摘要
目的:FLT3跨膜区内部串联重复突变是与白血病发生相关的FLT3基因中最常见的突变类型。本实验旨在探讨FLT3跨膜区内部串联重复突变与骨髓增生异常综合征预后的关系及其临床意义。方法:实验于中国刑警学院法医教研室、沈阳医学院生物化学及分子生物学教研室完成。选择1998-03/2005-10于中国医科大学及沈阳医学院附属医院血液科门诊或住院治疗的患者58例作为实验组,经免疫学、细胞遗传学和分子生物学检查确诊均为骨髓增生异常综合征患者。男37例,女21例,平均年龄19岁,均自愿参加。形态学分型中58例骨髓增生异常综合征患者中难治性贫血13例、难治性贫血伴环状铁粒幼细胞增多4例、难治性贫血伴原始细胞增多20例、转变中的难治性贫血伴原始细胞增多12例、慢性粒-单核细胞性白血病9例。对照组为同期就诊非白血病患者及健康供者10例。①DNA提取:患者骨髓或健康志愿者外周血标本采用淋巴细胞分离液分离单核细胞,传统酚/氯仿/异丙醇法提取DNA。②PCR引物设计:针对FLT3基因内部重复串联结构主要发生在11号外显子,在11号外显子近端和远端分别设计引物11F和11R,扩增产物为133bp。同时设计对照组β-actin引物actin F和actin R,扩增产物为305bp。③PCR扩增及电泳PCR:反应体系包括上、下游引物各0.2μmol/L,dNTPs 0.25mmol/L,Taq DNA聚合酶2.5U,模板DNA 200ng。反应条件为94℃变性3min后进行35个循环,最后72℃延伸10min。取扩增产物,经2%琼脂糖凝胶电泳观察结果。④测序及Blast分析。结果:骨髓增生异常综合征患者58例和对照组10例全部进入结果分析。①58例骨髓增生异常综合征患者中19例FLT3表达阳性,阳性率为32.78%。19例FLT3基因检测阳性患者,有5例出现FLT3跨膜区内部串联重复突变,阳性率为2.74%,其中慢性粒-单核细胞性白血病2例、难治性贫血伴原始细胞过多过度型3例。②测
AIM： FLT3/internal tandem duplication （FLT3/ITD） in transmembrane region is most common i.n leukemia related FLT3 mutation, In this study, we explore the relationship between FLT3/ITD mutation and myelodysplastic syndromes （MDS） prognosis as well as its clinical significance. METHODS： The experiment was conducted in the Department of Forensic Medicine, China Criminal Police University and Department of Biochemistry and Molecular Biology, Shenyang Medical College, Fifty-eight inpatients and outpatients with MDS diagnosed by examinations of immunology, cell heredity and molecular biology from Department of Hematology, Affiliated Hospital of Chinese Medical University and Shenyang Medical College were selected from March 1998 to October 2005, including 37 males and 21 females with the mean age of 19 years old, They all voluntarily participated in the experiment, Among the 58 MDS patients, there were 13 with refractory anemia （RA）, 4 refractory anemia with ring sideroblast （RAS）, 20 RA with an excess of blast （RAEB）, 12 RAEB in transformation （RAEB-T） and 9 chronic myelo-monocytic leukemia （CMML）, Ten health donors and non-leukemia patients treated in the corresponding period were selected as control. （1）DNA extraction： Monocytes in bone marrow of patients or whole peripheral blood of healthy volunteers were isolated by Lymphocyte Separation Medium, and then genomic DNA were extracted with the traditional phenol/chloroform method. （2）PCR primer design： Primer 11F and 11R were designed at the two end of exon 11 as the main occurrence location of FLT3/ITD, and 133 bp amplified product was desired. Meantime, primer actin F and actin R were designed for β-actin control group and 305 bp amplified product was desired. （3）PCR reaction and electrophoresis： The volume of reaction system included 0.2 μmol/L of each primer, 0.25 mmol/L dNTPs, 2,5 U Taq DNA polymerase, 200 ng template DNA. The PCR protocol involved 35 cycles of denaturation at 94 ℃for 3 minutes and then elong
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第3期518-521,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
