目的建立慢性粒细胞白血病(chronic myelogenous leukemia,CML)患者BCR-ABL融合基因检测及微小残留病变实时定量监测的诊断平台。方法依据GenBank中编码P210蛋白的融合基因M(b2a2,b3a3)及ABL的基因序列,分别设计引物及Taqman探针,以BCR-ABL阳性的CML患者cDNA为模板,通过PCR扩增出587 bp的基因片段,连入pMD 18-T载体,制备成标准品并绘制标准曲线,运用实时荧光定量PCR(real-time quantitative PCR,RQ-PCR)技术监测BCR-ABL转录水平的变化。结果成功构建BCR-ABL标准品。应用RQ-PCR探针法,以ABL为内参,对CML患者进行BCR-ABL融合基因的检测,得到比较稳定的定量数据,与定性结果一致。结论自行构建BCR-ABL质粒为标准品,运用RQ-PCR实时监测BCR-ABL融合基因的表达变化,敏感性好,稳定性高,对临床检验具有普遍意义。

Objective To improve clinical diagnosis and treatment of chronic myeloid leukemia ( CML) , we aim to establish a diagnosis platform for detecting BCR -ABL fusion gene and mornitoring minimal residual disease by constructing a circular plasmid using BCR-ABL fusion gene as a standard .Methods We designed primers and Taqman probes specific to BCR -ABL(b2-a2,b3-a2)and ABL, and used cDNA of the CML patient as the tamplate to amplify a BCR -ABL fragment.The 587bp PCR product of BCR-ABL was cloned into pMD 18T vector and used as a reference standard .The copy numbers was then determined and standard curve derived .The transcriptional expression of BCR/ABL in bone marrow samples from pa-tients was quantitated by real -time quantitative PCR ( RQ-PCR) .Results We constructed a circular plasmid with BCR -ABL fusion gene according to the method from cancer groups in Europe .With pMD 18T BCR-ABL plasmid as reference standard and ABL as an internal control , we accurately detected BCR -ABL expressio

目的 总结bcr-abl融合基因阳性急性淋巴细胞白血病(ALL)患儿临床特点,探讨其治疗及预后的相关因素.方法 对经反转录聚合酶链反应(RT-PCR)方法检测bcr-abl融合基因阳性的7例ALL患儿临床表现、治疗、预后进行回顾性分析.结果 bcr-abl融合基因阳性ALL患儿7例,平均发病年龄为8岁1个月,均为B细胞型ALL,治疗第33天骨髓完全缓解率为50％.结论 bcr-abl融合基因阳性ALL患儿化疗效果差,难缓解,复发率高,预后差.

Objective To analyze the clinical features of pediatric patients with acute lymphoblastic leukemia(ALL) with bcr-abl fusion gene transcript,and discuss the treatment,prognosis factors of this kind of ALL.Methods Clinical features,treatment and prognosis were studied retrospectively in 7 bcr-abl fusion gene positive ALL patients.bcr-abl fusion gene was detected by reverse transcription polymerase chain reaction (RT-PCR).Results The average age of the 7 patients was 8 years and 1 month old.All of them were common B-immunology ALL.The rate of complete response was 50 ％ after 33 days'' treatment.Conclusions The incidence rate of bcr-abl fusion gene positive ALL in pediatric is low.This type of ALL has poor remission,high relapse rate and poor prognosis.

目的：克隆并在293细胞中表达人Polo样激酶1（Plk1）基因，探索Plk1对非受体型酪氨酸激酶c-Abl表达水平的影响。方法：利用PCR法扩增Plk1基因，分别定向克隆至pcDNA3-Flag及pCMV-Myc真核表达载体，将上述质粒分别转染293细胞进行瞬时表达，Western印迹检测Plk1蛋白的表达；将上述质粒分别与c-Abl表达质粒共转293细胞，检测带有不同标签的Flag-Plk1或Myc-Plk1对细胞中c-Abl激酶表达的影响。结果：构建了Flag-Plk1和Myc-Plk1真核表达质粒，其在293细胞中均可表达，蛋白的相对分子质量为68×103；与其共转的c-Abl激酶表达水平显著下调。结论：在293细胞中表达了Flag-Plk1和Myc-Plk1蛋白，且初步发现Plk1可以抑制c-Abl的表达。

Objective: To clone and express human Polo-like kinase-1(Plk1) gene in 293 cell lines, and study its effect on the expression of non-receptor tyrosine kinase c-Abl. Methods: Plk1 gene was amplified by PCR, and then inserted into eukaryotic expression vector pcDNA3-Flag and pCMV-Myc respectively. Those recombinant expression plasmids were transiently transfected into 293 cell lines, and the expression of Plk1 was identified by Western blotting. Different tagged Plk1 and c-Abl were cotransfected into 293 cells to identify the role of Plk1 on the expression of c-Abl kinase. Results: The Flag-Plk1 and Myc-Plk1 eukaryotic expression plasmids were con?structed, and expressed in 293 cell lines with the molecular weight of 68 kD. The expression of c-Abl reduced significantly when contrandfected with tagged Plk1 or c-Abl plasmids into 293 cells. Conclusion: Flag-Plk1 and Myc-Plk1 have been expressed in 293 cells, and it was found that Plk1 can inhibit expression of c-Abl.

