miR-449b和miR-34c对卵巢癌细胞SKOV3-ipl周期相关蛋白的下调及细胞周期阻滞作用

目的:基于miR-449和miR-34在p53突变的卵巢癌细胞系SKOV3和SKOV3-ipl的表达差异,研究探讨这些miRNA对肿瘤细胞生长、细胞周期的影响及靶基因的表达变化.方法:通过反转录实时定量PCR方法测定mjR-449a/b和miR-34b,c在SKOV3和SKOV3-ipl的表达,通过转染使它们在极低表达的SKOV3-ipl中获得表达.用MTS方法测定细胞生长率变化、流式细胞术检测细胞周期改变、Western blot检测细胞周期相关蛋白表达.结果:miR-4496和niR-34c使SKOV3-ipl附性下降28%～34%,细胞周期阻滞:G1期细胞数量分别增加15.62%和15.71%;s期细胞数量分别减少15.96%和16.56%.细胞周期相关蛋白CDK6和CDC25A均表达下调,miR-449b使CDK6减少39%,CDC25A减少22% ;miR-34c使CDK6减少49%,CDC25A减少32%a;而miR-449b与miR-34.共同作用后,下调作用更明显,CDK6减少69% ,CDC25A减少86% ,CyclinA减少59%.结论:miR-449b和miR-34.使高恶性卵巢癌细胞系SKOV3-ipl发生细胞周期阻滞,CDK6,CDC25A和CyclinA表达均下调.     

伴NPM1基因突变的成年人急性髓系白血病临床研究

目的 探讨我国成年伴NPM1基因突变的急性髓系白血病患者(NPMc+AML)的临床特点,初步探讨定期定性检测该突变在早期判断AML复发中的意义.方法 采用聚合酶链反应(PCR)-毛细管电泳法对95例成年初治AML患者检测NPM1突变情况,并选取其中5例完全缓解患者定期检测该突变.结果 95例成年AML患者NPM1突变发生率为29.5%(28/95);≥40岁患者突变发生率[40.0%(22/55)]明显高于＜40岁患者[15.0%(6/40)](λ 2=6.963,P=0.012);正常核型AML患者突变发生率[51.1%(24/47)]明显高于异常核型患者[8.3%(4/48)](λ2=20.860,P=0.000).AML患者发生NPM1突变以M5[72.7%(16/22)]、M2[36.3%(8/22)]常见,在具有重现性染色体异常的AML中,未发现该突变.NPMc+AML患者白细胞、血小板计数及乳酸脱氢酶水平均明显高于NPMc-AML组(t值分别为4.132、4.603、4.069,均P＜0.05).NPMc+AML患者完全缓解率、无复发生存率及总生存率均明显高于NPMc-AML患者(λ2值分别为10.448、4.146、4.384,均P＜0.05).定期检测的患者血液学复发前1.5～2.0个月草新出现NPM1基因突变.结论 NPM1基因突变在成年AML患者中,尤其是正常核型AML患者中有较高的发生率,临床表现为患者年龄偏大,白细胞计数、血小板计数、乳酸脱氢酶均较高,NPM1基因突变是成年AML患者预后良好的指标.定期定性监测该突变可早期判断AML复发.     

多发性骨髓瘤中14q32易位与13q14缺失的相关性研究

目的 研究多发性骨髓瘤(MM)常见的分子遗传学异常14q32易位与13q14缺失及其与临床指标的关系.方法 采用间期荧光原位杂交(I-FISH)技术应用RB1、D13S319和LSI IGHC/IGHV探针检测49例MM患者骨髓标本中RB1基因、13q14.3缺失及14q32易位,结合临床资料作统计分析.结果 49例MM患者有26例(53.1%)检测到14q32易位,25例(51.02%)存在13q14缺失(其中18例检测到13q14.3缺失,9例存在RB1缺失).Spearman相关分析显示,14q32易位多见于浆细胞比例高的患者(r=0.316,P=0.27),与患者年龄、国际分期系统(ISS)分期、免疫球蛋白分型、β2微球蛋白及肾损害无相关性(P＞0.05).结论 13q14缺失及14q32相关的易位在MM中发生率均较高,两者有密切相关性;14q32易位的MM患者浆细胞百分比明显升高,14q32易位的检测可作为预测MM预后的指标.     

