动态测定急性白血病化疗过程中flt3配体水平变化及意义

目的 :研究急性白血病 (AL )化疗前后骨髓及外周血 FL含量变化及其来源。方法 :夹心酶联免疫吸附法 (Elisa法 )分析了 AL化疗前后骨髓及外周血 flt3配体 (FL )水平变化 ,并应用免疫荧光单标记和双标记流式细胞仪检测技术跟踪确定 FL 的主要产生细胞。结果 :T、B淋巴细胞及单核细胞膜表面仅表达少量膜型 FL;细胞经用皂素穿孔及双标记分析发现 FL 主要贮存在 CD3+细胞内 ;AL 患者化疗前骨髓及外周血 FL 含量略高于正常对照 ,与正常对照无区别 (P >0 .0 5 )。化疗后 FL 水平升高 ,与患者外周血象呈负相关 (r =- 0 .73,P <0 .0 5 ) ,流式细胞分析则提示细胞内 FL 含量减少 ,而细胞膜 FL 表达显著增加。结论 :AL 患者过程中 FL 水平升高与骨髓抑制程度相关 ,FL主要来自 CD3+ 细胞。     

1,25-(OH)_2维生素D3对再生障碍性贫血患者T细胞亚群的影响

目的:研究再生障碍性贫血(AA)患者外周血中的T细胞亚群变化与血清中1,25-(OH)_2维生素D3水平的关系。方法:利用流式细胞仪检测15例AA患者和25例健康对照者外周血T细胞亚群的变化,并用ELISA法检测AA患者和健康对照者血清中1,25-(OH)_2维生素D3的水平。结果:AA患者血清中1,25-(OH)_2维生素D3水平明显低于健康对照者[(35.5±11.8)pmol/L∶(51.8±12.6)pmol/L,P0.05]。与健康对照者比较,AA患者外周血中CD4在淋巴细胞中比率无明显变化[(31.8±4.7)%∶(33.6±4.2)%,P=0.217],但CD8在淋巴细胞中比率显著升高[(31.5±5.2)%∶(24.8±3.8)%,P0.01],故CD4/CD8比值降低(P0.01);其中在CD4~+细胞中,TH1/TH2比值较健康对照者明显升高[(4.09±1.05)∶(3.12±0.89),P=0.003]。结论:AA患者外周血清中1,25-(OH)_2维生素D3水平异常降低,可能影响AA患者CD4~+T细胞的分化。     

CD28/CTLA-4:B7在特发性血小板减少性紫癜患者外周血淋巴细胞中的表达及意义

目的:探讨特发性血小板减少性紫癜(ITP)患者外周血淋巴细胞CD28、CTLA-4(CD152)、B7-1(CD80)及B7-2(CD86)的表达及意义。方法:采用免疫荧光标记和流式细胞术检测41例ITP患者和40例健康对照者外周血CD3+CD28+细胞、CD3+CD152+细胞、CD80+CD19+细胞和CD86+CD19+细胞分别占淋巴细胞的比例及血小板表面相关抗体水平,进行2组对比、分析。结果:与正常对照组相比,急性ITP患者外周血CD3+CD28+细胞和CD3+CD152+细胞差异无统计学意义(P0.05),CD80+CD19+细胞增多(P0.05),CD86+CD19+细胞显著增多(P0.01),慢性ITP患者CD86+CD19+细胞增多(P0.05);急性ITP患者外周血CD86+CD19+细胞较慢性ITP患者增多(P0.05);与正常对照组相比,急性ITP患者PAIg's、PAIgG和PAIgM水平显著增高,慢性ITP患者PAIgG水平增高;CD80、CD86表达与PAIgG水平之间存在显著的相关性(均P0.01)。结论:ITP患者外周血B淋巴细胞上CD86和CD80表达均异常,可能与其发病相关。     

