用荧光原位杂交技术研究慢性粒细胞性白血病变异Ph染色体易位的形成

目的：研究慢性粒细胞性白血病（ｃｈｒｏｎｉｃｍｙｅｌｏｉｄｌｅｕｋｉｅｍｉａ,ＣＭＬ）变异Ｐｈ染色体的形成,方法：应用荧光原位杂交（ｆｌｕｏｒｅｓｃｅｎｃｅｉｎｓｉｔｕｈｙｂｒｉｄｉｚａｔｉｏｎ,ＦＩＳＨ）技术,以９号,１５号,２２号整条染色体ＤＮＡ探针和Ｍ－ｂｃｒ／ａｂｌ易位探针检测７例具有变异ｐｈ染色体易位ｔ（９;２２）（ｑ３４：ｑ１１）衍生而来,结论：ＦＩＳＨ技术比传统细胞遗传学方法更敏感。     

用荧光原位杂交技术研究慢性粒细胞性白血病变异Ph染色体易位的形成

目的：研究慢性粒细胞性白血病（ｃｈｒｏｎｉｃ ｍｙｅｌｏｉｄｌｅｕｋｅｍｉａ,ＣＭＬ）变异Ｐｈ 染色体的形成。方法：应用荧光原位杂交（ｆｌｕｏｒｅｓｃｅｎｃｅ ｉｎ ｓｉｔｕ ｈｙｂｒｉｄｉｚａｔｉｏｎ,ＦＩＳＨ）技术,以９ 号、１５ 号、２２ 号整条染色体ＤＮＡ探针和Ｍｂｃｒ／ａｂｌ 易位探针检测７ 例具有变异Ｐｈ 染色体的ＣＭＬ病人骨髓中期分裂相及间期细胞。结果：所有变异Ｐｈ 染色体易位至少涉及３ 条染色体,并由标准Ｐｈ 染色体易位ｔ（９ ;２２）（ｑ３４ ;ｑ１１） 衍生而来。结论：ＦＩＳＨ 技术比传统细胞遗传学方法更敏感,能准确分析变异Ｐｈ 染色体及其形成过程。     

H—2K^b cDNA的克隆及其在多种真核细胞中的表达

目的 克隆Ｈ－２Ｋ＾ｂｃＤＮＡ及研究其在多种真核细胞中的表达。 方法 ＲＴ－ＰＣＲ法扩增Ｈ－２Ｋ＾ｂｃＤＮＡ,克隆入双顺反子逆转录病毒载体ｐＧＣＥＮ,ＰＡ３１７包装出重组病毒后,分别感染ＮＩＨ３Ｔ３、ＣＯＳ７及ＭＭ４５Ｔ．Ｌｉ,ＰＣＲ和ＲＴ－ＰＣＲ分析目的基因表达,ＦＡＣＳ分析Ｈ－２Ｋ＾ｂ在细胞膜上的表达。结果 以双顺反子逆转录病毒载体ｐＧＣＥＮ介导,分别建立了Ｈ－２Ｋ＾ｂ基因转导细胞：ＮＩＨ３Ｔ     