双表型急性白血病流式细胞术免疫分型检测结果

目的 应用流式细胞术(FCM)检测双表型急性白血病(BAL)的免疫表型.方法 采用四色FCM检测23例BAL患者的免疫表型.结果 23例BAL患者中有10例(43.4%)表达cCD3,16例(69.6%)表达cCD79a,20例(87.0%)表达cMPO,14例(60.9%)表达TdT,19例(82.6%)表达CD34,20例(87.0%)表达CD117,同时表达髓系和B系抗原者13例(56.5%),均共同表达cCD79a和cMPO;同时表达髓系和T系抗原者7例(30.4%),均共同表达cCD3和cMPO;同时表达T系和B系抗原者3例(13.04%),均共同表达cCD3和cCD79a.结论 cCD3、cCD79a、cMPO为系列特异性抗原标志,对诊断及鉴别BAL具有重要意义,FCM是目前诊断BAL特异口靠的方法,在临床白血病治疗和预后方面有重要的指导意义.     

亲子鉴定中IdentifilerTM系统15个STR基因座突变分析

目的:观察和分析IdentifilerTM系统15个短串联重复序列(short tandem repeat,STR)基因座在亲子鉴定中的突变现象.方法:应用IdentifilerTM荧光标记复合扩增试剂盒检测710例亲子鉴定案,对其中发现突变基因座的案件加用STRtyper荧光标记复合扩增试剂盒进行等位基因检测,或6个mini Y-STR基因座检测.结果:在认定亲子关系的615例中,IdentifilerTM荧光标记复合扩增试剂盒中的15个基因座确定7例突变,其中vWA基因座2例,D13S17、FGA、D18S51、D21S11、D19S433基因座各1例;一步突变的6例,二步突变的1例.其突变均来自父亲,且年龄均在35岁以上.结论:在亲子鉴定中用IdentifileTM荧光标记复合扩增试剂盒检测到1～2个基因座不符合遗传规律时,有必要增加突变率低、稳定性好的STR基因座进行复核并排除近亲关系.     

急性白血病线粒体D-loop区微卫星D310和D16184不稳定性研究

目的 探讨线粒体D-loop区微卫星(MSI)D310和D16184在急性白血病(AL)中的不稳定性.方法 采用反转录聚合酶链-单链构象多态性分析(PCR-SSCP)法筛选100例初诊未治及治疗的AL患者D-loop区HV-1和HV-2.对迁移异常者进行测序,结果与剑桥标准序列(rCRS)和mtDB数据库进行比对,分析其突变情况.比较各组组中D310和D16184的突变率.结果 100例AL患者中,D310总突变49例(49.0%),未治疗组40例中突变13例(32.5%),治疗组60例中突变36例(60.0%),治疗组高于未治疗组(P＜0.05);D16184总突变32例(32.0%),未治疗组突变8例(20.0%),治疗组为24例(40.0%),治疗组高于未治疗组(P＜0.05).结论 D310和D16184在AL中存在较高的突变率和多种突变类型,具有较强的不稳定性;化疗可能增强微卫星的不稳定性.     

P53突变蛋白表达对弥漫大B细胞淋巴瘤预后的预测作用

目的 探讨p53突变蛋白表达对弥漫大B细胞淋巴瘤(DLBCL)预后的预测作用,指导个体化治疗.方法 随机选择初治DLBCL患者62例,应用免疫组织化学方法检测p53突变蛋白和CD10、bcl_6、MUM1的表达,分析p53突变蛋白表达与患者临床特征、分子分型以及预后的关系.结果 48.4%(30/62)的患者表达p53突变蛋白.p53突变蛋白表达与初始治疗反应有关(x2=20.365,P=0.040),阳性组的完全缓解率为33.3%(10/30),阴性组为59.4%(19/21);与分子分型有关(x2=31.023,P=0.021),阳性组非生发中心型比例显著高于阴性组,分别为83.3%和56.2%;与其他临床特征无关.多因素生存分析显示p53突变蛋白表达是独立的预后预测因子,阳性组的无进展生存期和中位生存期均短于阴性组(x2=36.784,P=0.005和x2=35.276,P=0.006).结论 p53突变蛋白表达是DLBCL独立的不良预后因子,能够用来指导个体化治疗.     

