急性淋巴细胞白血病85例免疫表型分析

分析儿童急性淋巴细胞白血病 (ALL)的免疫表型特点。方法 用一组系列相关单克隆抗体和流式细胞术间接免疫荧光法对 85例儿童ALL进行免疫分型。结果 T系ALL占 2 8.2 % ,B系ALL 6 1.2 % ,T B急性混合细胞白血病 (AMLL) 8.2 % ,其中髓系抗原表达率 2 1.2 % ;各组间L1 、L2 比率、染色体异常及治疗完全缓解 (CR)率均无显著性差异 (P >0 .0 5 ) ;T ALL中CD3平均阳性率较CD2 、CD7低 (P <0 .0 1,P <0 .0 5 ) ,B ALL中CD2 2 阳性率明显低于CD1 0 、CD1 9(P均 <0 .0 0 1)。结论 ALL免疫分型与FAB分型、染色体核型变化无相关性 ;系列特异性单抗CD3、CD2 2 在诊断T ALL、B ALL中起决定作用 ;ALL髓系抗原阳性与治疗CR率无关。

自分泌IL-4对K_(562)细胞诱导的CTL杀伤活性的影响

探讨ＩＬ－４基因修饰对Ｋ５６２细胞诱导的肿瘤特异性ＣＴＬ杀伤活性的促进作用及可能机理。采用逆转录病毒体ＬＸＳＮ将ＩＬ－４ｃＤＮＡ导入Ｋ５６２细胞，经Ｇ４１８筛选后获得表达ＩＬ－４蛋白的Ｋ５６２克隆。以野生型和载体修饰的相应细胞为对照，检测了ＩＬ－４基因修饰Ｋ５６２细胞诱导的Ｔ细胞增殖反应，用５１铬（５１Ｃｒ）释放法检测ＣＴＬ杀伤活性。结果发现ＩＬ－４基因修饰的Ｋ５６２细胞诱导的Ｔ细胞增殖反应及ＣＴＬ杀伤活性均明显高于对照组。提示ＩＬ－４基因修饰可能通过上调Ｔ细胞功能促进肿瘤特异性ＣＴＬ杀伤活性。

In vitro study on arsenic sulfide (realgar)-induced apoptosis of retinoic acid susceptible or resistant acute promyelocytic leukemia cell lines

Objective: To further understand the possible mechanisms of arsenic sulfide (realgar) in the treatment of acute promyelocytic leukemia (APL). Methods: All-trans retinoic acid (ATRA)-susceptible APL cell line (NB4 cells) and ATRA-resistant APL cell line (MR2 subclone) were used as models in vitro. At various times after incubated with various concentrations of realgar, NB4 and MR2 cells were observed by cell viability , cell proliferation and cell morphology; cell cycle and the expression of Annexin V were assayed by flow cytometry. Results: Cell viability and proliferation of NB4 and MR2 cells were inhibited after the treatment, to some extent, in a dose and time dependent manner. 177 - 711g/L of realgar treated NB4 and MR2 cell presented morphologically some features of apoptotic cells such as intact cell membrane, chromatin condensation and nuclear fragmentation, apoptosis body could be found by electron microscopy as well. Sub-Gl cells and cell cycle arrest were observed by flow cytometry. The proportion of Annexin V -FITC+/PI cells , which represent apoptotic cells, was up-regulated. Conclusion: Realgar could induce apoptosis of acute promyelocytic leukemia cell despite its susceptibility to retinoic acid in the way that may be different from retinoic acid.

Effect of realgar on telomerase activity and hTERT-mRNA expression in NB4 cells

Objective: To evaluate whether realgar could down-regulate human telomerase reverse transcriptase (hTERT) gene expression and telomerase activity in acute promyelocytic leukemia cell line-NB4 cells. Methods: The expression of hTERT-mRNA was analyzed by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). Flow cytometry using PI staining was applied to analyze the cell cycle and apoptosis. Results: Treatment of NB4 cells with 155, 300, 600 μg/L realgar reduced telomerase activity significantly accompanying with decrease of hTERT-mRNA and increasing cell apoptosis. G2/M phase arrest appeared when treated with realgar in 300, 600 μg/L. Conclusion: It is suggested that telomerase activity of NB4 cells can be specifically inhibited by realgar through the down-regulation of hTERT gene expression. G2/M phase arrest and apoptosis by realgar in NB4 cells might be related to the reduction of telomerase activity and hTERT-mRNA expression.