慢性粒细胞白血病反义核酸基因治疗的实验研究

目的单独应用ｂｃｒ－ａｂｌ反义硫代磷酸寡聚脱氧核糖核酸（ＡＳＰＯ）或ｃ－ｍｙｂＡＳＰＯ作用于慢性粒细胞白血病（ＣＭＬ）细胞的研究已有报道，但抑制的不彻底性是个主要问题。由于ＣＭＬ的发生是多癌基因协同作用及相互影响的结果，为进一步提高ＡＳ－ＰＯ对ＣＭＬ细胞的作用效果，用互补于两种类型（ｂ２ａ２及ｂ３ａ２）的ｂｃｒ－ａｂｌ基因融合位点两侧各１３个核苷酸的ＡＳＰＯ（ｂ２ＡＳＰＯ和ｂ３ＡＳＰＯ）及ｃ－ｍｙｂ基因起始密码子２－７互补的ＡＳＰＯ（ｍＡＳＰＯ）联合作用于Ｋ５６２细胞株和原代ＣＭＬ细胞。方法硫代磷酸寡核基酸（ＰＳ－ＯＤＮ）的合成由３９２－０５型ＤＮＡ－ＲＮＡ合成仪自动合成，经ＯＰＣ柱纯化；细胞与ＰＳ－ＯＤＮ共培养后２４，４８，９６，１２０ｈ倒置显微镜下活体观察，０．４％台盼蓝拒染法进行死活细胞计数；ＣＦＵ－Ｋ５６２、ＣＦＵ－ＧＭ、ＣＦＵ－ＧＥＭＭ采用０．８％甲基纤维素半固体培养法；采用两对引物ＲＴ－ＰＣＲ半定量法检测细胞ｂｃｒ－ａｂｌｍＲＮＡ表达；通过流式细胞仪检测及电镜观察细胞凋亡情况。结果（１）ＡＳＰＯ能够特异性抑制ｂｃｒ－ａｂｌ基因表达；经ＲＴ－ＰＣＲ的检测发现１０μｍｏｌＡＳＰＯ作用４８ｈ，两种反义     

反义bcr／abl寡核苷酸诱导K562细胞凋亡时Caspase3表达及？…

用反义ｂｃｒ／ａｂｌ寡核苷酸片段诱导Ｋ５６２细胞凋亡,观察Ｃａｓｐａｓｅ３表达及活性变化,以阐明Ｃａｓｐａｓｅ在慢性髓性白血病（ＣＭＬ）发病机制中的作用。方法：用反义ｂｃｒ／ａｂｌ寡核苷酸片段ＡＳ和对照无义片段ＮＳ自理５６２,ＳＷＩＭＸＬＥＱＵＭＦ ＹＮＶ ＥＹ display status     

小鼠造血干细胞因子基因克隆及在CHO细胞中的表达

克隆造血干细胞因子并在ＣＨＯ细胞中表达。方法：采用ＰＣＲ方法从ｐＲＣ／ＣＭＶ（含ＳＣＦｃＤＮＡ）中钓取ＳＣＦｃＤＮＡ膜外段活性区，克隆入真核表达载体ｐｃＤＮＡ３，转染入ＣＨＯ细胞；用ＲＴ－ＰＣＲ方法检测ｍＲＮＡ水平表达及通过协同刺激骨髓细胞集落形成来检测转染细胞上清的生物活性。     

肾细胞癌中脆性组氨酸三联体基因异常及其意义

目的 检测肾细胞癌（ＲＣＣ）组织脆性组氨酸三本（ｆｒａｇｉｌｅ ｈｉｓｔｉｄｉｎｅ ｔｒａｉｄ,ＦＨＩＴ）基因蛋白编码外显子及ｍＲＮＡ表达情况,了解ＲＣＣ组织中ＦＨＩＴ基因异常特征,探讨ＦＨＩＴ基因与ＲＣＣ发生发展的关系。方法 收集ＲＣＣ及相应癌周肾组织标本,提取组织ＤＮＡ及ＲＮＡ。ＤＩＧ标记ＦＨＩＴ全长ｃＤＮＡ探针。ＰＣＲ法扩增ＦＨＩＴ基因Ｅ５～Ｅ９。扩增产物电泳转膜后行Ｓｏｕｔｈｅｒｎ ｂｏｌ     

白血病 BCR-ABL 融合基因逆转录 PCR 检测中的实验因素探讨

建立白血病中费城（Ｐｈ）染色体上独特的ｂｃｒ－ａｂｌ融合基因的逆转录ＰＣＲ（ＲＴ－ＰＣＲ）快速检测方法。利用正交设计原理优化逆转录反应条件，探讨该法中引物设计和嵌套式ＰＣＲ对检测灵敏度的影响，利用扩增产物所含限制性酶切位点进行产物特异性鉴定。运用此法检测１２例慢性粒细胞白血病，除１例阴性外，其余１１例阳性标本含不同类型（ｂ３ａ２，ｂ２ａ２，Ｂ１ａ２）ｂｃｒ－ａｂｌｍＲＮＡ。讨论了用于临床标本检测中应考虑的问题。     