目的 建立聚合酶链反应限制性片段长度多态性(PCR-RFLP)联合等位基因特异性聚合酶链反应(AS-PCR)检测bcr/abl融合基因T315I点突变的方法,以提高bcr/abl基因T315I突变检出率.方法 方法学建立.构建abl基因野生型和T315I突变型重组质粒作为检测对象,设计一对野生型和突变型abl基因序列的通用引物,分别以野生型和T315I突变型质粒作为模板,PCR扩增出目的片段,经酶切初筛检测abl基因T315I突变情况,另设计一对仅针对T315I突变的特异性引物进行AS-PCR检测.并对具有该基因突变的1例慢性粒细胞白血病患者进行检测,同时对该方法的灵敏度和特异性进行检测.结果 PCR扩增的目的片段(273 bp)经限制性内切酶Dde Ⅰ酶切后,野生型abl基因片段产生141、68、46和18 bp4条片段,而T315I突变型abl基因片段酶切后产生159、68和46 bp3条片段.PCR-RFLP方法可以检测T315I突变,检测灵敏度为6％.再以酶切产物作为模板进行AS-PCR,优化反应条件,AS-PCR所能检测到的T315I最低突变浓度为0.18％.所测临床标本结果显示,该患者发生了bcr/abl融合基因T315I突变.结论 PCR-RFLP联合AS-PCR检测方法特异性好、灵敏度高,能够给临床检测abl基因T315I突变提供一种新型的检测方法.

Objective To develop a appropriate assay for identifying T315I point mutation of BCR/ABL fusion gene by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) followed mutant enriched allele specific (AS)-PCR,and to improve screening sentivity of BCR/ABL gene T315I mutant.Methods Recommbinant plasmids harboring wild-type ABL gene and the T315I mutant were constructed respectively and used as the detection objects.A pair of universal primers was designed according to wild-type and T315I mutant gene sequence.The wild-type and T315I mutant plasmids were served as template,the screening of ABL gene T315I mutation was carried out by RFLP after PCR amplification.The AS-PCR for T315I mutation detection was established based on a pair of specific primer.The method was used to detect the status of T315I gene mutation in one patient with chronic myeloid leukemia (CML).Also,the specificity and sensitivity of this method were evaluated.Results The PCR amplicons of the target

目的 探讨伴不典型BCR-ABL融合基因亚型el4a3和e19a2型慢性髓性白血病(CML)的临床和实验室特点.方法 对2004至2012年染色体核型分析有t(9;22) (q34;q11),荧光原位杂交(FISH)证实为BCR-ABL融合基因阳性,而常规实时定量PCR(RQ-PCR)检测常见BCR-ABL融合基因(b3a2、b2a2和e1a2)阴性的6例CML患者,重新设计引物进行PCR扩增,并将扩增产物测序,以明确不典型BCR-ABL融合基因类型.对BCR-ABL融合基因扩增产物进行突变检测.对患者的临床资料进行回顾性分析.结果 6例患者PCR扩增产物经测序分析,其中5例为e14a3型,1例为e19a2型.5例e14a3型CML患者中,男4例,女1例,中位年龄48岁,慢性期4例,加速期1例;1例e19a2型患者为女性,40岁,CML慢性期,PLT＞1 000× 109/L.5例e14a3型CML患者中4例在羟基脲或IFN治疗无效后予以伊马替尼(IM)治疗,1例行造血干细胞移植(HSCT).前4例患者中1例因有E255K突变而对IM耐药,改用达沙替尼后获完全细胞遗传学反应(CCyR);1例在IM治疗获CCyR后3个月复发并急变,最终死亡;2例IM治疗后获CCyR,目前状态稳定,仍处于CCyR,尽管其中1例伴有I293T突变.行HSCT治疗患者目前处于CCyR.1例e19a2型CML患者羟基脲治疗后获得完全血液学反应,后改用IM治疗,很快获得CCyR.结论 伴不典型BCR-ABL融合基因的CML发病率极低,酪氨酸激酶抑制剂或HSCT都可以取得疗效,常规RQ-PCR可能漏检少见的不典型BCR-ABL融合基因亚型.

Objective To explore the clinical and laboratory features of chronic myeloid leukemia (CML) with atypical e14a3 and e19a2 BCR-ABL fusion gene subtypes.Methods We retrospectively analyzed a cohort of CML patients with Ph chromosome positive confirmed by cytogenetic and FISH but classical el3a3 (b2a2),e14a2 (b3a2)and ela2 fusion transcripts negative identified by conventional realtime quantification RT-PCR (RQ-PCR).Further RQ-PCR was done with the forward primer and reverse primer designed to detect rare atypical BCR-ABL fusion genes including e14a3 and e19a2 transcripts.Direct sequencing analysis was performed on the PCR products and mutations in the BCR-ABL kinase domain were detected.The clinical data of patients were retrospectively analyzed.Results Six CML patients were found to carry t (9;22) abnormality and BCR-ABL rearrangement confirmed by FISH but classical BCR-ABL fusion genes negative detected by RQ-PCR.Further RQ-PCR and sequencing analysis confirmed the fusion of BCR exon 1