AML1-ETO 真核表达载体的构建及对 U937细胞增殖和分化的影响

目的 构建pcDNA3.1-AML1-ETO真核表达载体,并观察AML1-ETO融合蛋白在U937细胞内的表达并检测其对细胞增殖与分化的影响.方法 以原核表达载体pCMV5-AML1-ETO为模板扩增AML1-ETO目的 片段,将该基因重组于pcDNA3.1/V5-His-TOPO真核表达载体上,用脂质体转染技术将其导入 U937细胞,经G418筛选获得稳定转染的克隆,PCR检测AML1-ETO基因的整合,RT-PCR及 Western blot检测AML1-ETO mRNA和蛋白的表达,用锥虫蓝拒染人工计数法观察细胞增殖活性,流式细胞术检测髓系分化抗原表达的变化,瑞特染色法观察细胞形态改变.结果 pcDNA3.1-AML1-ETO经酶切鉴定及DNA测序证实序列完全正确,筛选出AML1-ETO基因高表达的亚克隆,证实AML1-ETO基因稳定转染到U937细胞中并得到表达;转染细胞增殖受抑(P<0.05),髓系分化抗原CD11b表达阳性率[(4.17±0.31)%]低于转染空载体和未转染对照组[(11.40±0.17)%、(11.03±0.15)%](P<0.001),形态呈低分化表现.转染细胞经TPA处理后,CD11b表达无明显变化(P>0.05).结论 成功构建pcDNA3.1-AML1-ETO表达载体,并在真核细胞中得到了正确表达,AML1-ETO 基因能抑制U937细胞的增殖与分化,为进一步研究该基因致白血病的机制奠定了基础.

Abstract：

Objective To construct a pcDNA3. 1 -AML1-ETO expression vector and investigate its effects on proliferation and differentiation of U937 leukemic cells. Methods AML1 -ETO gene was amplified by PCR from pCMV5-AML1-ETO and inserted into eukaryotic expression plasmid pcDNA3. 1/V5-His-TOPO. The recombinant plasmid was transfected into U937 cells by Lipofectamin 2000. Individual clones selected with G418 were isolated. The integration and the expression levels of AML1-ETO in transfectants were determined by PCR, RT-PCR and Western blot analysis respectively. Trypan blue refusal staining method was used to detect the proliferation of U937 cells. Light microscope was applied to observe the morphologic changes of the cell. The expression of myeloid cell differentiation antigen was detected using flow cytometry. Results The recombinant pcDNA3. 1-AML1-ETO was confirmed by enzyme digestion and sequencing. The highly expressing AML1-ETO subclone was established. AML1-ETO was expressed in U937 cells transfected with pcDNA3.1 -AML1-ETO. The growth of the monoclonal cells was inhibited evidently (P < 0. 05). The expression of CD11b in transfected group [(4. 17±0. 31)%] was lower than that in empty plasmid transfected group and non-transfected group [(11.40 ± 0. 17)% and (11.03 ± 0. 15) %] respectively (P < 0.001). Transfected cells displayed morphology of less differentiation. The expression level of CD11b was unchanged in transfected cells treated with TPA (P > 0. 05). Conclusion The eukaryotic expression vector for AML1ETO gene was successfully constructed and expressed in U937. AML1-ETO inhibits the proliferation and differentiation of transfected cells. It provides the basis for further study of mechanisms of AML1 -ETO in leukemogenesis.     