IFN—α及其联合GM—CSF对CML患者骨髓细胞凋亡相关基因？…

目的：探讨ＩＦＮ－α及ＩＦＮ－α联合ＧＭ－ＣＳＦ调节慢性期慢性粒细胞白血病（ＣＭＬ－ＣＰ）患者骨髓单个核细胞（ＭＮＣｓ）ｂｃｒ－ａｂｌ,ｂｃｌ－２和ｃ－ｍｙｃ基因表达的影响。方法：淋巴细胞分离液离心富集１４例ＣＭＬ－ＣＰ患者骨髓ＭＮＣｓ,经干扰素－α（ＩＦＮ－α）及ＩＦＮ－α联合粒细胞－巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ）培养２４ｈ后,采用相对定量逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）及扩增片段光密     

bcr／abl反义核酸体外净化自体骨髓移植的临床报告

报告了国内首例ｂｃｒ／ａｂｌ反义核酸体外 净化的慢性髓细胞白血病自体骨髓移植、净化后存留ＣＵＦ－ＧＭ的ｂｃｌ／ａｂｌｍＲＮＡＰＣＲ（一），移植后１２月仍保持５０％的Ｐｈ（一）造血重建，至今已无病生存３０个月。反义核酸体外净化尚未见明显毒副作用。     

双色ES探针检测慢性粒细胞白血病bcr/abl融合基因的应用研究

目的:探讨使用bcr/abl双色ES探针的荧光原位杂交(fluorescence in-situ hybridization,FISH)应用于慢性粒细胞白血病bcr/abl融合基因检测的意义.方法:使用荧光原位杂交方法对2005年1月至2006年2月本院收治的12例慢性粒细胞白血病患者之骨髓细胞进行检测分析.结果:12例CML均检出bcr/abl基因的存在,其中2例伴ASS基因缺失.结论:使用bcr/abl双色ES探针进行荧光原位杂交能为bcr/abl融合基因的检测提供准确直观的依据.     

双色双融合荧光原位杂交检测慢性粒细胞白血病的bcr/abl融合基因研究

目的：探讨双标双融合荧光原住杂交技术(dual-color and dual-fusion fluorescence in—situ hybridization，D—FISH)在慢性粒细胞白血病(chronic myeloid leukemia，CML)中检测bcr／abl融合基因的灵敏度及特异性。方法：应用细胞遗传学G显带(CCG)核型分析、双色荧光原住杂交(D—FISH)和染色体涂染技术，检测了29例CML患者骨髓中期分裂相和间期细胞。结果：CCG检出Ph( )24例，D—FISH检测均为bcr／abl( )；Ph(-)5例，其中4例核型为46，XY，经D—FISH检测2例bcr／abl( )，2例ber／abl(-)，另1例核型为46，XYt(20；22)，D—F1SH检测ber／abl( )，以9号，20号和22号全染色体探针涂染，确定该例核型为46，XYt(9；20；22)，D—FISH检测总阳性率为93．10％(27／29)，高于CCG检查时阳性率82．76％(24／29)(P=0．025)。结论：与CCG方法相比，D—FISH检测CML的ber／abl融合基因具有简便、快速、灵敏、特异性强等优点，可以作为CML临床诊断和微小残留病监测的一种重要检测方法。．     

双色FISH检测21号染色体及DSCR Cosmid克隆制图

应用Ｂｉｏｔｉｎ－１６－ｄＵＴＰ和Ｄｉｇｏｘｉｇｅｎｉｎ－１１－ｄＵＴＰ分别标记人２１／１３号染色体着丝粒区ＤＮＡ探针（Ｄ１３ＺＩ／Ｄ２１ＺＩ）和Ｄｏｗｎ综合征核心区（ＤＳＣＲ）ＣｏｓｍｉｄＤＮＡ探针，与人类正常二倍体染色体进行原位杂交（ＩＳＨ），并用相应的免疫荧光系统检测杂交信号，在两条１３号和两条２１号染色体着丝粒区显示绿色（ＦＩＴＣ）信号；在两条２１号染色体长臂（２１ｑ２２）显示红色（Ｒｈｏｄａｍｉｎｅ）信号。双色ＦＩＳＨ为检测２１号染色体数目和结构异常提供了可靠的手段，为基因在染色体上制图提供了有效的方法。     