外周血CD4~+CD25~+FOXP3~+调节性T细胞与炎症性肠病疾病活动度的关系

背景:炎症性肠病(IBD)的发病机制尚未完全阐明。调节性T细胞是一组具有免疫抑制作用的T细胞亚群,研究显示其改变与IBD的发病密切相关。目的:观察IBD患者外周血CD4~+CD25~+FOXP3~+调节性T细胞与疾病活动度的关系。方法:纳入克罗恩病(CD)和溃疡性结肠炎(UC)患者各31例,15例健康体检者作为正常对照。分别采用简化CD活动指数(CDAI)和临床活动度指数(CAI)评估CD和UC患者的疾病活动度,以流式细胞术检测外周血CD4~+CD25~+FOXP3~+调节性T细胞比例,同时检测ESR和血清CRP水平。结果:CD和UC患者外周血CD4~+CD25~+FOXP3~+调节性T细胞比例显著低于正常对照组(P0.05),并分别与简化CDAI评分和CAI评分呈负相关(P0.05),与ESR和血清CRP水平之间则无明显相关性。活动期CD和UC患者的ESR和血清CRP水平明显高于缓解期,但差异无统计学意义。ESR和CRP与疾病活动度评分之间亦无明显相关性。结论:CD4~+CD25~+FOXP3~+调节性T细胞在IBD的发生、发展中起重要作用,外周血调节性T细胞数量减少可能是IBD复发的重要因素。     

胃癌患者CD4~+/8~+T细胞免疫球蛋白黏蛋白分子3表达水平及意义

目的观察胃癌患者外周血、癌旁组织和癌组织中CD4~+T细胞和CD8~+T细胞表面T细胞免疫球蛋白黏蛋白分子3(Tim3)的表达情况及其对CD4~+T细胞和CD8~+T细胞功能的影响。方法抽取胃癌患者外周血,通过密度梯度离心法分选获取外周血单个核细胞(PBMC)。获取的肿瘤组织及癌旁组织标本经研磨法获取肿瘤浸润T细胞。通过流式细胞染色的方法检测胃癌患者外周血、癌旁组织和癌组织中CD4~+T细胞和CD8~+T细胞表面Tim3的表达情况。激活Tim3信号后,通过流式细胞术胞内染色的方法检测CD4~+T细胞和CD8~+T细胞表达肿瘤坏死因子(TNF)-α和穿孔素的能力。结果与胃癌患者外周血CD4~+/8~+T细胞表面Tim3表达水平相比,胃癌患者癌旁组织和癌组织浸润CD4~+/8~+T细胞表面Tim3表达水平显著升高(P0.01);且与胃癌患者癌旁组织浸润CD4~+/8~+T细胞表面Tim3表达水平相比,胃癌患者癌组织浸润CD4~+/8~+T细胞表面Tim3表达水平显著升高(P0.01)。激活Tim3信号后,CD4~+T细胞分泌TNF-α的能力显著降低(P0.01),同时CD8~+T细胞分泌穿孔素的能力亦显著降低(P0.01)。结论胃癌组织CD4~+T细胞和CD8~+T细胞表面Tim3表达水平的升高可能与胃癌的进展密切相关。     

糖皮质激素对系统性红斑狼疮CD4+Foxp3+T细胞水平的影响

目的 探讨糖皮质激素治疗对系统性红斑狼疮(SLE)患者外周血CD4+ Foxp3+ T细胞水平的影响以及CD4+Foxp3+T细胞与SLE疾病活动的相关性.方法 采用流式细胞术检测26例SLE患者和5名正常人外周血CD4+Foxp3+T细胞百分率,Spearman相关分析法分析CD4+Foxp3+T细胞与SLE疾病活动指标及糖皮质激素的相关性.结果 活动期SLE患者外周血CD4+Foxp3+T细胞百分率(2.4±1.6)%低于正常人(3.3±0.8)%,但差异无统计学意义;非活动期SLE患者外周血CD4+Foxp3+T细胞百分率(3.3±0.7)%与正常人比较差异无统计学意义.活动期SLE患者经糖皮质激素治疗后外周血CD4+Foxp3+T细胞(6.1±3.5)%较治疗前(3.9±2.4)%升高(P＜0.05).SLE患者外周血CD4+Foxp3+T细胞水平与年龄、病程、红细胞沉降率、尿蛋白定量(24 h)、血清补体C3浓度、血清抗双链DNA(anti-dsDNA)抗体水平及SLE疾病活动指数(SLEDAI)无相关性,但与糖皮质激素每日用量呈显著正相关(r=0.51,P＜0.05).结论 SLE患者外周血CD4+Foxp3+T细胞水平不能作为狼疮活动指标.糖皮质激素治疗可上调SLE患者外周血CD4+Foxp3+T细胞水平,CD4+Foxp3+T细胞水平上调可能是糖皮质激素治疗SLE有效的机制之一.     

