣，可出現(xiàn)副瘤免疫現(xiàn)象，如血管炎，關(guān)節(jié)炎，Sweet綜合癥，Crohns病等。這些免疫紊亂的表現(xiàn)可出現(xiàn)在MDS發(fā)病之前、之后或同時(shí)發(fā)生。以上事實(shí)提示了MDS可能與免疫功能異常有關(guān)，但確切的機(jī)理尚不清楚1。本研究通過流式細(xì)胞儀三色熒光標(biāo)記的方法對(duì)MDS患者外周血（PB）淋巴細(xì)胞功能及激活狀態(tài)進(jìn)行了分析，并探討其臨床意義。材料和方法研究對(duì)象所有病例均系我院2003年5月至2004年12月住院或門診的患者，共30例，根據(jù)FAB分型，

12、難治性貧血RA 20例，RAS 2例，RAEB 6例，RAEBT 2例。其中女性13例，男性17例，中位年齡58歲（16-87歲），均為初治的病例。將RA和RAS分為低危組，RAEB和RAEBT分為高危組，病人情況詳見表1。對(duì)照組15例，男性8例，女性7例，為我院工作人員和正常獻(xiàn)血者，中位年齡45歲。Table 1. Clinical characteristics of the MDS patients     方法取肘靜脈血2 ml，2EDTA-Na2抗凝，然后取抗凝血100  l，加入放有三色熒光標(biāo)記單克隆抗體組合(PE/FITC/CY5

13、)的試管：CD3/CD4/CD8，CD3/CD(16+56)/CD19，CD3/CD4/CD28，CD3/CD8/CD28，CD4/CD45RA/CD45RO，CD8/CD45RA/CD45RO，CD3/HLA-DR/CD69（法國Coulter公司），混勻后室溫閉光溫育30分鐘，用Coulter公司Immuno-PREP全血標(biāo)本處理儀，經(jīng)配套產(chǎn)品進(jìn)行溶血，稀釋和固定后，4小時(shí)內(nèi)上流式細(xì)胞儀檢測(cè)，配套軟件取數(shù)和分析，以CD3+細(xì)胞設(shè)門圈定淋巴細(xì)胞進(jìn)行分析（圖1）。每個(gè)樣本分析20 000個(gè)細(xì)胞，結(jié)果以陽性細(xì)胞的百分率（）表示。統(tǒng)計(jì)學(xué)處理所有資料均采用SPSS  11.5統(tǒng)計(jì)軟件進(jìn)行分

14、析。數(shù)據(jù)經(jīng)過正態(tài)性檢驗(yàn)符合正態(tài)分布，故采用t檢驗(yàn)分析，數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差（X±SD）表示。結(jié) 果MDS和對(duì)照組外周血中不同淋巴細(xì)胞亞群表型比較MDS患者外周血中CD3+細(xì)胞（T淋巴細(xì)胞）的百分率低于正常對(duì)照組(P<0.05)，但絕對(duì)值無顯著差異（表2），CD3+CD4+CD8-細(xì)胞（T輔助細(xì)胞）和CD3+CD4-CD8+細(xì)胞（細(xì)胞毒性/抑制細(xì)胞）的百分率和絕對(duì)值以及CD4/CD8的比值與正常對(duì)照組相比無顯著差異(P>0.05)，其中CD4+CD45RA+細(xì)胞（未致敏CD4+細(xì)胞）的數(shù)值明顯低于正常對(duì)照組(P<0.01)。CD4+CD45RO+細(xì)胞（活化和記

15、憶CD4+細(xì)胞）、CD8+CD45RA+（未致敏CD8+細(xì)胞）和CD8+CD45RO+（活化和記憶CD8+細(xì)胞）的數(shù)值與正常對(duì)照組相比無顯著差異(P>0.05)。CD3-CD19+細(xì)胞（B細(xì)胞）的數(shù)值低于正常對(duì)照組。CD3-CD(16+56)+ 細(xì)胞（NK細(xì)胞）、T細(xì)胞共刺激因子的表達(dá)（CD3+CD4+CD28+，CD3+CD8+CD28+）與正常對(duì)照組相比無顯著差異(P>0.05)。早期活化T細(xì)胞（CD3+CD69+細(xì)胞）和晚期活化T細(xì)胞（CD3+HLA-DR+細(xì)胞）的絕對(duì)值均顯著高于正常對(duì)照組(P<0.01)。Table 2. Comparison of the expr

