一步法RT-PCR检测慢性粒细胞白血病BCR-ABL融合基因

目的：建立一步法RT-PCR检测CML患者BCR—ABL融合基因的实验方法。方法：Trizol试剂提取细胞RNA,用特异引物一步法RT—PCR检测BCR—ABL融合基因．并作灵敏度试验。结果：本方法的灵敏度可达到10pg水平。对25例CML患者检测BCR-ABL融合基因,阳性率为100％．并可检测出BCR—ABL融合基因各种融合方式。结论：一步法RT—PCR检测BCR-ABL融合基因有很好的灵敏度和特异性,操作简便、快速,特别适用于临床检测。     

慢性髓系白血病中prame基因转录本的定量研究

本研究旨在分析慢性髓系白血病(CML)患者中黑色素瘤优先表达抗原(prame)的基因转录本表达状况及其临床意义。用实时定量PCR(RQ-PCR)方法对30例CML患者和15例缺铁性贫血(IDA)患者骨髓单个核细胞中prame基因转录本水平进行检测。结果表明:15例缺铁性贫血患者骨髓单个核细胞(BMMNC)中prame转录本水平为0%-1.46%(中位0.19%),30例CML患者中BMMNC中prame基因转录本水平为0%-772.25%(中位8.28%),二组之间具有显著性差异(p0.001)。CML患者中prame基因转录本含量与bcr-abl融合基因转录本水平显著相关(r=0.708,p0.001);6例CML加速期(AP)和急变期(BC)患者中prame基因转录本水平明显高于24例慢性期(CP)患者(p=0.007)。动态检测2例CML患者在不同病程中prame基因转录本水平变化显示,AP及BC时prame基因转录本水平增高,经治疗后恢复至CP时prame基因转录本水平降低,与bcr-abl转录本变化趋势一致。结论:CML患者中prame基因转录本表达水平增高,且与病情发展相关,可用于病情监测。     

慢性粒细胞白血病一线治疗药物研究进展

第一代BCR-ABL酪氨酸激酶抑制剂(tyrosine kinase inhibitors,TKIs)伊马替尼(Imatinib)是慢性粒细胞白血病(chronic myeloid leukemia,CML)慢性期的一线标准治疗.伊马替尼通过直接靶向作用于Bcr-Abl激酶,极大改善了CML病程.近期,第二代TKIs药物尼洛替尼(Nilotinib)和达沙替尼(Dasatinib)又被美国FDA批准为CML的一线治疗.本文主要就CML一线治疗的进展作一综述.     

p15^INK4B基因甲基化在急性普细胞白血病和慢性粒细胞白血病中的研究

为探索ｐ１５＾ＩＮＫ４Ｂ基因在ＣｐＧ岛高甲基化对白血病发病机制的重要作用,应用敏感的甲基化特异的ＭＳＰ－ＰＣＲ的测定方法,测定了ｐ１５＾ＩＮＫ４Ｂ基因在急性粒细胞白血病（ＡＭＬ）和慢性粒细胞白血病（ＣＭＬ）中甲基化的表达变化,研究结果表明,ｐ１５＾ＩＮＫ４Ｂ基因操纵区在ＡＭＬ和ＣＭＬ中的甲基化发生率分别为８３．９％（２６／３１）和０％（０／２８）。结论提示：甲基化是ｐ１５＾ＩＮＫ４Ｂ基因在ＡＭＬ中主要的失活方式之一,并可在病程进展中出现甲基化,使病情加重,在ＣＭＬ中无基化的发生,说明ｐ１５＾ＩＮＫ４Ｂ基因的功能在ＣＭＬ中可能是完整的。     

慢性髓系白血病不同病期基因表达谱研究

本研究旨在探讨慢性髓系白血病(chronic myeloid leukemia,CML)急变的分子机制。采用cDNA微重排方法对4例CML急变期、4例CML慢性期患者进行基因差异表达分析。结果显示,共筛选出至少在3张芯片有差异表达的基因74条,其中下调52条,上调22条;差异表达基因包括:细胞骨架/运动相关基因、信号传导相关基因、转录因子相关基因、免疫相关基因、代谢相关基因、细胞周期相关基因、原癌和抑癌基因、细胞受体相关基因、蛋白质翻译合成相关基因及功能未知的基因等。结论:急变是多基因异常相互作用的结果,其中功能异常的信号转导、细胞周期调控、细胞分化及免疫的相关基因可能是导致CML急变的关键基因。     