Effect of arsenic sulfide on tissue factor expression in acute promyelocytic leukemia cell lines

Objective: To investigate the effect of arsenic sulfide (tetra-arsenic tetra-sulfide As4S4; diarsenic trisulfide As2S3) on tissue factor (TF) expression and procoagulant activity (PCA) of acute promyelocytic leukemia( APL) cell lines ( NB4 and MR2) and the basic mechanism of their role. Methods: NB4 and MR2 cells were respectively treated with As4S4 , As2S3, As4S4 and Cyclohexamide( CHX). PCA of the cells was detected using one-stage clotting assay. TF antigen was detected by ELISA. TF and PML/RARa fusion gene mRNA by semi-quantitive RT-PCR. The PCA and TF antigen of HL-60 and K562 cells were also examined. Results: The PCA and TF antigen level in NB4 and MR2 cells were significantly higher than that in HL-60 and K562 cells. Both As4S4 and As2S3 can down-regulate the TF antigen , TF mRNA transcription and membrane PCA of NB4 and MR2 cells in vitro in a time-dependent manner. The role of As4S4 was stronger than that of As2S3. Both As4S4 and As2S3 had no effect on PML/RARa fusion gene transcription. CHX treatment completely suppressed the down-regulate effect of As4S4 on the TF mRNA expression. Conclusion: As4S4 and As2S3 may down regulate tissue factor expression and PCA of NB4 and MR2 cells. By down-regulating TF expression, As4S4 and As2S3 might be used to improve the DIC-related hemorrhage in APL patients. Elevated TF antigen level of NB4 and MR2 cells may be related to the fusion gene PML/RARa. The modulation of the TF mRNA expression in NB4 and MR2 cells by As4S4 and As2S3 might be indirect and might not involve PML/RARa fusion gene.

Effects of anti-CXCR_4 monoclonal antibody 12G5 on proliferation and apoptosis of human acute myelocytic leukemia cell line HL-60

Objective：To investigate the expression of CXCR4 on HL-60 cell line and the proliferation, apoptosis of HL-60 cell line cocultured with bone marrow stromal cells, so as to assess the possibility of 12G5, an anti-CXCR4 monoclonal antibody, in eradicating the minimal residual disease. Methods：The activity of SDF-1 was inhibited by 10 μg/ml 12G5. After treatment with 12G5, the status of adhesion was observed, and the adhesion rates, apoptosis and cell cycles were detected after 24 h of treatment. Cell growth rates were measured by trypan blue exclusion. Cell growth curve was plotted, and the expression of PCNA and apoptosis related protein including PCNA, Bcl-2 and Fas were detected with immunohistochemical technique. Results：（1） There was middling degree expression of CXCR4 on HL-B0 membrane. From 0 h to 6 h, as the time of 12G5 incubation along, the expression of CXCR4 decreased gradually. （2） After treatment for 24 h, the adhesion rates in the experiment group and the control were （39.4±7.9）% and （51.4±5.9）%, respectively. （3）After treatment for 24 h, the percentage of HL-60 cells in G0/G1 phase were （55.21±4.9）%, and that in S phase and G2/M phase were （30.40±4.1）% and （14.39± 5.2）%, respectively, with the corresponding proportions being （44. 67±2.2）%, （45.30±3.7）%, and （10. 03±2.6）% in the control. （4） The percentage of apoptotic HL-60 cells was （8.95±1.7）% in the experiment group, compared to （3. 97±2. 4）% in the control. （5）The survival rates of HL-60 cells decreased markedly at 48 h to 96 h, and the proliferation slowed down at this time duration. （6）The expression of PCNA and Bcl-2 down-regulated significantly, but the Fas protein expression was up-regulated. Conclusion：12G5 could inhibit the capability of adhesion and proliferation of HL-60 cells and it can induce more cells to enter G0/G1 phase and promote apoptosis. It may be helpful by inhibiting the bioactivity of SDF-1 with 12G5 in the therapy of marrow residual disease.

Analyses of therapeutic effects using rludarabine and cytarabine on acute myeloid leukemia at different stages during treatment

Objective: To evaluate the therapeutic effect of the fludarabine and cytarabine (FA) regimen on acute myeloid leukemia (AML) at different phases during treatment. Methods: A total of 185 patients with AML were divided into 4 groups based on the outcome of previous treatments. Patients in Group 1 had no remission after the first course of induction chemotherapy (n = 55). Patients in Group 2 had no remission after no less than two courses of induction chemotherapy (n = 41). Patients in Group 3 had early relapse (n = 40). Patients in Group 4 had late relapse (n = 49). Patients in groups 2, 3 and 4 had refractory AML or AML with relapse. We assessed the efficacy and toxicity of FA combination chemotherapy in each of these 4 groups. Results: The complete remission (CR) rates of Groups 1, 2, 3 and 4 were 74.5% (41/55), 45.9% (19/41), 17.5% (7/40) and 38.8% (19/49), respectively. The CR rate was higher in Group 1 than in the other 3 groups (34.6%, 45/130) (P = 0.000). A significant correlation was found between CR rate and the number of chemotherapeutic courses (P = 0.023). The main adverse reactions included bone marrow suppression and secondary infection. Conclusion: FA regimen is a good choice for patients with AML, especially those who have failed to achieve CR after the first course of induction chemotherapy.