支气管镜刷取细胞染色体异倍性的研究

目的：探讨间期核荧光原位杂交（FISH）检测支拨管镜刷取细胞染色体异倍体和将其应用于肺癌早期诊断的可行性。方法：选取7、8、9和12号染色体特异着丝探针,用FISH技术检测14例肺癌患者支气管镜刷取细胞上述染色体异倍体。结果：在细胞诊断为癌或可疑癌细胞的7例中均发现染色体异倍体,主要特征为7、8和12号染色体增多及9号染色体丢失;而在细胞学未发现恶性证据的7例中,有3例存在7、8和9号染色体异倍体。结论：间期核FISH可以检测出支气管刷取细胞中染色体异倍体,在传统细胞学诊断标准下不能定为恶性的细胞中发现染色体异倍体,可将这种方法用于肺癌的早期诊断。     

人肺癌染色体异倍性的初步研究

目的 探讨间期核荧光原位杂交（FISH）检测肺癌印片标本染色体异倍体的可行性和其临床应用前景。方法 选取7、8、9和12号染色体特异着丝粒DNA探针,用FISH技术检测46例肺癌印片标本的染色体异倍体。结果 46例肺癌印片标本均检出染色体异倍体,主要特征为7、8、和12号染色体增多及9号染色体丢失。7、8、9和12号染色体异倍体总检出率分别为：67．4％（31／46）、60．9％（28／46）、28．3％（13／46）和56．5％（26／31）。结论 间期核FISH可以检测出肺癌印片标本中染色体异倍体,这些改变有望应用于肺癌早期诊断和预后判断。     

增强子结合蛋白家庭的一个新基因HP8定位于人19号染色体长臂13.1-13.2区带

目的:探讨增强子结合蛋白(C/EBP)家族一个新基因HP8的人染色体定位.方法:按常规方法制备分裂期人淋巴细胞切片,用生物素标记HP8基因 3’-端 2.1kb cDNA作探针,进行荧光原位杂交(FISH).对FISH信号及染色体上DAPI结合条带同时拍照,二者重迭信号进行计数,结果:100个有丝分裂图中,有72个显示阳性信号位于同一对染色体,并且无其他非特异性杂交带,杂交效率约为70%.参照DAPI结合条带,阳性信号位于人19号染色体长臂13.1-13.2区带.结论:HP8基因定位于人19号染色体长臂 13.1-13.2区带.     

Chromosomal changes detected by fluorescence in situ hybridization in patients with acute lymphoblastic leukemia