VD方案联合自体造血干细胞移植治疗难治性不分泌型多发性骨髓瘤一例并文献复习

目的 观察硼替佐米+地塞米松(VD)方案联合自体造血干细胞移植(ASCT)治疗一例难治性不分泌型多发性骨髓瘤(MM)患者的疗效.方法 通过对1例难治性不分泌型MM患者给予VD方案化疗4个周期,在疾病得到有效控制、临床体征改善后,进行ASCT(预处理方案为美法仑200 mg/m2),并对国内外相关文献复习.结果 患者经过化疗达到接近完全缓解(nCR),之后联合ASCT后达到完全缓解(CR).结论 VD方案联合ASCT可明显改善难治性不分泌型MM患者的临床预后.     

t(8;21)急性髓系白血病患者MICM分型及预后的回顾性分析

目的 分析t(8;21)急性髓系白血病(AML)患者的细胞形态学、免疫表型、遗传学、分子生物学(MICM)分型及临床治疗疗效.方法 运用瑞特染色法、FAB细胞形态分类标准、流式细胞术(FCM)直接免疫荧光标记技术、遗传学染色体吉姆萨显带技术及RT-PCR技术对70例确认有t(8;21)与AML1-ETO融合基因双阳性的AML患者及70例正常染色体核型的AML患者进行分析和比较.结果 70例t(8;21)AML患者中M11例,M2 64例,M4 3例,无法分型的急性白血病(AL)2例;免疫表型分析发现CD13、CD33、CD34、CD117高表达,40%表达CD19,11%表达CD15,10%表达CD11b,7%表达CD7;遗传学显示50%的t(8;21)AML患者有附加染色体异常,主要为性染色体丢失、9q-及超二倍体;RT-PCR检测AML1-ETO融合基因100%阳性.CD+19t(8;21)AML患者完全缓解(CR)率72%,CD+19伴CD+7t(8;21)AML患者CR率为0,正常核型CR率31%.结论 t(8;21)AML患者主要在M2中集中出现,附加染色体异常较多见.CD19表达较高,而CD7表达极低,CD34、CD117高表达,这些抗原的表达可能与核型密切相关.CD+19是预后良好的指标,但同时出现CD+7,则预后不良.     

多发性骨髓瘤患者基质细胞衍生因子-1α、CD44v6表达的研究

目的 探讨基质细胞衍生因子-1α(SDF-1α)、CD44v6(一种变异的CD44受体)在多发性骨髓瘤(MM)中的表达水平及其与病情进展的关系.方法 用酶联免疫吸附试验(ELISA)检测24例MM患者[14例初发和复发MM患者(初发和复发MM组),10例病情稳定MM患者(病情稳定MM组)]和15位健康骨髓移植供者或非肿瘤良性贫血患者(对照组)的骨髓单个核细胞(MNC)和骨髓基质细胞(BMSC)培养上清的SDF-1 α、CD44v6水平.结果 初发和复发MM组MNC培养上清的SDF-1α、CD44v6表达水平[(7232.41±2644.97)pg/ml和(34.34±13.20)ng/ml]显著高于病情稳定MM组[(2315.49±748.29)pg/ml和(15.69±5.28)ng/m1](t=6.25、t=7.82;均P＜0.05)和对照组[(1149.52±636.06)pg/ml和(4.85±3.62)ng/ml](t=4.60、t=7.61;均P＜0.05).病情稳定MM组SDF-1α、CD44v6水平显著高于对照绀(t=2.99、t=4.87;均P＜0.05).9例初发和复发MM组的BMSC与人类骨髓瘤细胞系细胞U266加入rhIL-6进行混合培养后,SDF-1 α水平[(6180.25±5925.38)pg/ml]显著高于5例对照组BMSC[(1021.13±358.65)pg/ml]和9例初发和复发MM组[(1004.07±727.36)pg/ml](t=2.66、t=2.42;均P＜0.05).而其他BMSC各组问的SDF-1α水平差异无统计学意义(P＞0.05).SDF-1 α与CD44v6两者表达水平呈正相关(r=0.51,P=0.03).结论 SDF-1 α、CD44v6水平升高与MM的病情进展或发病有关,也可能与MM的肿瘤浸润过程有关;而这些体内过程可能需骨髓瘤细胞和BMSC与IL-6、SDF-1α和CD44v6等因素协同完成.     

