调节性T细胞在慢性粒细胞白血病中的表达及临床意义

  目的   分析CD4+CD25+Treg细胞在慢性粒细胞白血病（CML）患者不同病程中的表达,探讨其与该疾病的疗效及预后的关系,从免疫学方面揭示CML发生及转归的可能机制。   方法   收集23例不同病程分期CML患者及10例健康对照的外周血,采用流式细胞仪技术对CD4+CD25+Treg细胞进行检测,比较CML患者与对照之间及不同病程CML患者之间CD4+CD25+Treg细胞表达的差异;比较CD4+CD25+Treg细胞表达水平与对应的临床参数间的关系。   结果   与正常对照比较,CML初治患者、加速急变期患者外周血单个核细胞中CD4+CD25+Treg细胞百分比呈显著性下降（P < 0.05）;而CML慢性治疗期患者外周血单个核细胞中CD4+ CD25+Treg细胞百分比与正常对照比较无显著性差异（P>0.05）;与正常对照比较,CML初治患者、加速急变期患者外周血单个核细胞中CD4+Treg细胞百分比呈显著性下降（P < 0.05）;而CML慢性治疗期患者外周血单个核细胞中CD4+Treg细胞百分比与正常对照比较无显著性差异（P>0.05）;不同病程CML患者外周血单个核细胞中CD4+CD25+Treg细胞百分比相比较,慢性稳定期患者较初治患者有明显上升,差异有统计学意义（P < 0.05）;而其他组之间无显著性差异（P>0.05）;初治CML患者外周血单个核细胞中CD4+ CD25+Treg细胞百分比与外周血血红蛋白量呈正相关（P=0.010）;与骨髓中幼稚细胞（原始粒细胞+早幼粒细胞）百分比呈负相关（P=0.022）。   结论   CML患者外周血单个核细胞中CD4+CD25+Treg细胞占外周血单个核细胞的比例下降,提示患者进入加速急变期CD4+CD25+Treg细胞在CML患者中起到一定的免疫调节作用。      

中西医结合治疗慢性粒细胞白血病现状及进展

慢性粒细胞白血病(CML)是一种起源于多能造血干细胞的恶性增殖性疾病.它的细胞遗传学特征是有Ph染色体,即t(9;22)(q34;q11).9号染色体上的abl基因易位到22号染色体的bcr上,形成bcr/abl融合基因.bcr/abl融合基因是慢性粒细胞白血病的分子生物学标志.bcr/abl融合基因表达P210(bcr/abl),P190(bcr/abl)或P230(bcr/abl)蛋白,该蛋白具有异常增高的酪氨酸激酶活性,使造血干细胞发生异常转化而导致CML发生.近几十年来,由于异基因造血干细胞移植、干扰素及联合化疗的临床应用,尤其是bcr/abl酪氨酸激酶抑制剂(STI571)及中药提取物三氧化二砷等的应用,慢性粒细胞白血病的治疗取得了很大进步.     

急性白血病患者外周血Treg细胞与Th17细胞的相关性

目的:检测急性白血病患者外周血中Treg细胞与Th17细胞的比例以及相关细胞因子如IL-17、IL-6、TGF-β的变化,分析其相关性。方法:选取兰州大学第二医院血液科急性白血病初诊患者15例,另取15名健康志愿者为对照。流式细胞术检测外周血中CD3+CD4+TIL-17+辅助性T细胞(Th17细胞)、CD4+TCD25+Foxp3+调节性T细胞(Treg细胞)占CD4+T细胞的比例,ELISA法检测血清中细胞因子IL-17、TGF-β、IL-6的水平。结果:急性白血病患者外周血中Th17细胞占CD4+T细胞的(1.39±0.24)%,高于对照组的(0.26±0.11)%(P<0.05);急性白血病患者外周血Treg细胞占CD4+T细胞的(11.58±2.17)%,高于对照组的(2.47±0.72)%(P<0.05);且Treg细胞与Th17细胞呈正相关(γ=0.37)。急性白血病患者血清中TGF-β、IL-6、IL-17的水平分别为(26.06±2.43)、(14.66±2.47)、(18.63±2.38)pg/ml,高于对照组的(13.41±1.92)、(1.44±0.29)、(10.34±1.71)pg/ml(均P<0.05)。结论:急性白血病患者外周血中Treg、Th17细胞比例升高,且两者呈正相关;急性白血病患者血清中TGF-β、IL-6、IL-17水平升高,可能影响Treg与Th17细胞的平衡。     