新型高通量肺癌相关基因甲基化检测方法的建立

Objective To explore a new high-throughput method with internal standards for analyzing the methylation profiles of lung cancer related genes. Methods The promoter sequences of 7 lung cancer related genes were cloned into plasmids and the target segments were amplified by their special primers respectively. The products were treated with M. Sss Ⅰ methylase and bisulfite. The multiplex ligation PCR method was established by designing probes containing CpGpCpG(for methylatedsequence) at the 3' ends and choosing the optimal ligation enzyme, annealing and ligation temperatures. The standard calibrators and clinic samples were tested by fluid chip platform. The results were validated by methylationspecific PCR. Results We successfully set up the standard calibrators for methylation and unmethylaiton of 7 lung cancer related genes and established a multiplex ligation PCR combined with fluid chip method, which was used to detect methylation status of 7 genes simultaneously. The fluorescence value of p16INK4A, APC,DAPK, RARIβ, RASSF1 A, MGMT and GSTP1 methylation standard calibrators were 863,909,703,701,901,1 060 and 885, much higher than that of unmethylation standard calibrators. The results were consistent with the results of methylation-specific PCP. ConclusionThe new high-throughput method can be used to evaluate the methylation status of 7 lung cancer related genes simultaneously and might be useful for clinical practice.     

新型高通量肺癌相关基因甲基化检测方法的建立

Objective To explore a new high-throughput method with internal standards for analyzing the methylation profiles of lung cancer related genes. Methods The promoter sequences of 7 lung cancer related genes were cloned into plasmids and the target segments were amplified by their special primers respectively. The products were treated with M. Sss Ⅰ methylase and bisulfite. The multiplex ligation PCR method was established by designing probes containing CpGpCpG(for methylatedsequence) at the 3' ends and choosing the optimal ligation enzyme, annealing and ligation temperatures. The standard calibrators and clinic samples were tested by fluid chip platform. The results were validated by methylationspecific PCR. Results We successfully set up the standard calibrators for methylation and unmethylaiton of 7 lung cancer related genes and established a multiplex ligation PCR combined with fluid chip method, which was used to detect methylation status of 7 genes simultaneously. The fluorescence value of p16INK4A, APC,DAPK, RARIβ, RASSF1 A, MGMT and GSTP1 methylation standard calibrators were 863,909,703,701,901,1 060 and 885, much higher than that of unmethylation standard calibrators. The results were consistent with the results of methylation-specific PCP. ConclusionThe new high-throughput method can be used to evaluate the methylation status of 7 lung cancer related genes simultaneously and might be useful for clinical practice.     

典型与不典型免疫表型慢性淋巴细胞白血病的预后相关因素分析

Objective To analyze the proguostic factors for chronic lymphocytic leukemia (CLL) with typical and atypical immunophenotype. The parameters analyzed included sex, age, Binet stages, abso-lute lymphocyte count (ALC), immunoglobulin heavy-chain variable region (IgVH) gene mutation status, ZAP-70 protein, CD38 expression and cytogenetic aberrations. Methods According to the clinical guideline and scoring system for CLL in Britain, among 77 patients, 61 patients with score 5 called typical immunophe-notype CLL, 16 with score 4 or 3 were atypical immunophenotype CI,L. Multiparameter flow cytometry was employed for immunophenotypic analysis in 77 CLL patients for CD5, CD19, CD23, FMC7, slg, CD20, CD79h expression and ZAP-70 protein and CD38. IgVH mutation status was detected by multiplex RT-PCR and sequencing of the purified PCR amplification products. Fluorescence in situ hybridization (FISH) and a panel of probes were used to detect cytogenetic aberrations. Results There was no significant difference be-tween the two groups in sex, age, ZAP-70 and IgVH mutation status (P =0.398, P =0. 189, P =0.268 and P =0. 131, respectively). The incidence of ALC≥50 × 109/L, Binet B + C, CD38 ≥30% in atypical CLL patients(43.8%, 87.5% and 43.8%, respectively) were higher than that in typical group (16.4%, 36.1% and 16.4%, respectively) (P = 0. 026, P < 0. 01 and P = 0. 026, respectively). The proportion of typical patients (26. 8%) with a 13q14 deletion as sole abnormality was higher than that of atypical patients (7.6%), and that with deletion of 11q22 or 17p13 was lower than that of atypical patients (12.2% vs 46.2%) (P = 0. 022). Conclusion There were obvious differences between the typical immunophenotype CLL and atypical CLL in ALC, Binet stages, CD38 expression level and cytogenetic aberrations.     