16、ession rates of T cell subsets of each groups （略）低危組和對(duì)照組PB中不同淋巴細(xì)胞亞群表型比較低危患者PB中CD3+細(xì)胞（T淋巴細(xì)胞），CD3+CD4+CD8-細(xì)胞（T輔助細(xì)胞）和CD3+CD4-CD8+細(xì)胞（細(xì)胞毒性/抑制細(xì)胞）的絕對(duì)值以及CD4/CD8的比值與正常對(duì)照組無顯著差異(P>0.05)。但CD4+CD45RA+細(xì)胞（未致敏CD4+細(xì)胞）的數(shù)值低于正常對(duì)照組(P<0.01)（圖2），CD4+CD45RO+細(xì)胞（活化和記憶CD4+細(xì)胞）、CD8+CD45RA+（未致敏CD8+細(xì)胞）和CD8+CD45RO+（活化和記憶CD8+

17、細(xì)胞）的數(shù)值與正常對(duì)照組無顯著差異(P>0.05) （表2）。CD3-CD(16+56)+ 細(xì)胞（NK細(xì)胞）的數(shù)值低于正常對(duì)照組。CD3-CD19+細(xì)胞（B細(xì)胞），T細(xì)胞共刺激因子的表達(dá)（CD3+CD4+CD28+，CD3+CD8+CD28+）與正常對(duì)照組相比無顯著差異(P>0.05)。早期活化T細(xì)胞（CD3+CD69+細(xì)胞）和晚期活化T細(xì)胞（CD3+高危組和對(duì)照組PB不同淋巴細(xì)胞亞群表型比較高危患者PB中CD3+細(xì)胞（T淋巴細(xì)胞）總數(shù)明顯低于正常對(duì)照組(P<0.01) （表2），CD3+CD4+CD8-細(xì)胞（T輔助細(xì)胞）絕對(duì)值明顯低于正常對(duì)照組(P<0.05)(圖4)

18、，CD3+CD4-CD8+細(xì)胞（細(xì)胞毒性/抑制細(xì)胞）的數(shù)值與正常對(duì)照組相比無顯著差異(P>0.05)，CD4+CD45RA+細(xì)胞（未致敏CD4+細(xì)胞）數(shù)值明顯低于正常對(duì)照組(P<0.01)，CD4+CD45RO+細(xì)胞（活化和記憶CD4+細(xì)胞），CD8+CD45RA+（未致敏CD8+細(xì)胞），CD8+CD45RO+（活化和記憶CD8+細(xì)胞）的絕對(duì)值與正常對(duì)照組相比無顯著差異(P>0.05)。CD3-CD(16+56)+ 細(xì)胞（NK細(xì)胞）的絕對(duì)值明顯低于正常對(duì)照組，CD4+細(xì)胞共刺激因子的表達(dá)（CD3+CD4+CD28+）明顯低于正常對(duì)照組。CD3-CD19+細(xì)胞（B細(xì)胞）和 CD

19、3+CD8+CD28+細(xì)胞與正常對(duì)照組相比無顯著差異(P>0.05)。早期活化T細(xì)胞（CD3+CD69+細(xì)胞）和晚期活化T細(xì)胞（CD3+HLA-DR+細(xì)胞）的絕對(duì)值與正常對(duì)照組相比無顯著差異(P>0.05)。討論目前，國內(nèi)外對(duì)MDS免疫功能的研究報(bào)道大多數(shù)為T淋巴細(xì)胞亞群變化的研究，而對(duì)細(xì)胞的功能狀態(tài)的研究報(bào)道較少見。靜止的T細(xì)胞只有通過內(nèi)源性或外源性激活物刺激激活后才能發(fā)揮免疫調(diào)節(jié)和造血調(diào)控作用。我們對(duì)MDS患者外周血淋巴細(xì)胞亞群的檢測(cè)觀察到CD3+細(xì)胞的百分率數(shù)較正常對(duì)照組減少，但CD3+細(xì)胞、T輔助細(xì)胞（CD3+CD4+CD8-細(xì)胞）和T細(xì)胞毒性/抑制細(xì)胞（CD3+CD4-C

20、D8+細(xì)胞）數(shù)量無明顯改變。我們對(duì)MDS病人進(jìn)一步分組后發(fā)現(xiàn)，高危組病人CD3+細(xì)胞的總數(shù)和T輔助細(xì)胞（CD3+CD4+CD8-細(xì)胞）數(shù)量均低于正常對(duì)照組，而低危組病人則與正常對(duì)照組無明顯差異，這一結(jié)果提示MDS患者T淋巴細(xì)胞絕對(duì)數(shù)值的變化可能是MDS惡性轉(zhuǎn)化的前兆。有文獻(xiàn)報(bào)道，MDS病人存在NK細(xì)胞功能的異常，主要表現(xiàn)為NKT細(xì)胞功能的活化2，而本研究發(fā)現(xiàn)高危組病人NK細(xì)胞的數(shù)量明顯減少，而低危組病人的NK細(xì)胞數(shù)量無明顯異常。此外，低危組病人B細(xì)胞的數(shù)量是明顯低于正常對(duì)照組，而高危組病人中無明顯差異，這提示低危組病人可能存在有體液免疫的異常。     