竞争性内源性RNA在急性髓系白血病中的研究进展

急性髓系白血病 (acute myeloid leukemia, AML) 是多种因素引起的造血系统恶性疾病,具有很高的复发率及死亡率。非编码 RNA在 AML的发生发展过程中发挥着重要作用。竞争性内源性 RNA(competing endogenous RNA, ceRNA)观点指出,不同的非编码 RNA可通过作用于相同的 miRNA反应元件 (MRE)从而调控基因的表达。目前越来越多的研究表明,非编码 RNA作为 ceRNA在调控 AML的增殖、凋亡、侵袭和耐药等生物学过程中发挥关键作用。该文主要对 ceRNA在 AML生物学过程中的调控作用,以及治疗和预后中的临床意义作一综述。     

362例慢性髓系白血病的细胞遗传学分析

为探讨慢性髓系白血病（CML）的细胞遗传学特点及临床意义,对362例CML患者采用24小时短期培养法制备骨髓染色体,用R显带技术进行染色体核型分析。将患者分为慢性期和急变期两组。结果表明：附加染色体异常、变异易位、Ph（-）bcr／abl（＋）并伴有染色体异常者在两组中的比例分别为：70／268（26．1％）、19／268（7．1％）、4／268（1．5％）;50／94（53．2％）、8／94（8．5％）、4／94、（4．3％）。362例标本中检出Ph阳性标本324例（89．5％）,其中典型t（9;22）（q34;q11）易位297例（91．7％）,变异易位27例（8．3％）。在27例变异易位中单纯变异易位13例,复杂变异易位13例,隐匿Ph1例。362例标本中共发现120例特殊核型异常。对上述异常分析显示,出现频率较高的数目异常有：＋Ph：26例（21．7％）;＋8：12例（10．0％）;＋21：12例（10．0％）;＋19：9例（7．5％）。结构异常中以i（17q）最多,有16例（13．3％）。结论：与慢性期相比,急变期附加染色体、变异易位等异常率均明显增加,染色体核型分析有助于疾病进展的判断。     

血府逐瘀汤影响慢性粒细胞白血病VEGF表达的体外研究

目的:体外研究血府逐瘀汤含药血清对慢性粒细胞白血病细胞VEGF表达的影响。方法:采用酶联免疫吸附法(ELISA)检测20例初治CML患者、20例正常人骨髓细胞以及人慢性粒红白血病急性变K562细胞上清液的VEGF含量,并测定CML原代细胞和K562细胞加入不同浓度血府逐瘀汤含药血清共同培养后上清液的VEGF含量。结果:CML原代细胞上清液VEGF浓度为(389.27±65.77)pg/mL、K562细胞VEGF浓度为(461.24±137.57)pg/mL,均比正常人(125.45±28.24)pg/mL显著增高(P<0.05),加入不同浓度的含药血清,在中、高浓度范围,CML原代细胞、K562细胞VEGF表达均明显下降,分别为(152.43±26.29)pg/mL、(80.31±19.73)pg/mL和(188.86±32.77)pg/mL、(114.81±14.16)pg/mL(P<0.05)。结论:异常血管新生可能参与CML发病,血府逐瘀汤含药血清在体外能显著抑制CML原代细胞和K562白血病细胞表达VEGF,该方有参与抑制CML异常血管形成的作用。     

格列卫在1例慢性粒细胞白血病慢性期患者中的循证应用

目的探讨格列卫在1例慢性粒细胞性白血病慢性期的患者的最佳应用方案.方法计算机检索ACP Journal Club (1991～2005年4月)、Cochrane图书馆(2005年第2期)和MEDLINE(1990～2005年4月),收集格列卫治疗慢性粒细胞白血病慢性期的系统评价、临床随机对照试验、卫生经济学评价等,并对所获证据质量进行评价.结果高质量的临床证据表明:在血液学和细胞遗传学缓解率以及生存质量方面,格列卫均优于传统疗法,且副作用相对较少.结合我们的临床经验,并考虑患者及其家属的意愿,最后为患者制定治疗方案为格列卫400 mg qd,随访3个月血液指标学完全缓解,ph染色体1/30( ),达到主要细胞遗传学缓解,且无明显不良反应发生.结论格列卫是治疗慢性粒细胞性白血病慢性期的有效药物,可作为临床一线用药.但其长期疗效和不良反应以及经济学评价仍有待进一步研究.     