典型与不典型免疫表型慢性淋巴细胞白血病的预后相关因素分析

Objective To analyze the proguostic factors for chronic lymphocytic leukemia (CLL) with typical and atypical immunophenotype. The parameters analyzed included sex, age, Binet stages, abso-lute lymphocyte count (ALC), immunoglobulin heavy-chain variable region (IgVH) gene mutation status, ZAP-70 protein, CD38 expression and cytogenetic aberrations. Methods According to the clinical guideline and scoring system for CLL in Britain, among 77 patients, 61 patients with score 5 called typical immunophe-notype CLL, 16 with score 4 or 3 were atypical immunophenotype CI,L. Multiparameter flow cytometry was employed for immunophenotypic analysis in 77 CLL patients for CD5, CD19, CD23, FMC7, slg, CD20, CD79h expression and ZAP-70 protein and CD38. IgVH mutation status was detected by multiplex RT-PCR and sequencing of the purified PCR amplification products. Fluorescence in situ hybridization (FISH) and a panel of probes were used to detect cytogenetic aberrations. Results There was no significant difference be-tween the two groups in sex, age, ZAP-70 and IgVH mutation status (P =0.398, P =0. 189, P =0.268 and P =0. 131, respectively). The incidence of ALC≥50 × 109/L, Binet B + C, CD38 ≥30% in atypical CLL patients(43.8%, 87.5% and 43.8%, respectively) were higher than that in typical group (16.4%, 36.1% and 16.4%, respectively) (P = 0. 026, P < 0. 01 and P = 0. 026, respectively). The proportion of typical patients (26. 8%) with a 13q14 deletion as sole abnormality was higher than that of atypical patients (7.6%), and that with deletion of 11q22 or 17p13 was lower than that of atypical patients (12.2% vs 46.2%) (P = 0. 022). Conclusion There were obvious differences between the typical immunophenotype CLL and atypical CLL in ALC, Binet stages, CD38 expression level and cytogenetic aberrations.     

典型与不典型免疫表型慢性淋巴细胞白血病的预后相关因素分析

Objective To analyze the proguostic factors for chronic lymphocytic leukemia (CLL) with typical and atypical immunophenotype. The parameters analyzed included sex, age, Binet stages, abso-lute lymphocyte count (ALC), immunoglobulin heavy-chain variable region (IgVH) gene mutation status, ZAP-70 protein, CD38 expression and cytogenetic aberrations. Methods According to the clinical guideline and scoring system for CLL in Britain, among 77 patients, 61 patients with score 5 called typical immunophe-notype CLL, 16 with score 4 or 3 were atypical immunophenotype CI,L. Multiparameter flow cytometry was employed for immunophenotypic analysis in 77 CLL patients for CD5, CD19, CD23, FMC7, slg, CD20, CD79h expression and ZAP-70 protein and CD38. IgVH mutation status was detected by multiplex RT-PCR and sequencing of the purified PCR amplification products. Fluorescence in situ hybridization (FISH) and a panel of probes were used to detect cytogenetic aberrations. Results There was no significant difference be-tween the two groups in sex, age, ZAP-70 and IgVH mutation status (P =0.398, P =0. 189, P =0.268 and P =0. 131, respectively). The incidence of ALC≥50 × 109/L, Binet B + C, CD38 ≥30% in atypical CLL patients(43.8%, 87.5% and 43.8%, respectively) were higher than that in typical group (16.4%, 36.1% and 16.4%, respectively) (P = 0. 026, P < 0. 01 and P = 0. 026, respectively). The proportion of typical patients (26. 8%) with a 13q14 deletion as sole abnormality was higher than that of atypical patients (7.6%), and that with deletion of 11q22 or 17p13 was lower than that of atypical patients (12.2% vs 46.2%) (P = 0. 022). Conclusion There were obvious differences between the typical immunophenotype CLL and atypical CLL in ALC, Binet stages, CD38 expression level and cytogenetic aberrations.     