阵发性睡眠性血红蛋白尿症与再生障碍性贫血患者干细胞因子和FLT3配体及其受体表达的研究

目的:检测干细胞因子(SCF)与FLT3配体(FL)及其受体在阵发性睡眠性血红蛋白尿(PNH)与再生障碍性贫血(AA)的表达情况,探讨2者在PNH与AA发病中作用。方法:用ELISA法检测PNH与AA患者骨髓SCF和可溶性FL的含量,用流式细胞术对AA与PNH骨髓单个核细胞c kit与FLT3的表达进行分析。结果:PNH、慢性AA患者SCF含量分别为(456.8±115.2)、(372.6±111.5),与正常对照组(389.2±123.3)比较差异无统计学意义(P>0.05);PNH、慢性AA患者c kit表达分别为(48.8±15.6)、(39.6±11.5),低于正常对照组(75.2±23.3)。PNH患者FL水平为226.3±50.6,明显高于正常对照组(89.4±20.8),但低于慢性AA(658.2±125.5)(P<0.01);PNH、慢性AA患者FLT3表达分别为(13.2±5.8)、(8.5±2.7),与正常对照相比差异无统计学意义(P>0.05)。结论:干细胞因子、FLT3配体及其受体参与PNH与AA的发病,2者共同的发病机制可能为干细胞因子受体缺陷、免疫紊乱。     

TRAIL和TRAIL-R2在重型再生障碍性贫血患者外周血CD8~+T细胞的表达变化

目的:研究肿瘤坏死因子相关凋亡诱导配体(TRAIL)和TRAIL受体2(TRAIL-R2)在重型再生障碍性贫血(SAA)患者外周血CD8~+T细胞的表达变化。方法:将初诊为SAA的47例患者作为初诊组,同期免疫抑制治疗的41例SAA患者作为缓解组,34例健康者作为对照组。采用流式细胞术检测3组外周血CD8~+T细胞中TRAIL和TRAIL-R2表达,采用RT-PCR和Western Blot检测3组外周血CD8~+T细胞中TRAIL、TRAILR2mRNA和蛋白表达水平,分析TRAIL和TRAIL-R2表达水平与SAA患者临床指标的关系。结果:初诊组CD8~+T细胞中TRAIL、TRAIL-R2阳性表达率均显著低于对照组和缓解组(P0.05);3组CD8~+T细胞中TRAIL、TRAIL-R2mRNA相对表达量比较差异均无统计学意义(P0.05);初诊组和缓解组CD8~+T细胞中TRAIL和TRAIL-R2蛋白表达量显著低于对照组(P0.05);SAA患者CD8~+T细胞中TRAIL表达水平与中性粒细胞计数、网织红细胞百分比分别呈显著正相关关系(r=0.615、0.703,P=0.000、0.000)。结论:SAA患者外周血CD8~+T细胞中TRAIL和TRAIL-R2表达降低,可能与自身免疫性T细胞过度激活有关,在骨髓造血功能损伤及全血细胞减少过程中发挥重要调控作用。     

滤泡辅助性T细胞在肝棘球蚴病患者外周血中的表达

目的检测肝棘球蚴病患者和健康对照者外周血中滤泡辅助性T细胞(Tfh)和白细胞介素-21(IL-21)表达水平,探讨它们与肝棘球蚴病进展的关系。方法收集青海省人民医院肝棘球蚴病患者和健康体检者各50例,采用流式细胞术检测和比较肝棘球蚴病患者和健康对照者外周血中Tfh细胞表达水平,采用酶联免疫吸附试验检测和比较肝棘球蚴病患者和健康对照者血清中IL-21水平,分析肝棘球蚴病患者外周血Tfh细胞表达水平和血清IL-21含量之间的相关性。结果肝棘球蚴病患者外周血中CD4+CXCR5+T细胞(18.49%±5.67%vs. 16.18%±4.04%,P0.05)、CD4+CXCR5+PD-1+T细胞(4.94%±1.91%vs. 2.29%±0.79%,P0.05)和CD4+CRCR5+ICOS+PD-1+T细胞比例(30.93%±24.10%vs.21.07%±14.25%,P0.05)均显著高于健康对照者,但肝棘球蚴病患者和健康对照者外周血CD4+CRCR5+ICOS+T细胞比例差异无统计学意义(0.29%±0.32%vs. 0.25%±0.31%,P 0.05)。肝棘球蚴病患者血清IL-21含量显著高于健康对照者([293.35±2 03.65)pg/mL vs.(192.72±70.09)pg/mL,P0.05],但肝棘球蚴病患者外周血Tfh细胞表达水平和血清IL-21含量无相关性(P 0.05)。结论肝棘球蚴病患者外周血Tfh细胞及血清IL-21表达水平升高,Tfh细胞和IL-21可能参与了肝棘球蚴病进展。     