21、    CD28是T細(xì)胞的表面標(biāo)記物之一，CD28與它的配體（抗原呈遞細(xì)胞上的B7）相結(jié)合使T細(xì)胞活化，一旦T細(xì)胞表面不表達(dá)CD28，則T細(xì)胞將處于無能狀態(tài)3，4。本實(shí)驗(yàn)結(jié)果顯示，高危組病人的CD4陽性細(xì)胞CD28的表達(dá)明顯減少，提示此組病人可能存在有CD4表達(dá)的無能。    根據(jù)表達(dá)CD45分子的不同，可將T細(xì)胞分為CD45RA+細(xì)胞（未致敏T細(xì)胞）和CD45RO+細(xì)胞（活化和記憶T細(xì)胞），MDS患者的CD4+CD45RA+細(xì)胞數(shù)下降，原因可能是在抗原刺激下轉(zhuǎn)化為CD4+CD45RO+細(xì)胞5，6。   

22、  CD69抗原，也叫早期激活抗原（EA-1），是由同型二聚體組成的型糖基化細(xì)胞膜蛋白，屬于C-植物凝血素家族，分子量為60  kD，在B淋巴細(xì)胞，NK細(xì)胞和激活的T細(xì)胞上均可表達(dá)，在靜止的T細(xì)胞上不表達(dá)，在絲裂原和抗原刺激激活的淋巴細(xì)胞表面表達(dá)，這是T細(xì)胞激活后最早表達(dá)的表面抗原7，8。HLA-DR屬于主要組織相容復(fù)合體MHC類分子，受體是CD4，由和兩條重鏈通過非共價(jià)鍵連接分子量分別為34 kD和29 kD，是免疫反應(yīng)中重要的參與者，在抗原呈遞和胸腺選擇中起作用，通常表達(dá)于免疫系統(tǒng)的細(xì)胞，如淋巴細(xì)胞和巨噬細(xì)胞。HLA-DR在炎性刺激24小時(shí)后表達(dá)升高，持續(xù)數(shù)周，MHC限

23、制性是免疫細(xì)胞間相互識(shí)別和協(xié)作的前提，是淋巴細(xì)胞激活的晚期標(biāo)志9，10。目前，國內(nèi)外對(duì)MDS淋巴細(xì)胞激活方面的研究多數(shù)是研究CD25，而對(duì)CD69和HLA-DR的研究較少見。本研究觀察到MDS低危組患者體內(nèi)的T細(xì)胞CD69和HLA-DR的表達(dá)均明顯升高，這可能是機(jī)體的免疫系統(tǒng)對(duì)MDS異?？寺≡鲋车姆磻?yīng)，此反應(yīng)為持續(xù)性的，從而使CD69和HLA-DR的表達(dá)升高。而高危組病人CD3+HLA-DR+細(xì)胞和CD3+CD69+細(xì)胞并不增高，但伴有CD3細(xì)胞數(shù)量的減少，提示隨著疾病的進(jìn)展T細(xì)胞亞群出現(xiàn)功能的異?；驒C(jī)體免疫能力下降。臨床上，對(duì)早期的病人，尤其是RA病人，由于T細(xì)胞處于激活狀態(tài)，故使用一些免疫

24、抑制劑（如環(huán)孢菌素，ATG等）可能對(duì)其有效，此實(shí)驗(yàn)的結(jié)果為免疫抑制劑的臨床使用提供了理論的依據(jù)。而對(duì)于高危期MDS病人由于T細(xì)胞的活化功能在疾病的發(fā)病中已經(jīng)不起主要作用，表現(xiàn)為T細(xì)胞亞群功能的異常，所以用免疫抑制劑對(duì)此類病人可能無效，需要采取更積極的治療措施（如化療或骨髓移植等）來防止疾病向白血病轉(zhuǎn)化。    綜上所述，T細(xì)胞亞群及活化功能的研究對(duì)判斷疾病進(jìn)程，預(yù)測(cè)疾病的轉(zhuǎn)歸，指導(dǎo)臨床用藥，判斷預(yù)后具有重要的價(jià)值，是目前測(cè)定體內(nèi)免疫功能的最適指標(biāo)。【參考文獻(xiàn)】  1 Roy-Peaud F，Paccalin M，Le-Moal G，et al. Asso

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