典型与不典型免疫表型慢性淋巴细胞白血病的预后相关因素分析

Objective To analyze the proguostic factors for chronic lymphocytic leukemia (CLL) with typical and atypical immunophenotype. The parameters analyzed included sex, age, Binet stages, abso-lute lymphocyte count (ALC), immunoglobulin heavy-chain variable region (IgVH) gene mutation status, ZAP-70 protein, CD38 expression and cytogenetic aberrations. Methods According to the clinical guideline and scoring system for CLL in Britain, among 77 patients, 61 patients with score 5 called typical immunophe-notype CLL, 16 with score 4 or 3 were atypical immunophenotype CI,L. Multiparameter flow cytometry was employed for immunophenotypic analysis in 77 CLL patients for CD5, CD19, CD23, FMC7, slg, CD20, CD79h expression and ZAP-70 protein and CD38. IgVH mutation status was detected by multiplex RT-PCR and sequencing of the purified PCR amplification products. Fluorescence in situ hybridization (FISH) and a panel of probes were used to detect cytogenetic aberrations. Results There was no significant difference be-tween the two groups in sex, age, ZAP-70 and IgVH mutation status (P =0.398, P =0. 189, P =0.268 and P =0. 131, respectively). The incidence of ALC≥50 × 109/L, Binet B + C, CD38 ≥30% in atypical CLL patients(43.8%, 87.5% and 43.8%, respectively) were higher than that in typical group (16.4%, 36.1% and 16.4%, respectively) (P = 0. 026, P < 0. 01 and P = 0. 026, respectively). The proportion of typical patients (26. 8%) with a 13q14 deletion as sole abnormality was higher than that of atypical patients (7.6%), and that with deletion of 11q22 or 17p13 was lower than that of atypical patients (12.2% vs 46.2%) (P = 0. 022). Conclusion There were obvious differences between the typical immunophenotype CLL and atypical CLL in ALC, Binet stages, CD38 expression level and cytogenetic aberrations.     

典型与不典型免疫表型慢性淋巴细胞白血病的预后相关因素分析

Objective To analyze the proguostic factors for chronic lymphocytic leukemia (CLL) with typical and atypical immunophenotype. The parameters analyzed included sex, age, Binet stages, abso-lute lymphocyte count (ALC), immunoglobulin heavy-chain variable region (IgVH) gene mutation status, ZAP-70 protein, CD38 expression and cytogenetic aberrations. Methods According to the clinical guideline and scoring system for CLL in Britain, among 77 patients, 61 patients with score 5 called typical immunophe-notype CLL, 16 with score 4 or 3 were atypical immunophenotype CI,L. Multiparameter flow cytometry was employed for immunophenotypic analysis in 77 CLL patients for CD5, CD19, CD23, FMC7, slg, CD20, CD79h expression and ZAP-70 protein and CD38. IgVH mutation status was detected by multiplex RT-PCR and sequencing of the purified PCR amplification products. Fluorescence in situ hybridization (FISH) and a panel of probes were used to detect cytogenetic aberrations. Results There was no significant difference be-tween the two groups in sex, age, ZAP-70 and IgVH mutation status (P =0.398, P =0. 189, P =0.268 and P =0. 131, respectively). The incidence of ALC≥50 × 109/L, Binet B + C, CD38 ≥30% in atypical CLL patients(43.8%, 87.5% and 43.8%, respectively) were higher than that in typical group (16.4%, 36.1% and 16.4%, respectively) (P = 0. 026, P < 0. 01 and P = 0. 026, respectively). The proportion of typical patients (26. 8%) with a 13q14 deletion as sole abnormality was higher than that of atypical patients (7.6%), and that with deletion of 11q22 or 17p13 was lower than that of atypical patients (12.2% vs 46.2%) (P = 0. 022). Conclusion There were obvious differences between the typical immunophenotype CLL and atypical CLL in ALC, Binet stages, CD38 expression level and cytogenetic aberrations.     