再生障碍性贫血雄激素受体与T细胞亚群的变化

目的:检测慢性再生障碍性贫血(CAA)患者骨髓单个核细胞(MNC)雄激素受体(AR)以及骨髓中T细胞亚群水平,探讨AR在CAA免疫病理机制中的作用。方法:①免疫细胞化学SP法检测CAA患者骨髓MNC内AR的表达水平;②流式细胞术检测CAA骨髓中T细胞亚群(CD3 CD4 细胞、CD3 CD8 细胞)的含量。结果:①CAA患者骨髓MNC中AR阳性水平[(35．18±8．78)个／200个MNC]显著低于对照组[(48．46±9．82)个／200个MNC];不同性别间AR的含量差异无统计学意义。②CAA患者骨髓中的CD3 CD8 细胞含量为(28．54±7．57)％,显著高于对照组。③CAA患者骨髓中的AR阳性水平与骨髓中的CD3 CD8 细胞呈负相关(r=-0．576,P<0．01);而与CD3 CD4 细胞含量未见明显的直线相关关系。结论:CAA患者骨髓AR的表达减少,从而使雄激素刺激造血的作用减弱。患者AR的表达与CD3 CD8 细胞含量呈明显负相关,表明AR的异常可能在一定程度上参与了CAA发病机制中的细胞免疫。     

Chronic overexpression of membrane-bound flt3 ligand by T lymphocytes in severe aplastic anaemia

Aplastic anaemia (AA) is an immune-mediated bone marrow failure associated with high serum levels of flt3 ligand (FL). We examined expression of the membrane-bound isoform of FL in peripheral blood and bone marrow cells from AA patients at diagnosis (n = 16) and after immunosuppressive (IS) treatment (n = 36). Flow cytometry demonstrated strongly increased FL levels on the cell surface of T lymphocytes in AA relative to normal controls (P < 0.0001). T-cell-specific expression of membrane-bound FL was confirmed by confocal microscopy. FL mRNA and total cellular FL protein levels were increased about threefold. Overexpression of FL in AA was observed for up to 20 years after IS treatment. FL levels correlated inversely with CD34+ cell numbers and the colony-forming ability of AA bone marrow (R = -0.68 and -0.85 respectively). Histological examination of spleen specimens and bone marrow biopsies gave no evidence of degeneration or fibrosis due to prolonged exposure to high FL. Levels of membrane-bound FL were not increased in autoimmune diseases (n = 23), including rheumatoid arthritis and lupus erythematosus, nor in graft-versus-host disease (n = 8). Chronic overexpression of FL on the surface of T lymphocytes in AA, but not in other T-cell-mediated disorders, suggests that membrane-bound FL plays a role in cell-cell interactions in bone marrow failure and may be important for long-term haemopoietic recovery.     

Plasma levels and production of soluble stem cell factor by marrow stromal cells in patients with aplastic anaemia

Defective marrow stroma or microenvironment have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA). Stem cell factor (SCF), the ligand for the c-kit receptor, is produced mainly by marrow stromal cells and seems to reflect the haemopoietic function of bone marrow stroma. We measured the plasma levels of soluble SCF in 87 patients with AA and investigated the production of soluble SCF by the marrow stromal cells of 46 patients with acquired AA. The mean plasma SCF concentrations in the AA patients and normal controls were not significantly different (1098 ± 398 pg/ml versus 1160 ± 316 pg/ml, respectively), and there was no significant correlation between the peripheral blood counts and the SCF concentrations. However, the mean SCF concentration in patients who received prednisolone ± anabolic steroids at the time of sampling was significantly lower than that in the patients who did not receive both agents. We did not find any correlation between the changes in SCF concentrations and the response to immunosuppressive therapy, although it did increase significantly after bone marrow transplantation. The ability of marrow stromal cells to release soluble SCF did not differ significantly between the patients with AA and normal controls. We conclude that soluble SCF production does not appear to be altered in patients with AA and that defective production of soluble SCF is unlikely to be the cause of AA in most patients.     