人mda-7/IL-24对慢性粒细胞白血病K562细胞的抑制作用

摘 要 目的: 探索人黑素瘤分化相关基因-7（melanoma differentiation associated gene7，mda7；又称IL-24）对慢性粒细胞白血病K562细胞的抑制作用。方法：用RTPCR法检测11个造血系统恶性肿瘤细胞系mda-7受体的转录情况。用脂质体转染法将构建的重组真核表达载体pTargetIL24转染K562细胞。以RTPCR和Western blotting方法检测转染的K562细胞mda7的表达情况；通过MTT法、集落形成实验、流式细胞术、AnnexinⅤ/PI检测mda7/IL24对K562细胞增殖、集落形成、细胞周期和凋亡的影响；以裸鼠移植瘤模型观察mda-7对K562细胞移植瘤的治疗作用。结果：在11个造血系统恶性肿瘤细胞系（NB4、HL60、CEM、Namalwa、Jurkat、KG1a、U937、K562、Ramos、J61、HEL细胞）中没有检测到完整mda7受体的表达。转染mda7的K562细胞能显著表达mda-7mRNA及其蛋白；其与转染空载体和未转染的K562细胞相比，细胞增殖活力以及集落形成能力明显下降（P＜0.05），细胞周期阻滞于G0/G1期（P＜0.05），但细胞凋亡率没有明显变化。裸鼠体内实验证实了mda7/IL24明显抑制K562细胞移植瘤生长（P＜0.01）。结论：mda-7/IL-24对白血病细胞系K562具有明显的体内外抑制作用，该作用与mda7/IL24引起细胞周期G0/G1期阻滞有关。     

肺癌患者外周血CD+4 CDHi25 CDLo127 调节性T细胞检测的临床意义

 目的　探讨肺癌患者外周血中CD+4 CDHi25 CDLo127 调节性T细胞（Treg）占CD+4 T淋巴细胞比例的变化及其意义。方法　采用流式细胞术检测肺癌患者30例（鳞癌20例、腺癌10例）及健康人（对照组）20名外周血中Treg的变化;ELISA法检测同一标本血清中白细胞介素10（IL-10）,转化生长因子-β（TGF-β）的表达水平。结果　鳞癌、腺癌患者外周血中Treg 占CD+4 T淋巴细胞的比例分别为（6.81±1.52）％、（7.08±1.17）％,两组间比较差异无统计学意义（P＞0.05）,二者均高于对照组的（4.91±1.24）％ ,差异有统计学意义（P＜0.05）;肺癌组血清中IL-10水平为（24.36±4.02）ng／ml,高于健康对照组的（21.53±4.30）ng／ml;肺癌组血清中TGF-β水平为（218.49±35.23）ng／ml,高于健康对照组的（124.31±20.32）ng／ml。结论　肺癌患者外周血中Treg比例明显升高,可能是导致免疫抑制的原因之一,并且可能通过分泌IL-10和TGF-β,发挥免疫抑制的效应。     

调节性B细胞与肿瘤免疫的关系

B细胞介导的体液免疫在肿瘤免疫系统中发挥着重要作用。研究发现,确实存在一种名为调节性B细胞（Bregs）的B淋巴细胞亚群,可与肿瘤细胞相互作用,通过分泌白细胞介素10和转化生长因子β等细胞因子,在肿瘤免疫系统中承担其特殊的调节性作用。针对Bregs的肿瘤诊断思路和治疗方法的研究也初步展开,并显示出其重要的研究价值和广阔的应用前景。     

HLA-A0201阳性慢性粒细胞白血病PR1特异性细胞毒性T细胞的初步研究

目的 探讨PR1特异性细胞毒性T细胞(CTL)与HLA-A0201阳性慢性粒细胞白血病(CML)患者疗效及预后的关系,探讨是否可以应用PR1肽作为疗效达到停药试验标准患者的后续免疫治疗,从而维持长期缓解状态.方法 收集28例HLA-A0201阳性CML患者外周血,利用可溶性PR1-MHC-Ⅰ类四聚体技术及流式细胞术检测PR1特异性CTL存在与否、频率高低;比较不同治疗时间CML患者PR1特异性CTL表达的差异.结果 PR1特异性CTL频率与同期PCR(bcr-abl/abl)IS(IS:国际标准值)之间呈负相关(r=-0.658,P＜0.001).接受治疗3、6、9个月及1、2、3、4、5、6年的PR1特异性CTL频率分别为(0.06±0.02)％、(0.10±0.02)％、(0.14土0.02)％、(0.16±0.02)％、(0.20±0.03)％、(0.18±0.03)％、(0.18±0.01)％、(0.17±0.05)％、(0.18±0.03)％.治疗3、6、9个月的频率分别与其他时间点频率相比,差异均有统计学意义(均P＜ 0.05).治疗1年及以上的频率两两相比,差异均无统计学意义(均P＞0.05).伊马替尼400 mg 1次/d治疗3、6、9、12个月,Sokal评分高危组患者PR1特异性CTL频率低于低危组、中危组.结论 治疗效果显著的CML患者体内可持续检测到PR1特异性CTL,其与肿瘤负荷呈负相关,提示PR1特异性CTL可能与抗白血病细胞作用相关,为疗效获得持续稳定MR4-5及MR5.0的CML患者使用PR1肽疫苗作为后续免疫治疗提供了依据.     