典型与不典型免疫表型慢性淋巴细胞白血病的预后相关因素分析

Objective To analyze the proguostic factors for chronic lymphocytic leukemia (CLL) with typical and atypical immunophenotype. The parameters analyzed included sex, age, Binet stages, abso-lute lymphocyte count (ALC), immunoglobulin heavy-chain variable region (IgVH) gene mutation status, ZAP-70 protein, CD38 expression and cytogenetic aberrations. Methods According to the clinical guideline and scoring system for CLL in Britain, among 77 patients, 61 patients with score 5 called typical immunophe-notype CLL, 16 with score 4 or 3 were atypical immunophenotype CI,L. Multiparameter flow cytometry was employed for immunophenotypic analysis in 77 CLL patients for CD5, CD19, CD23, FMC7, slg, CD20, CD79h expression and ZAP-70 protein and CD38. IgVH mutation status was detected by multiplex RT-PCR and sequencing of the purified PCR amplification products. Fluorescence in situ hybridization (FISH) and a panel of probes were used to detect cytogenetic aberrations. Results There was no significant difference be-tween the two groups in sex, age, ZAP-70 and IgVH mutation status (P =0.398, P =0. 189, P =0.268 and P =0. 131, respectively). The incidence of ALC≥50 × 109/L, Binet B + C, CD38 ≥30% in atypical CLL patients(43.8%, 87.5% and 43.8%, respectively) were higher than that in typical group (16.4%, 36.1% and 16.4%, respectively) (P = 0. 026, P < 0. 01 and P = 0. 026, respectively). The proportion of typical patients (26. 8%) with a 13q14 deletion as sole abnormality was higher than that of atypical patients (7.6%), and that with deletion of 11q22 or 17p13 was lower than that of atypical patients (12.2% vs 46.2%) (P = 0. 022). Conclusion There were obvious differences between the typical immunophenotype CLL and atypical CLL in ALC, Binet stages, CD38 expression level and cytogenetic aberrations.     

典型与不典型免疫表型慢性淋巴细胞白血病的预后相关因素分析

Objective To analyze the proguostic factors for chronic lymphocytic leukemia (CLL) with typical and atypical immunophenotype. The parameters analyzed included sex, age, Binet stages, abso-lute lymphocyte count (ALC), immunoglobulin heavy-chain variable region (IgVH) gene mutation status, ZAP-70 protein, CD38 expression and cytogenetic aberrations. Methods According to the clinical guideline and scoring system for CLL in Britain, among 77 patients, 61 patients with score 5 called typical immunophe-notype CLL, 16 with score 4 or 3 were atypical immunophenotype CI,L. Multiparameter flow cytometry was employed for immunophenotypic analysis in 77 CLL patients for CD5, CD19, CD23, FMC7, slg, CD20, CD79h expression and ZAP-70 protein and CD38. IgVH mutation status was detected by multiplex RT-PCR and sequencing of the purified PCR amplification products. Fluorescence in situ hybridization (FISH) and a panel of probes were used to detect cytogenetic aberrations. Results There was no significant difference be-tween the two groups in sex, age, ZAP-70 and IgVH mutation status (P =0.398, P =0. 189, P =0.268 and P =0. 131, respectively). The incidence of ALC≥50 × 109/L, Binet B + C, CD38 ≥30% in atypical CLL patients(43.8%, 87.5% and 43.8%, respectively) were higher than that in typical group (16.4%, 36.1% and 16.4%, respectively) (P = 0. 026, P < 0. 01 and P = 0. 026, respectively). The proportion of typical patients (26. 8%) with a 13q14 deletion as sole abnormality was higher than that of atypical patients (7.6%), and that with deletion of 11q22 or 17p13 was lower than that of atypical patients (12.2% vs 46.2%) (P = 0. 022). Conclusion There were obvious differences between the typical immunophenotype CLL and atypical CLL in ALC, Binet stages, CD38 expression level and cytogenetic aberrations.     