Characterization of p16(INK4A) expression in multiple myeloma and plasma cell leukemia

Loss of p16(INK4A) (p16) expression is frequently associated with the development of epithelial and lymphoid malignancies. However, the frequency and significance of p16 abnormalities in multiple myeloma (MM) and the more aggressive phase of plasma cell leukemia (PCL) have not been well defined. Accordingly, the goal of this study was to define the expression and function of p16 in fresh samples of MM and PCL. We found that p16 protein was highly expressed in primary MM cells, although it was undetectable in fresh samples of PCL. Additionally, p16 protein was also absent in four of four MM-derived cell lines. To determine the mechanism for p16 underexpression in PCL and MM-derived cell lines, we performed PCR analysis to evaluate both gene deletion and the presence of methylation. Interestingly, the p16 gene was present and methylated in all patient PCL cells and MM cell lines, whereas it was unmethylated in patient MM cells and normal B cells. Furthermore, treatment with the demethylating agent 5-deoxyazacytidine or p16 retrofection restored p16 protein expression and induced G1 growth arrest in patient PCL cells and MM cell lines. These results suggest that inactivation of the p16 gene by methylation may be associated with decreased growth control and the development of PCL in a subset of patients with MM.     

Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers

We describe a gene encoding p73, a protein that shares considerable homology with the tumor suppressor p53. p73 maps to 1p36, a region frequently deleted in neuroblastoma and other tumors and thought to contain multiple tumor suppressor genes. Our analysis of neuroblastoma cell lines with 1p and p73 loss of heterozygosity failed to detect coding sequence mutations in remaining p73 alleles. However, the demonstration that p73 is monoallelically expressed supports the notion that it is a candidate gene in neuroblastoma. p73 also has the potential to activate p53 target genes and to interact with p53. We propose that the disregulation of p73 contributes to tumorigenesis and that p53-related proteins operate in a network of developmental and cell cycle controls.     

The arts and humanities: a creative approach to developing nurse leaders

There is still much controversy about the precursor cell type in multiple myeloma (MM). Some authors claim that it is a pre-B cell, others state that it is a memory B cell or plasmablast. We have recently shown that the VDJ region of the MM immunoglobulin heavy chain gene is somatically hypermutated and antigen selected, without intraclonal variation or evolution in time. By using a patient-specific PCR approach we have now obtained evidence that the premyeloma cell can be situated in the pre-switched B-cell compartment and that heavy chain switching can occur without further somatic mutation. Based on the MM immunoglobulin sequences derived from the bone marrow, patient-specific CDR2 and CDR3 oligonucleotides were designed. B lymphocytes were separated from plasma cells based on the expression of CD19 and HLA class II or surface bound IgM using immunomagnetic beads. The expressed Ig sequences were amplified by RT-PCR using patient specific CDR2 primers and isotype specific primers (C mu, C gamma, and C alpha). Myeloma-specific Ig sequences were detected by a myeloma-specific CDR3 probe and sequenced. In one out of five cases we found in the peripheral blood clonally related IgM and IgA sequences with the same somatic mutations as the MM-IgG sequence. In another case of an IgG MM we found in the bone marrow clonally related IgA sequences with the same somatic mutations. These findings, together with the fact that myeloma-Ig genes contain somatic mutations without intraclonal variation, suggest that the clonogenic cell in multiple myeloma can originate from a pre-switched but somatically mutated B cell.     

Evidence that multiple myeloma Ig heavy chain VDJ genes contain somatic mutations but show no intraclonal variation

To investigate whether somatic hypermutation occurs in multiple myeloma (MM) Ig VH region genes, we have cloned and sequenced the expressed VH genes from five cases of MM. The sequences were obtained after polymerase chain reaction (PCR) on total RNA isolated from the bone marrow, using 5' VH family-specific leader and 3' C gamma- or C alpha-specific primers. MM-specific CDR3 oligonucleotides were produced to isolate VH genes expressed by the malignant plasma cells. In all five cases, the productive Ig gene used the VH3 family. Extensive sequence analysis of multiple independent M13 clones showed no intraclonal variation with no evidence for ongoing somatic hypermutation in MM VH region genes. We were able to identify possible germline counterparts of the expressed VH genes in two cases. Comparison of these genes shows that the MM VH region genes have somatic mutations characteristic for an antigen-driven process. In the other three cases, no close homology could be found with published VH3 sequences. These findings implicate that, in MM, clonal proliferation takes place in a cell type that has already passed through the phase of somatic hypermutation.     

Cloning and characterization of a novel Rab-family gene, Rab36, within the region at 22q11.2 that is homozygously deleted in malignant rhabdoid tumors

Malignant rhabdoid tumors (MRTs) are rare, pediatric soft-tissue tumors. Homozygous deletions at chromosome 22q11.2 are a recurrent cytogenetic characteristic of MRTs, an indication that this locus may harbor one or more genes conferring tumor-suppressor activity. We constructed a deletion map of the relevant part of 22q11.2 from a panel of seven MRT cell lines, and isolated a novel gene from the center of the region. As it showed a high degree of sequence homology to genes of the Rab family, we designated it Rab36. The protein encoded by Rab36 was localized at the Golgi body. Sequencing of Rab36 cDNAs from three cell lines that retained at least one allele of this gene revealed no nonsense or frameshift mutations. Experiments to induce over-expression of Rab36 by transfection to an MRT cell line similarly failed to justify designation of this gene as a tumor suppressor that would contribute to tumorigenesis by a loss-of-function mechanism.     

