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关键词 急性白血病 血小板减少 肿瘤坏死因子-α
The serum of acute leukemia patients inhibit human
normal megakaryocytic progenitor cells
Chen Yu,Wu Weili,Wu Wen,et al.
Shanghai Institute of Hematology,Ruijin Hospital,Shanghai Second Medical University,Shanghai200025.
【Abstract】 Objective To investigate the inhibitory activities,which in the serum of patients with acute leukemia(AL)for the human normal megakaryocyticprogenitor cells,and to clarify the relationship between thromboˉcytopenia ofthose patients and the inhibitory activities.Methods Semi-solid culture in vitro,ELISA and RT-PCR were used.81novel patients with AL and40healthy volunteers were involved.Results The inhibitory rate of serum(serum IP)from the patients with AL on the growth of human normal CFU-MK is(25.71±30.13)%,which is sigˉnificantly higher than that of controls(P<0.01).The serum IR was reversely correlated with the number of megakaryocytes on the bone marrow slides and plateletsin the peripheral blood(r=-0.223、-0.236,P<0.05).The level of TNF-αin the serum of the patients with ALwas significantly higher than that of conrtrols,andwas correˉlated with serum IR(r=0.782,P<0.01).TNF-αmRNA were detected in the leukemic cells from5patients with AL.Conclusion The inhibitory activities exist in the serum of AL patients and may cause thrombocytopenia.Tumor necrosis factor-α(TNF-α)may play one of the major roles in the occurence of inhibition of megakaryocytic hematopoiessis and thrombocytopenia.It may be one of the reasons why the levels of TNF-αin AL patients increase that leukemic cells can secrete TNF-α.
Key words acute leukemia thrombocytopenia tumor necrosis factor-α
出血是急性白血病(AL)的常见症状,主要原因是血小板减少。以前研究发现急性早幼粒细胞白血病患者血小板减少与血清存在对正常巨核系造血抑制活性有关,该活性可能与肿瘤坏死因子相关 [1] 。本文进一步研究AL患者血小板减少的发生机制。
1 资料与方法
1.1 一般资料 为1999年6月~2001年2月我院住院初发未治的AL患者,共81例,其中男44例,女37例;中位年龄37(9~79)岁。根据患者的临床表现、外周血象、骨髓象,按FAB分型标准确立诊断,其中急性淋巴细胞白血病ALL)24例,急性非淋巴细胞白血病57例,FAB分型见表1。
表1 81例AL患者FAB分型
正常人(对照组)为同期我院健康职工,男、女各20人,中位年龄36(19~58)岁。
1.2 血清制备 分别抽取患者或正常人静脉血5ml,注入无菌试管中,静置3h后2500rpm离心20min,吸取血清加入无菌冻存管中,-20℃分装保存。
1.3 体外AL患者血清对正常人骨髓巨核系造血祖细胞(CFU-MK)增殖的影响
1.3.1 正常人骨髓单个核细胞悬液制备 取自胸外科手术中截取的非血液病患者的正常肋骨,加以挤压,冲洗出骨髓细胞,制成细胞悬液,并使用含肝素的α培养液充分洗涤,然后沿管壁缓缓注到2ml淋巴细胞分离液(比重1.077)液面上,离心(2500rpm,20min)后吸出单个核细胞层,再用α培养液洗涤2次,制成单个核细胞悬液,计数后备用。
1.3.2 血清抑制CFU-MK增殖实验
1.3.2.1 CFU-MK培养体系 调整正常人骨髓单个核细胞浓度为5×10 6 /ml。取24孔板,每孔培养体系0.5ml,分别含10%BSA(牛血清白蛋白)0.05ml,细胞悬液0.05ml(含细胞2.5×10 5 ),AB型血清0.05ml,牛血浆0.05ml,0.034%氯化钙0.05ml,10 -3 M二巯基乙醇0.05ml,α培养液补足至0.5ml。
1.3.2.2 血清抑制实验 设置空白对照组、实验(AL患者血清)组、对照(正常人血清)组。空白对照组设4孔,其他组每个标本设复孔。在巨核系造血祖细胞培养体系中分别加入患者或正常人血清0.05ml(空白对照加α培养液0.05ml)。置37℃、5%CO 2 、饱和湿度培养箱中培养,14天后取出固定染色。甲醇/丙酮(1:3)原位固定,经PBS充分洗涤后用抗Gp(血小板糖蛋白)Ⅲa的单克隆抗体、生物素-抗生物素反应盒(VECTASTAIN ABC试剂盒,购自Gibco公司)进行染色,步骤严格按说明书操作(略)。以染成棕色或棕黄色,≥3个细胞组成的细胞团为CFU-MK集落。
1.3.2.3 抑制率(IR) 按下列公式计算IR=(1- 实验组集落均数空白对照组集落均数)×100%
1.4 血清中肿瘤坏死因子α(TNF-α)和γ干扰素(IFN-γ)浓度测定(ELISA法) 严格按照TNF-α和IFN-γ试剂盒(双抗体夹心法,试剂盒购自晶美公司)说明书进行操作。
1.5 AL患者骨髓单个核细胞TNF-αmRNA表达检测 采用RT-PCR法。
1.5.1 骨髓标本 为同期未经治疗的5例急性白血病患者,中位骨髓白血病细胞百分比为88%(56%~96.5%),其中ALL-L 2 3例,ANLL-M 2 1例,ANLL-M 3 1例。对照为1例20岁男性正常志愿者骨髓者。
1.5.2 RT-PCR
1.5.2.1 骨髓单个核细胞分离 同前。
1.5.2.2 总RNA提取 按TRIZOL试剂盒说明书的方法进行。
1.5.2.3 RNA定量和纯度鉴定 取1μl5.2.2中制备的RNA,用DEPC水稀释100倍,紫外分光光度计(BECKMAN DU-600)测RNA量和260/280nm的OD值。
1.5.2.4 引物设计(参照文献2) 人TNF-α基因引物序列如下:
Forward Primer:5′ATGAGCACTGAAAGCATGATCCGG3′Reverse Primer:5′GCAATGATCCCAAAGTAGACCTGCCC3′ PCR产物692bp。
内参照采用GAPDH,引物序列如下:
Forward Primer:5′GAAGGTGAAAGGTCGGAGTCA