白血病细胞系中抑癌基因PTEN启动子区甲基化状态检测及其去甲基化研究

目的 探讨抑癌基因PTEN表达沉默的机制及诱导PTEN表达对白血病细胞的作用.方法 应用甲基化特异性聚合酶链反应(Methylation specific PCR,MSP)检测白血病细胞系HL-60、Nalm-6、Raji、KG-1a、U937、NB4、K562中PTEN基因启动子区域甲基化状态;用甲基化转移酶抑制剂5-氮-2'-脱氧胞苷(5-Aza-CdR)处理白血病细胞,MSP检测甲基化状态的改变、RT-PCR检测PTEN mRNA水平的改变、瑞特染色观察细胞形态学改变、膜联蛋白V/碘化丙锭(Annexin V/PI)标记染色检测细胞凋亡.结果 检测的白血病细胞系中HL-60、Nalm-6、Raji、KG-1a、U937细胞PTEN基因显示超甲基化状态,而NB4和K562细胞显示低甲基化状态;5-Aza-CdR处理HL-60和Nalm-6细胞后,PTEN基因甲基化降低、mRNA表达水平则逐渐增高,并呈剂量依赖性,细胞出现凋亡现象.结论PTEN基因启动子区异常甲基化可能导致该基因转录表达失活或沉默,甲基化抑制剂可以诱导PTEN表达,并引起白血病细胞凋亡.     

急性白血病死亡相关蛋白激酶基因表达及启动子区甲基化状态研究简

本研究通过对急性白血病病人及体外肿瘤细胞株的DNA甲基化及基因表达的实验,探讨死亡相关蛋白激酶（death-associated protein kinase,DAPK）基因在急性白血病中的表达情况及其启动子区域甲基化的状态,并分析两者之间的相关程度.应用RT-PCR检测DAPK基因在白血病细胞及正常骨髓细胞中的表达;用巢式甲基化特异性PCR（n-MSP）法检测急性白血病患者及细胞株DAPK基因启动子甲基化状况;从第二轮巢式MSP扩增出的随机产物中挑选2个随机引物,交予专业机构克隆后进行序列分析.结果表明：DAPK基因在10例正常对照者骨髓标本中均有表达,DAPKmRNA在急性白血病病人骨髓标本中表达平均值为0.61 ±0.40,低于正常对照者,差异有显著性（P＜0.05）,其中52.94％（9/17例）的急性淋巴细胞白血病病人骨髓标本DAPKmRNA表达降低或缺失,急性髓系白血病病人数据为41.18％ （42/102）;细胞株U-937显示基因表达正常,HL-60表达缺失;102例急性髓系白血病病人骨髓标本中33例存在DAPK启动子区甲基化,甲基化率为32.4％（33/102）;17例急性淋巴细胞白血病病人骨髓标本中8例存在DAPK启动子区甲基化,甲基化率为47％ （8/17）;7例正常人骨髓标本DAPK启动子区均为非甲基化;细胞株U-937 DAPK启动子区非甲基化,HL-60 DAPK启动子区甲基化.ALL组与AML组患者骨髓细胞DAPKmRNA表达与其启动子甲基化均呈显著负相关（ALL组r=-0.855,P＜0.05;AML组r=-0.343,P＜0.05）,提示两者有密切的关联.结论：急性白血病患者中DAPK基因启动区甲基化与其mRNA的异常表达或失表达有关.     