Isolation and identification of DNA fragments frequently deleted from tumor cell lines of human esophageal cancer

OBJECTIVE: To clone tumor suppressor genes associated with human esophageal cancer (EC). METHODS: DNA fragments deleted from cell lines of esophageal cancer EC8712 and EC8733 were isolated by using modified genomic substractive hybridization. Twenty EC tissues paired with adjacent normal mucosa were analysed by Southern blot and PCR with these fragments as probes. RESULTS: Seven DNA fragments deleted from EC cell lines obtained were named as 12H1, 12H2, 33H3, 33H4, 12B1, 33B2 and 33B3. Sequencing revealed their size of 512bp, 428bp, 509bp, 355bp, 680bp (partially sequencing), 519bp and 425bp, respectively. 12H1 and 33H3 showed high homology (98%) to a new repeated sequence in human genome. 33H4 displayed 87% nucleotide identity with human alphoid-like repetitive sequence located on chromosome 17. 12B1 was homologous (90%) to LINE 1 transoposon containing two open reading frames. 12H2, 33B2 and 33B3 were novel sequences. These DNA fragments were deleted in 20%-60% of EC tissues and in 10%-20% of adjacent mucosa. CONCLUSIONS: Loss of 33H4 may be accompanied by deletion of an adjacent gene. 12B1 as transposon may suppress the tumorigenesis by activating suppressor genes or/and by inactivating oncogenes. 12H1/33H3, 12H2, 33B2 and 33B3 may be candidates of tumor suppressor genes associated with the development and the progression of esophageal cancer.     

Compound heterozygosity for two novel missense mutations in the prothrombin gene in a patient with a severe bleeding tendency

The abnormal prothrombin gene of an Italian patient with a severe bleeding tendency and hypoprothrombinemia was selected for study and compared with the prothrombin genes of healthy controls. All the coding and their flanking regions and the 5'- and 3'-UT regions of the prothrombin gene were screened by analyzing the nucleotide sequence of the corresponding PCR products. The patient was found to be heterozygous for two novel point mutations: one at nucleotide 4251 in exon 6, which changes the codon for cysteine-138 (TGC) in the kringle 1 domain to that for tyrosine (TAC), and one at nucleotide 8812 in exon 10, which results in the replacement of tryptophan-357 (TGG) by cysteine (TGT) in the catalytic domain. Her mother was heterozygous for the Cys-138 Tyr mutation and her father heterozygous for the Trp-357 Cys mutation. Several other sequence variations were identified in the prothrombin genes from control individuals. Only the variations at nucleotide 4203 and 10253 could be established as polymorphisms.     

PTEN mutations in gliomas and glioneuronal tumors

Cytogenetic and loss of heterozygosity studies have suggested the presence of at least one tumor suppressor gene on chromosome 10 involved in the formation of high grade gliomas. Recently, the PTEN gene, also termed MMAC1 or TEP1, on chromosomal band 10q23 has been identified. Initial studies revealed mutations of PTEN in limited series of glioma cell lines and glioblastomas. In order to systematically evaluate the involvement of PTEN in gliomas, we have analysed the entire PTEN coding sequence by SSCP and direct sequencing in a series of 331 gliomas and glioneuronal tumors. PTEN mutations were detected in 20/142 glioblastomas, 1/7 giant cell glioblastomas, 1/2 gliosarcomas, 1/30 pilocytic astrocytomas and 2/22 oligodendrogliomas. No PTEN mutations were detected in 52 astrocytomas, 37 oligoastrocytomas, three subependymal giant cell astrocytomas, four pleomorphic xanthoastrocytomas, 15 ependymomas, 16 gangliogliomas and one dysembryoplastic neuroepithelial tumor. In addition, all tumors were examined for the presence of homozygous deletions of the PTEN gene; these were detected in 7 glioblastomas that did not have PTEN mutations. Therefore, PTEN mutations occur in approximately 20% of glioblastomas but are rare in lower grade gliomas. These findings confirm that PTEN is one of the chromosome 10 tumor suppressor genes involved in the development of glioblastomas.