THE STUDY ON AUTOIMMUNE PATHOLOGY IN OSTEOARTHRITIS

Sixteen patients with osteoarthritis (13 knees and 3 hips), 3 patients with rheumatoid arthritis (RA) and 4 cadaver were studied for evidence of immune complex in the destroyed articular cartilage tissues. Frozen sections of the articular cartilage from arthroplasty were stained with fluoresceinated antibodies to human immunoglobulins IgG, IgA, IgM and complement C3. The results showed: 1. There were immune complexes linear deposited in the surface of the irregular articular cartilage tissues and on some chondro- cytes remained in most patients with osteoarthritis (14/16). The patterns of immune complexes are IgA, complement C3, IgG and IgM, their percentage is 81. 25%, 75%, 75% and 50% respectively. 2. In all of 3 patients with RA, the surfaces of articular tissues were seen with patchy diffusely positive areas for IgA, IgG, IgM (excepting negative in I case) and complement C3. 3. There were no immune complexes deposited in the surfaces of 4 cases of normal articular tissues. The presence of immune complexes in the cartilages suggested that an autoimmune reaction participated in the pathological process of osteoarthritis and that the autoimmunity may be responsible for the continuous degeneration of the osteoarthritis.

All-trans retinoic acid as a single agent induces complete remission in a patient with acute leukemia of M_(2a) subtype

Objective To present a special case with the karyotype and molecular marker of acute myeloid leukemia (AML)-M 2 who was induced to complete remission by all-trans retinoic acid (ATRA) alone.Methods A recently hospitalized young female patient with acute leukemia was initially diagnosed as M 3 subtype based on morphological French-American-British (FAB) classification. Karyotype analysis using standard G and R banding techniques and RT-PCR were applied to further define the diagnosis. After primarily cultured bone marrow cells from the iliac aspiration were tested for in vitro induced differentiation, the patient was treated with oral all-trans retinoic acid alone, 60?mg per day until complete remission was achieved. Peripheral blood and bone marrow changes were monitored over the whole treatment course.Results The characteristic chromosomal aberration for M 3, the t(15;17) reciprocal translocation, was not found while a t(8;21) translocation was verified. Furthermore, an amplified product of the AML-1/ETO fusion gene instead of the PML/RARα fusion gene was detected by RT-PCR and the diagnosis was corrected from M 3 to M 2. Primary cultured bone marrow cells can be fully induced to terminal differentiation after 4 days exposure to ATRA. A hematological complete remission was achieved after 40 days treatment with ATRA as a single therapeutic agent, suggesting an alternative pathway mediating ATRA-induced myeloid differentiation. Conclusion A leukemia patient with a subtype other than M 3, such as M 2 in this case, may also be induced to complete remission by the mechanism of ATRA-induced terminal differentiation. This implies that there may be a pathway other than PML/RARα fusion gene product which mediates ATRA-induced myeloid maturation in leukemia cells.

Detection of human herpesvirus 8 DNA in acute leukemia patients

Objective To determine the prevalence of human herpesvirus 8 (HHV-8) DNA in acute leukemia (AL) patients. Methods The presence of HHV-8 DNA sequences in peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC) from 50 AL patients was examined using polymerase chain reaction (PCR). Nine human hematopoietic cell lines and PBMC from 30 normal donors were also included. Results HHV-8 DNA sequences were detected in one case of acute myelogenous leukemia (AML). The specimens from the bone marrow aspirate, peripheral blood and serum of this patient were all positive. None of the normal donors and human hematopoietic cell lines showed evidence of HHV-8 DNA. Conclusion The results suggest that the prevalence of HHV-8 is low in AL in China.

Fighting against hematological malignancy in China: from unique system to global impact

During recent decades, substantial progress has been made in clinical strategies for treating hematological malignancies. Not only did China benefit from the global progression in the management of acute promyelocytic leukemia, risk-stratification-directed strategies for acute or chronic leukemia and haploidentical hematopoietic stem cell transplantation, the unique system developed by Chinese doctors has also become inspiration for refining global clinical practice. The multicenter trials and collaborations adhering to international standards might further strengthen the global impact and lead the way in specific fields of research worldwide.