流感疫苗对慢性粒细胞白血病源性树突状细胞功能影响的研究

 目的 探讨流感疫苗对白血病源性树突状细胞（DC）功能的影响。方法 分离慢性粒细胞白血病（CML）患者骨髓单个核细胞,加入GM-CSF和IL-4诱导培养7 d,将所得细胞分为4组：完整失活流感疫苗（WIV）组;裂解流感疫苗（SIV）组;TNF-α组;对照组,用不同培养方法再培育24 h后,流式细胞仪分析DC免疫表型,ELISA法检测上清液中IL-12浓度。结果 CML源性DC细胞表面分子表达、上清液中IL-12浓度在WIV及SIV组较TNF-α和对照组显著增高,差异有统计学意义（P＜0.05）;其中WIV组高于SIV组（P＜0.05）,而TNF-α组与对照组差异无统计学意义（P＞0.05）。结论 流感疫苗具有刺激CML源DC成熟的功能。     

慢性粒细胞白血病的治疗新进展

慢性粒细胞白血病(chronicmyelogenousleukemia,CML)是以粒系增生为主要表现的恶性克隆性疾病。以往CML的常规治疗包括羟基脲、马利兰、靛玉红及甲异靛、干扰素、联合化疗及造血干细胞移植等。近年来,CML的治疗在以往的基础上有了长足的进展,其中以bcr-ab融合基因及其具有酪氨酸激酶活性的蛋白产物为靶向的酪氨酸激酶抑制剂Imatinib是近年来CML治疗最重要的进展,此外在干扰素及干细胞移植治疗方面亦有了新的认识。CML的治疗已发展为一个复杂而有序的系统。本文就近年来CML治疗方面的进展进行综述。     

Isolated central nervous system blast crisis in chronic myeloid leukemia

Chronic myeloid leukemia is a myeloproliferative disorder characterized by the presence of the Philadelphia chromosome, t(9:22). Extramedullary blast crisis is a rare event. Imatinib mesylate has become the treatment of choice, especially for patients for whom allogenic stem cell transplantation is not an option. Imatinib produces complete cytogenetic responses in excess of 80%. However, the penetration of the drug and its metabolites into the CNS (Central Nervous System) is poor. Hence for patients who are on prolonged imatinib therapy and continue to have complete cytogenetic responses, the central nervous system may become a sanctuary site. We report a patient who had a complete hematologic and cytogenetic response and presented with headache and vomiting. The MRI showed meningeal enhancement and the CSF (Cerebro Spinal Fluid) examination was positive for blasts. He was started on cranial radiotherapy and triple intrathecal chemotherapy. He showed good symptomatic improvement and cleared the blasts in the CSF. At the end of radiation, he was in complete hematological remission but had 50% marrow metaphases positive for Philadelphia chromosome. As he did not have a matched sibling donor, the dose of imatinib was increased to 600 mg daily. He continues to be in complete hematologic remission at the time of this report.     

p21 Confers resistance to imatinib in human chronic myeloid leukemia cells

Imatinib is a Bcr-Abl inhibitor used as first-line therapy of chronic myeloid leukemia (CML). p21^Cip1, initially described as a cell cycle inhibitor, also protects from apoptosis in some models. We describe that imatinib down-regulates p21^Cip1 expression in CML cells. Using K562 cells with inducible p21 expression and transient transfections we found that p21 confers partial resistance to imatinib-induced apoptosis. This protection is not related to the G2-arrest provoked by p21, a decrease in the imatinib activity against Bcr-Abl or a cytoplasmic localization of p21. The results suggest an involvement of p21^Cip1 in the response to imatinib in CML.     