甲基化PCR作为白血病微量残留病检测方法的探索

本实验研究探讨以甲基化特异性聚合酶链反应（MS—PCR）作为白血病微量残留病（MRD）检测方法的可行性。将Id4基因呈完全甲基化的HL-60细胞和Id4基因呈完全非甲基化的Hek937细胞按不同比例混合,实验分为3组：A组为10％HL-60＋9％Hek937．B组为1％HL-60＋99％Hek937,C组为0．1％HL-60＋99．9％Hek937。用MS-PCR方法检测不同白血病细胞比例下Id4基因甲基化情况。结果表明,HL-60细胞仅有155bp的Id4基因甲基化特异性扩增,呈Id4基因完全甲基化;Hek937细胞仅有156bp的Id4基因非甲基化特异性扩增,呈Id4基因完全非甲基化。A、B、C组同时有155bp的Id4基因甲基化和156bp的Id4基因非甲基化特异性扩增,显示Id4基因甲基化表达。结论：MS-PCR方法可从0．1％比例含量的白血病细胞样本中检测到Id4基因甲基化,加之Id4基因甲基化在不同类型白血病中差异不明显,因此用MS-PCR检测Id4基因甲基化状态可以作为一种检测各种类型白血病微量残留病的方法。     

造血系统肿瘤WT1基因启动子区域DNA甲基化及其调控的研究

目的 应用聚合酶链反应（PCR）的实验方法研究白血病细胞系中WT1基因启动了区域的DNA甲基化水平，及其与WT1基因表达的关系。方法 ①采用RT-PCR技术及甲基化特异性PCR（Methylation-spectific PCR，MSP）技术检测8226、HL-60、Jurkat、KG-1及Raji等血液系统肿瘤细胞系中WT1基因mRNA表达水平及其启动子区域的DNA甲基化状态；②以5-杂氮脱氧胞嘧啶（5-aza-CdR）对U937细胞系进行去甲基化处理，并观察WT1基因表达水平的改变。结果 ①HL-60、K562、KG-1、NB4及SHI-1细胞系中WT1表达水平高，而8226、Jurkat、Raji、U266和U937细胞系WT1表达水平则极低，同时检测到在8226、Jurkat、Raji、U266和U937这5个细胞系存和WT1基因启动子区域DNA高甲基化；②经去甲基化处理后，U937细胞系的WT1基因表达水平较未处理者上升，同时伴随着WT1启动子区域DNA甲基化水平的下降和未甲基化水平的升高。结论 WT1基因启动子区域DNA高甲基化是抑制其表达的机制之一。     

白血病U937细胞系WT1基因静默的机制研究

本研究探讨白血病细胞系WT1基因的甲基化状况及其与表达的关系.用甲基化抑制剂和组蛋白脱乙酰基酶抑制剂成功诱导WTI表达.用地西他滨、曲古抑菌素处理白血病U937、HL-60、K562细胞系,用RT-PCR、改良的硫化PCR结合限制性内切酶技术检测mRNA表达.结果表明,U937细胞不表达WT1基因,而HL-60、K562和KG1细胞高表达WT1基因;HL-60细胞WT1基因没有甲基化,而K562和U937细胞发生了甲基化.地西他滨、曲古抑菌素单药处理U937细胞不能诱导WT1基因表达,而二药联合作用可以诱导U937细胞系WT1基因重新表达.结论:单纯DNA甲基化不能抑制白血病细胞WT1基因表达;DNA甲基化与组蛋白脱乙酰基共同抑制了WT1基因表达;地西他滨、曲古抑菌素联合作用可以重新诱导WT1基因表达.     

白血病细胞WT1基因启动子区DNA甲基化研究

目的：研究白血病细胞WT1启动子区DNA甲基化与其表达的关系。方法：采用RT-PCR技术、硫化PCR结合限制性内切酶技术检测白血病细胞系及正常人外周血单个核细胞WT1基因的表达及其启动子区DNA甲基化水平。结果：正常人外周血单个核细胞及U937细胞不表达WT1基因，而HL-60、K562和KG1细胞高表达WT1基因，HL-60细胞WT1基因无甲基化。结论：WT1基因启动子区DNA甲基化不能抑制其表达，尚存在其他调节WT1基因表达的因素。     

DR4去甲基化对髓性白血病K562细胞增殖凋亡作用的研究

目的:研究肿瘤坏死因子死亡受体(DR)4去甲基化对髓性白血病K562细胞增殖、凋亡的影响.方法:取对数期K562细胞,采用地西他滨(DCA)0、0.8、1.6、3.2 μmol/L干预,分别设为对照、DCA低剂量、DCA中剂量和DCA高剂量4组.取对照组细胞,采用肿瘤坏死因子相关凋亡诱导配体(TRAIL)0.5 μg/...     