骨髓增生异常综合征和再生障碍性贫血患者T淋巴亚群及CD34阳性细胞的研究

目的：对骨髓增生异常综合征（MDS）和再生障碍性贫血（AA）患者的外周血T淋巴细胞亚群、NK细胞及骨髓CD34阳性细胞占单个核细胞（MNC）的比率进行测定,探讨两者细胞免疫异常及骨髓CD34阳性细胞与发病机制的关系。方法：用流式细胞术（FCM）对15例MDS患者,11例AA患者,12例正常对照组T淋巴细胞亚群、NK细胞及CD34阳性细胞占MNC的比率进行测定。结果：MDS患者外周血CD3＋T、CD4＋ T、CD8＋T淋巴细胞均较正常人组明显降低,CD4＋/CD8＋倒置。NK细胞CD16＋56也较正常人组低。AA患者CD4＋ T淋巴细胞稍低于正常人组,但CD8＋T淋巴细胞明显升高,表现为CD4＋/CD8＋也倒置。NK细胞正常。MDS患者CD34＋占骨髓MNC的比率明显高于正常组,AA患者骨髓MNC的比率低于正常组。结论：MDS和AA患者均存在免疫功能状态紊乱,二者的发病机制不同。CD34＋率的测定有助于二者的鉴别诊断,同时是判断MDS预后的简便、可靠的方法。     

再生障碍性贫血和骨髓增生异常综合征患者骨髓CD34+细胞及其粒细胞集落刺激因子受体的研究

目的:检测再生障碍性贫血(AA)和骨髓增生异常综合征(MDS)患者CD34 细胞占骨髓单个核细胞(BMMNC)的比率及其表面粒细胞集落刺激因子受体(G-CSFR)的表达率。方法:用流式细胞术(FCM)检测13例AA、22例MDS及12例非血液病患者CD34 细胞占BMMNC的比率及其表面G-CSFR的表达率。结果:AA组与对照组、AA组与MDS组、MDS-难治性贫血(RA)组与难治性贫血伴原始细胞增多(RAEB)组BMMNC中CD34 细胞的比率比较差异有统计学意义(P<0.05),但G-CSFR的表达率差异无统计学意义(P>0.05)。多数重型AA(SAA)患者(3/4)及少数慢性AA(CAA)患者(1/9)BMMNC中的CD34 细胞少于0.1%。多数G-CSFR表达率低(<14%)的患者(7/9)外周血中性粒细胞减少;而表达率正常(14.0%~28.9%)的患者(1/6)很少见;表达率高(>28.9%)的患者(3/7)也可存在中性粒细胞减少。结论:造血干细胞减少是AA的主要发病机制之一,其表面G-CSFR的表达率不是影响AA的主要因素;MDS患者CD34 细胞比率升高是一个预后不良的指标,G-CSFR的检测可部分解释MDS患者外周血中性粒细胞减少的原因。     

Levels of soluble stem cell factor in serum of patients with aplastic anemia

Aplastic anemia (AA) is a rare bone marrow (BM) disorder characterized by an unexplained failure of hematopoietic precursors to proliferate. In vitro growth of AA BM cells can be improved by the addition of the hematopoietic growth factor SCF (stem cell factor), which suggests that deficiency of SCF may be one of the underlying causes of the disease. In this study, we measured the concentration of SCF in sera of patients with severe AA. One hundred twenty-eight serum samples from 32 patients, at diagnosis and following therapy, were analyzed. Before treatment, SCF levels varied between 0.33 and 6.1 ng/mL; no correlation between hematopoietic function and SCF serum levels was apparent. Therapy with antilymphocyte globulin (ALG) or bone marrow transplantation (BMT) did not result in a recognizable pattern of changes in SCF levels. However, serum concentration of SCF in many patients with AA was at the low range of control serum levels determined in healthy blood donors. Of 128 AA serum samples tested before and after therapy, 107 were below the mean normal value of 3.3 ng/mL, including 26 samples below the minimum normal value of 1.3 ng/mL, as estimated in 267 controls. We also found that SCF levels in peripheral blood serum correlate well with factor concentrations in the BM plasma. Clinical observations suggest that higher SCF serum levels are often associated with a better clinical status of the patients in terms of survival and transfusion requirements. The data indicate that a deficient production of soluble SCF may contribute to AA in some patients; thus, suggesting a potential therapeutic benefit of SCF in this disorder.     