RHBDD1基因在慢性髓系白血病患者骨髓细胞中的表达及临床意义

本研究旨在检测RHBDD1（Rhomboid domain containing 1）基因在慢性髓系白血病（CML）患者骨髓细胞中的表达水平并初步探讨其临床意义。采用实时定量PCR的方法检测RHBDD1在正常人和CML患者骨髓单个核细胞中的相对表达水平。结果表明,CML患者RHBDD1的表达水平明显高于正常人;BCR／ABL p210表达阴性的患者RHBDD1的表达水平显著高于BCR／ABLp210阳性的患者;≥50岁的患者RHBDD1表达水平低于〈50岁的患者;RHBDD1的表达水平与患者的性别无明显相关性。结论：RHBDD1基因可能参与了CML的发生与发展,尤其在BCR／ABL p210阴性患者的发病中可能发挥重要作用。     

线粒体途径在慢性髓系白血病信号转导中的作用

本研究旨在探讨线粒体途径在慢性髓系白血病（CML）信号转导中的作用。用脂质体转染法将bcr3／abl2反义寡核苷酸（ASO）导入CML细胞系K562细胞中;用MTr法检测bcr3／abl2 ASO对K562细胞活力的影响;流式细胞术（FCM）分析测定细胞凋亡率;荧光染料罗丹明123染色分析细胞线粒体跨膜电位（△ψm）的变化;Western blot检测线粒体凋亡信号转导通路相关蛋白细胞色素C（CytC）的表达。结果表明,2μmol／Lbcr3／abl2 ASO与K562细胞作用24小时后,可明显抑制K562细胞活力,其抑制率为65．7％;可诱导细胞凋亡,凋亡率为16．9％;可下调K562细胞线粒体△ψm,有38．33％的细胞出现线粒体△ψm下降;可增强CytC的表达,激光光度扫描仪测得其表达光密度值由2．33±0．3升高到4．78±0．1。结论：线粒体途径通过介导凋亡信号而在CML信号转导中发挥重要作用。     

慢性髓系白血病DNA依赖蛋白激酶催化亚基基因的研究

本研究旨在探讨髓系粒细胞白血病（CML）的DNA依赖蛋白激酶催化亚基（DNA-PKcs）基因表达水平、调控机制及其在CML急性变中的作用。用半定量RT-PCR、Western blot方法分别检测62例CML患者及K562细胞的DNA-PKcs mRNA和DNA-PKcs蛋白表达,并与23例正常人作对照;对26例接受同种异基因外周血干细胞移植（allo-PBSCT）及4例使用伊马替尼治疗的CML患者用RT-PCR、Western blot方法分别动态检测bcr-abl mRNA和DNA-PKcs蛋白表达水平;用伊马替尼体外作用CML患者的单个核细胞（MNC）及K562细胞后,用RT-PCR、Western blot方法分别检测DNA-PKcs mRNA和DNA-PKcs蛋白表达及bcr-abl融合蛋白的酪氨酸磷酸化水平。结果表明：与正常人比较,CML患者及K562细胞的DNA-PKcs蛋白表达量明显降低（P〈0.05）;26例allo-PBSCT及4例使用伊马替尼治疗的CML患者,DNA-PKcs蛋白表达量随着bcr-abl mRNA表达量的降低而升高;伊马替尼体外作用于CML患者MNC及K562细胞后,DNA-PKcs蛋白表达量随着bcr-abl融合蛋白酪氨酸磷酸化水平的降低而升高。结论：bcr-abl融合基因通过转录后机制下调DNA-PKcs蛋白的表达;DNA-PKcs蛋白表达下降可能是CML急性变的机制之一。     

Microsphere-Based Multiplex Analysis of DNA Methylation in Acute Myeloid Leukemia