四种恶性血液病细胞系的Id4基因启动子甲基化

本研究探讨4种白血病和淋巴瘤细胞系以及非肿瘤细胞系和正常人骨髓Id4基因启动子甲基化状态及其差异。采用甲基化特异性聚合酶链反应（MS-PCR）对白血病细胞系K562和HL-60、淋巴瘤细胞系Ramous和CA46以及良性细胞系Hek937和正常人骨髓细胞进行Id4基因甲基化状态检测。结果表明：Id4基因在正常人骨髓细胞和Hek937细胞系中呈完全非甲基化状态，在4个血液肿瘤细胞系K562、HL-60、Ramous和CA46中均呈甲基化状态。结论：所检测的4种血液肿瘤细胞系中，Id4基因的甲基化状态与正常人骨髓细胞和非肿瘤细胞系完全不同，Id4基因甲基化模式的改变与造血细胞恶变的发生密切相关。     

Apaf-1基因启动子甲基化与抑凋亡蛋白Apollon表达在成人急性白血病发生发展中的意义

目的 探讨Apaf-1基因启动子甲基化与抑凋亡蛋白Apollon在成人急性白血病(AL)发生发展中的作用.方法 采用甲基化特异性PCR(MS-PCR)检测53例AL患者骨髓细胞基因启动子区域甲基化情况,RT-PCR方法 检测Apaf-1 mRNA表达水平.免疫细胞化学方法 检测Apollon蛋白表达情况,正常骨髓对照为10名正常人和非恶性血液病患者.结果 18例(33.9%)AL患者Apaf-1基因启动子存在异常甲基化,甲基化检测阳性患者均未检测到Apaf-1 mRNA的表达,对照组仅1份Apaf-1 mRNA表达缺失,MS-PCR检测未发现Apaf-1基因启动子的异常甲基化,AL患者Apaf-1基因甲基化阳性率高于对照组,差异有统计学意义(P＜0.05),AL患者骨髓细胞Apollon蛋白表达水平高于对照组(P＜0.05),AL患者外周血WBC＞10×109/L的患者Apaf-1基因启动子甲基化阳性率及Apollon蛋白表达水平高于WBC≤10×109/L患者,差异有统计学意义(P＜0.05),AL患者中Apaf-1基因启动子甲基化阳性率与Apollon蛋白表达呈正相关.结论 Apaf-1基因启动子的异常甲基化与抑凋亡蛋白Apollon的高表达在白血病的发生发展中可能起协同作用,共同促进了白血病的发生、发展.     

急性髓性白血病中hMLH1基因启动子区甲基化与表达水平检测及意义

目的检测急性髓性白血病（acute myeloid leukaemia,AML）细胞hMLH1基因启动子甲基化状态及转录水平,探讨其与AML发生发展的关系。方法运用甲基特异性PCR和Real-Time PCR法检测62例AML患者外周血有核细胞中hM-LH1基因启动子甲基化状态及其转录水平,分析白血病细胞hMLH1基因启动子区甲基化与临床资料的关系。结果 62例急性髓性白血病hMLH1基因启动子甲基化频率为14.52%（9/62）,甲基化白血病细胞与非甲基化白血病细胞hMLH1 mRNA的相对含量分别是0.51±0.19和0.87±0.24,两者的hMLH1转录水平有明显差异（P〈0.05）;AML细胞中hMLH1基因启动子甲基化与患者性别及FAB分型无明显相关性,与发病年龄及难治/复发显著相关。结论在AML中hMLH1基因转录水平受基因启动子甲基化调控,且hMLH1基因甲基化差异与白血病发生及进展恶化显著相关。