急性髓系白血病细胞对树突状细胞分化、成熟和调节性T细胞生成的影响

Objective To investigate the effects of soluble factors secreted by acute myeloid leukemia (AML) cells on the phenotypical and functional properties of DCs derived from normal mononuclear cells. Methods Mononuclear cells were cultured with interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF), in the presence or absence of 24 h culture supernatants from fresh primary AML cells, to generate immature DCs. The maturation of DCs was induced by cytokines IL-1beta, IL-6, tumor necrosis factor-alpha (TNF-alpha), and prostaglandin-2 (PGE-2). The phenotypic alterations of DCs and DCs-primed CD4 T cells were evaluated using flow cytometry. Precursor frequency (PF) was calculated to monitor the allostimulatory effects of DCs on CD4 and CD8 T cells. ResultsAML cell supernatant-treated DCs showed significantly lower expression of co-stimulatory molecules CD80 and CD86, and reduced response to cytokines IL-1beta, IL-6, TNF-alpha,and PGE-2. The allostimulatory effects of AML cell supernatant-treated DCs on CD4 and CD8 T cells were significantly lower than those of normal mature DCs [PF (1.8±0.5)% vs. (5.2±1.6)% for CD4 T cells, (2.1±0.6)% vs. (6.5±2.0)%for CD8 T cells, P＜0.01]. These AML supematant-induced DCs could also induce allogeneic CD4 T cells to differentiate into CD4 CD25high T cells, which had immunophenotyping characteristics of regulatory T cells, i.e. they expressed Foxp3 but not active maker CD69. Conclusion This study demonstrates that soluble factors secreted by AML cells can inhibit development and functions of DCs. In addition, AML supematant-induced DCs can induce the generation of CD4 CD25high T cells from CD4 T cells, which may be a mechanism of increased prevalence of CD4 CD25high regulatory T cells and immune dysfunction in AML patients.     

miR-221对慢性粒细胞白血病K562细胞增殖和凋亡的影响及其作用机制

目的：探讨下调miR-221 对慢性粒细胞白血病（CML）K562 细胞增殖和凋亡的影响及其相关的调控机制。方法：将K562 细胞分为对照组、miRNA阴性对照（miR-NC）组、miR-221 inhibitor 组、miR-221 inhibitor+阴性对照siRNA（NC siRNA）组和miR-221 inhibitor+SOCS3 siRNA 组。其中,对照组细胞不进行另外处理;miR-NC 组和miR-221 inhibitor 组分别采用miR-NC 和miR-221 inhibitor 转染至细胞;miR-221 inhibitor+NC siRNA 组和miR-221 inhibitor+SOCS3 siRNA 组采用已稳定转染miR-221 inhibitor的细胞再分别转染NC siRNA 和SOCS3 siRNA。用qPCR鉴定miR-221 inhibitor 的转染效率,CCK-8 法检测各组细胞的增殖活性,Annexin V-FITC/PI 双染色流式术检测各组细胞的凋亡水平,WB实验检测各组细胞的SOCS3、p-JAK1、p-JAK2、p-STAT3和survivin 的蛋白表达水平。结果：与对照组比较,miR-221 inhibitor 组细胞内miR-221 的表达显著下调（P<0.01）,细胞增殖活性在转染后48、72 h 时明显降低（P<0.05 或P<0.01）,凋亡细胞数量明显增加（P<0.01）,细胞内SOCS3 的表达水平明显增加（P<0.01）,而p-JAK1、p-JAK2、p-STAT3 和survivin 的表达水平均明显降低（均P<0.01）。与miR-221 inhibitor 组比较,miR-221 inhibitor+SOCS3 siRNA组细胞增殖活性在转染后24、48 和72 h 时明显增加（P<0.05 或P<0.01）,凋亡细胞数量明显减少（P<0.01）,细胞内p-JAK1、p-JAK2、p-STAT3 和survivin 的表达水平均明显增加（均P<0.01）。结论：下调miR-221 可能通过上调SOCS3 表达抑制JAK-STAT3 信号通路,从而抑制K562 细胞增殖并促进其凋亡。     

Histone demethylase RBP2 decreases miR-21 in blast crisis of chronic myeloid leukemia

Chronic myeloid leukemia in the blastic phase (CML-BP) responds poorly to clinical treatments and is usually fatal. In this study, we found that the histone H3 lysine 4 (H3K4) demethylase RBP2 (also called JARID1A and KDM5A) is underexpressed in CML-BP. The RBP2 histone demethylase stimulates leukemia cell differentiation and inhibits cell proliferation. We identified miR-21 was directly downregulated by RBP2 and found that miR-21 downregulated PDCD4 expression in leukemia cells. By binding to miR-21 promoter and by demethylating of trimethylated H3K4 at the miR-21 locus, RBP2 downregulated miR-21 expression. This in turn activated PDCD4. In conclusion, RBP2 epigenetically downregulated miR-21 in blast transformation of CML.