Aberrant regulation of DNA methylation is characteristic of cancer cells and clearly influences phenotypes of various malignancies. Despite clear correlations between DNA methylation and patient outcome, tests that directly measure multiple-locus DNA methylation are typically expensive and technically challenging. Previous studies have demonstrated that the prognosis of patients with acute myeloid leukemia can be predicted by the DNA methylation pattern of 18 loci. We have developed a novel strategy, termed microsphere HpaII tiny fragment enrichment by ligation-mediated PCR (MELP), to simultaneously analyze the DNA methylation pattern at these loci using methylation-specific DNA digestion, fluorescently labeled microspheres, and branched DNA hybridization. The method uses techniques that are inexpensive and easily performed in a molecular laboratory. MELP accurately reflects the methylation levels at each locus analyzed and segregates patients with acute myeloid leukemia into prognostic subgroups. Our results demonstrate the usefulness of MELP as a platform for simultaneous evaluation of DNA methylation of multiple loci.Cancer has been traditionally considered a genetic disease, involving a series of mutations that activate oncogenes and inactivate tumor suppressors.¹ Although mutagenic events are critical for carcinogenesis, recent work has unequivocally demonstrated that cancer is also characterized by dysregulation of chromatin structure that involves the DNA itself (eg, CpG methylation) and its associated histones.² Because these epigenetic modifications are at least partially responsible for influencing tumorigenic processes, it is not surprising that several studies have demonstrated that prognosis of certain tumors can be predicted from analysis of epigenetic features.^3–5An example of a tumor type that shows clear dysregulation of epigenetic modifications is acute myeloid leukemia (AML).^6,7 Many of the recurrent mutations seen in AML, including those in DNMT3A, IDH1, IDH2, MLL, and EZH2, influence DNA methylation, DNA hydroxymethylation, or histone modification.^8–11 Dysregulated DNA methylation at specific loci, such as CDKN2B and MGMT, has been found in many cases of AML.¹² Moreover, studies of global methylation have shown DNA methylation in leukemic blasts is distinct from that seen in normal CD34⁺ cells, and that DNA methylation patterns alone can segregate AML samples into categories with significant clinical and biological features.³ Indeed, a DNA methylation analysis using only 18 loci was shown to distinguish prognostic subgroups of AML, and this methylation-based classifier retained significance in a multivariate analysis that included factors used clinically for determining patient prognosis.³Despite the clear implications of epigenetics for tumor biological features and patient prognosis, studies involving multiple-locus DNA methylation of cancers have lagged behind those assessing DNA sequence variations. One reason is the lack of robust multiplex assay platforms that are amenable for high-throughput laboratory use. Most assays that probe DNA methylation are technically challenging because they use sodium bisulfite treatment of DNA, which can cause sample degradation.^13,14 In addition, examination of methylation at multiple loci requires either nucleotide microarrays or high-throughput sequencing technologies, both of which require extensive investment for materials and equipment.To circumvent the technical challenges involved in routine epigenetic analysis, we have developed a novel method to determine DNA methylation status that uses analytical techniques commonly used in molecular laboratories. As a proof of principle of the utility of this assay and to directly compare it with well-established tests for DNA methylation, we have applied our novel technique to measure DNA methylation at 18 loci previously shown to carry prognostic significance in patients with AML.³ Our method, conceptually based on the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay, does not use bisulfite treatment. Rather, it uses methylation-sensitive restriction digestion, followed by oligonucleotide ligation and PCR.¹⁵ Examination of methylation levels is performed by flow cytometric analysis of fluorescent microspheres, thereby alleviating the need for microarrays or high-throughput sequencing technologies. We demonstrate that this methylation assay, designated microsphere HELP (MELP), accurately recapitulates genome-wide HELP of AML samples, both in terms of DNA methylation status at individual loci and with a global classifier relating DNA methylation to patient outcome. Thus, MELP may prove to be an appropriate technology for evaluation of DNA methylation in diseases associated with dysregulated epigenetic status.     

血管增生与慢性髓细胞性白血病治疗效果的关系

目的：探讨血管增生在CML(慢性髓细胞性白血病)病程中的变化，为CML抗血管新生药物治疗提供实验依据。方法：应用ELISA法检测CML患者与正常人群血浆中VEGF、TXB2、GMP-140的水平，并观察CML患者血浆作用下内皮细胞株在纤维蛋白、Ⅳ型胶原上迁移能力的改变。结果：CML患者血浆中VEGF、TXB2、GMP-140水平较正常对照显著增高，其中治疗前水平又较治疗后高。CML患者血浆作用下内皮细胞株迁移能力增强，以治疗前为甚。结论：CML患者存在血管增生，病情缓解后，血管增生也得到了缓解。提示不仅血管增生检测指标可以作为CML等恶性肿瘤疗效及预后判断参数，而且抗血管新生治疗将为恶性肿瘤治疗开拓新的领域。