Expression of the multidrug resistance-associated protein (MRP) mRNA and protein in normal peripheral blood and bone marrow haemopoietic cells

We studied the expression of multidrug resistance-associated protein (MRP) in normal haemopoietic cells from peripheral blood and bone marrow. The MRP mRNA levels were estimated by RT/PCR and in situ hybridization (ISH) assay, and the protein levels by flow cytometry. 21 samples of peripheral blood and 21 samples of bone marrow (11 normal bone marrow donors, 10 patients in complete remission after chemotherapy for large cell lymphoma or acute myeloid leukaemia) were analysed. In peripheral blood the mean MRP mRNA level in CD3⁺ cells was statistically higher than in the other cells (3-fold by the methods used). The levels of MRP in CD3⁺ varied from one individual to another (4.5–34.8 units by RT/PCR and 5–23 grains/cell by ISH); however, this was proportional to the variation in all the cell lineages of same individual ( r  = 0.84). In bone marrow the mean MRP levels of the various cell lineages (including CD34⁺) were similar to the basal level in HL60 cells. Individual expression levels were again variable; however, there was no difference between untreated normal bone marrow and post chemotherapy normal bone marrow. MRP protein expression was determined by flow cytometry with the monoclonal antibody MRPm6. The CD4⁺ lymphocytes exhibited a higher MRP protein expression than the other cell lineages, including CD8⁺ cells. There was a good correlation between the three methods used (RT/PCR and ISH, P  = 0.0001, r  = 0.87; RT/PCR and flow cytometry, P  = 0.0001, r  = 0.85; ISH and flow cytometry, P  = 0.002, r  = 0.67).     

Impaired in-vitro growth of megakaryocytic colonies derived from CD34 cells of HIV-1-infected patients with active viral replication

OBJECTIVE: To address the mechanisms of the thrombocytopoietic dysfunction that may follow HIV infection and to compare peripheral blood and bone marrow as sources of CD34 progenitor cells in HIV-infected patients. METHODS: The study used CD34 progenitor cells from 20 previously untreated HIV-infected individuals, 20 HIV-infected individuals treated with antiretroviral therapy and a control group of 20 HIV-uninfected healthy individuals to examine in-vitro megakaryocytopoiesis. There were no hematological abnormalities at baseline in the study groups. CD34 progenitor cells derived from peripheral blood and bone marrow were purified and cultured in medium containing thrombopoietin, interleukin-3, and interleukin-6. HIV-1 plasma viral load was determined by b-DNA technique. Expression of receptors for thrombopoietin, interleukin-3, and interleukin-6 was assessed on CD34 cells by flow cytometry, and numbers of receptors per single cell were calculated by Quanticalc software. RESULTS: Growth of megakaryocytopoietic colony-forming units (CFU-MK) were impaired in untreated HIV-infected individuals despite normal platelet counts. Viral load levels inversely correlate with CFU-MK growth and platelet counts. Antiretroviral drug-treated individuals showed normal megakaryocyte development. Similar results were obtained whether the CD34 progenitor cells derived from peripheral blood or bone marrow. CONCLUSIONS: These findings suggest that megakaryocyte differentiation is impaired before the onset of overt thrombocytopenia in HIV-infected patients and provide evidence for a direct link between viral replication and perturbed megakaryocytopoiesis, which appears to be prevented and/or restored by antiretroviral therapy. The results indicate that peripheral blood represents a suitable source of CD34 hematopoietic progenitors for studies of megakaryocytopoiesis in HIV disease.     

Fas-mediated apoptosis with normal expression of bcl-2 and p53 in lymphocytes from aplastic anaemia

In order to investigate the involvement of apoptosis in the pathogenesis of aplastic anaemia (AA) we measured the expression of the Fas receptor (membrane protein that triggers apoptosis), Fas ligand (FasL), bcl-2 (cytoplasmatic protein that blocks apoptosis) and p53 (nuclear protein that induces apoptosis) in CD3 and CD19 lymphocytes from the peripheral blood or bone marrow of controls, patients with AA, aplastic anaemia in complete remission (AA-CR) and multiply transfused patients without aplastic anaemia. The Fas receptor was overexpressed in both T and B lymphocytes from the peripheral blood and bone marrow from patients with AA. These abnormalities were not detected in AA-CR or multiply transfused patients. CD3/FasL cells were not increased and no FasL expression was detected in B lymphocytes. Bcl-2 was highly expressed in lymphocytes from controls, AA, AA-CR and multiply transfused patients (> 99% of positive cells) whereas p53 was not detected in any group. To further characterize the functional activity of the Fas receptor we performed a Fas-induced apoptosis assay in peripheral blood lymphocytes using an anti-Fas monoclonal antibody. The crosslinking of the Fas receptor transduced an increased apoptotic signal in lymphocytes from AA patients, but not in lymphocytes from controls, AA-CR patients or multiply transfused patients. Taken together, these data suggest that a Fas-based mediated apoptosis without the apparent participation of bcl-2 or p53 is a possible mechanism of lymphocyte depletion in patients with AA. In addition, these findings suggest that Fas expression is a continuous event occurring from progenitor bone marrow cells to mature cells.     

The FLT3 ligand is a direct and potent stimulator of the growth of primitive and committed human CD34+ bone marrow progenitor cells in vitro

The present studies investigated the effects of the recently cloned flt3 ligand (FL) on the in vitro growth and differentiation of primitive and committed subsets of human CD34+ bone marrow (BM) progenitor cells. FL alone was a weak growth stimulator of CD34+ BM cells, but synergistically and directly enhanced colony formation in combination with interleukin (IL) 3, granulocyte colony-stimulating factor (G-CSF), CSF-1, granulocyte macrophage (GM) CSF stem cell factor (SCF), and IL-6. FL and SCF were equally effective in stimulating colony formation in combination with IL-3. However, the tri-factor combination of FL + IL-3 + SCF stimulated 2.3-fold and 2.5-fold more colonies than FL + IL-3 and SCF + IL-3, respectively. These additional recruited progenitors appeared to be predominantly located in a primitive (CD71-) subset of the CD34+ progenitors, as 4.5-fold more colonies were formed by CD34+CD71- cells in response to FL + IL-3 + SCF than to FL + IL-3 or SCF + IL-3. Similar findings were observed in serum-containing and serum-deprived cultures. Whereas FL did not enhance burst-forming unit-erythroid (BFU-E) colony formation of CD34+ BM cells in the presence of serum, a low number of BFU-E colonies were formed in response to FL plus erythropoietin (Epo) under serum-deprived conditions. In addition, FL both in serum-containing and serum-deprived cultures stimulated colony formation of more committed myeloid progenitors in CD34+CD71+ BM cells. Thus, FL potently stimulates the growth of primitive and more committed human BM progenitor cells.     

AA和MDS患者骨髓CD+34细胞及G-CSFR的表达及意义

目的检测再生障碍性贫血(AA)和骨髓增生异常综合征(MDS)患者骨髓CD+34细胞占单个核细胞(MNC)的比率及其表面粒细胞集落刺激因子受体(G-CSFR)的表达率,以探讨二者可能的发病机制.方法用流式细胞术(FCM)检测13例AA、22例MDS及12例非血液病患者骨髓CD+34细胞占MNC的比率及其表面G-CSFR的表达率.结果 AA组与对照组、AA组与MDS组、MDS-难治性贫血(RA)组与MDS-难治性贫血伴原始细胞增多(RAEB)组的骨髓MNC中CD+34细胞比率比较有显著性差异(P＜0.05),但G-CSFR的表达率比较无显著性差异(P＞0.05).大多数重型AA(SAA)患者(3/4)及很少慢性AA(CAA)患者(1/9)的骨髓MNC中CD+34细胞比率小于0.1%.大多数G-CSFR表达率低(＜14%)的MDS患者(7/9)外周血中性粒细胞减少;中性粒细胞减少在G-CSFR表达率正常(14%～28.9%)的患者(1/6)很少见;G-CSFR表达率高(＞28.9%)的患者(3/7)也存在中性粒细胞减少.结论骨髓CD+34细胞检测有助于判断AA患者病情及MDS患者的预后,亦可用于鉴别AA和MDS.