Objectives To investigate patients with acute lymphoblastic leukemia (ALL) for TEL/AML1 fusion,BCR/ABL fusion, MLL gene rearrangements, and numerical changes of chromosomes 4, 10, 17 and 21 by fluorescence in situ hybridization (FISH) and to determine the relationship and the significance of those findings.Methods Fifty-one American patients (34 men and 17 women) were included in this study. Of them there were 41 patients with pro-B cell type ALL, 9 with B cell type ALL and 1 with T cell type ALL.Chromosome metaphases of each sample were prepared according to standard protocols.Fluorescence in situ hybridization was performed using commercially available DNA probes, including whole chromosome painting probes, locus specific probes, specific chromosome centromere probes and dual color/multiple color translocation fusion probes. The digital image analysis was carried out using Cytovision and Quips FISH programs.Results An overall incidence of chromosomal anomalies, including t (9; 22 ), MLL gene rearrangements, t (12;21), and numerical chromosomal anomalies of chromosomes 4, 10, 17 and 21 was found in 33 patients (65%). Thirty-one of them were pediatric patients and two adults. The t(12;21) was the commonest chromosomal anomaly detected in this population; 14 out of the 45pediatric patients (31%) were positive for TEL/AML1 fusion, among which three had an additionalderivative 21[t (12;21) ], four had a deletion of 12p and two had an extra copy of chromosome 21.All 14 patients with positive TEL/AML1 fusion had ALL pre-B cell or B-cell lineage according to standard immunotyping. The percentage of cells with fusion signals ranged from 20% to 80%. All fourteen patients positive for TEL/AML1 gene fusion were mosaic. Three out of the 14 patients positive for the TEL/AML1 gene fusion were originally reported to be culture failures and none of the remaining eleven samples had been found to have chromosome 12 abnormalities by conventional cytogenetic techniques. All pediatric patients with pre-T or T cell lineage and the six adults were negative for TEL/AML1 fusion. One patient had double Philadelphia chromosomes, three had a rearranaement or a deletion of the MLL aene. one had t (4;11)and two had a deletion of the MLL One of the patients with an MLL deletion also had a large ring of chromosome 21, and r (21) was caused by AML1 gene tandemly duplicated at least five times. The second case with the MLL deletion was also unique, the patient had a t (12;21) as well. A total of 20 patients had numerical changes( gain or loss) of chromosomes 4, 10, 17 and 21. Eight patients were found to have trisomies of three or four different chromosomes. Interestingly, seven of these patients did not have TEL/AML1, BCR/ABL or the MLL aene rearranaement, one did have the TEL/AML1 aene fusion. Eleven patients with pro-B cell or B cell type ALL (9 children with ALL, 2 adults with ALL) had numerical changes of chromosome 21 (gain 1 or 2 chromosome 21 ), among them, 10 patients had no structural alteration of chromosome 21, and one was combined by t (12;21 ). Four patients had a monosomy of chromosome 17 and three out of these patients with monosomy 17 also had a fusion signal of TEL/AML1.     

Diagnostic role of conventional cytogenetics and fluorescence in situ hybridization (FISH) in chronic myeloid leukemia patients

Introduction: The limitation of cytogenetic analysis is that the Ph chromosome cannot be detected in clumped metaphase or in interphase cells. Fluorescence in situ hybridization (FISH) is a highly sensitive molecular genetic technique, which enables to detect break point cluster region - Abelson (BCR-ABL) complex and minimal residual disease in all Ph positive CML patients not only in metaphase but also in interphase cells. Aims: To detect Ph chromosome in CML patients by the use of conventional cytogenetics and FISH. Material and methods: The bone marrow samples were collected in heparinised syringe from 35 diagnosed CML patients and transported to cytogenetic laboratory for chromosomal analysis. Conventional karyotype was prepared by direct harvesting and short-term culture. The FISH analysis was carried out on interphase cells of two patients to confirm the cytogenetic diagnosis. Results: Out of 35 CML patients, 17 (49.9%) were 100% Philadelphia positive, 10(28.5%) were 50-70% Ph+ mosaics and 3(9%) were 100% Ph negative. In 5 patients (14.25%) cytogenetic analysis failed to confirm the presence or absence of Ph chromosome. FISH was carried out in interphase cells from bone marrow preparations of two patients. The signals for BCR-ABL fusion gene was absent in Ph- negative CML patients. In Ph positive patients, the FISH analysis detected BCR-ABL fusion gene seen as a yellow signal on interphase cells. Conclusion: Conventional cytogenetics is a useful method for detection of Ph chromosome in metaphase stage of cell division. FISH can be used in interphase stage of cell division for the same purpose. Key words: CML, FISH, Chromosomal analysis, Philadelphia chromosome.     

应用荧光原位杂交检测HBV基因在转基因小鼠染色体上的整合

目的应用荧光原位杂交技术（fluorescence in situ hybridization,FISH）,检测本研究中心制备的乙型肝炎病毒（HBV）转基因小鼠目的基因的整合位点。方法采用荧光原位杂交技术,以正常小鼠染色体标本为对照,通过荧光标记的探针与转基因小鼠染色体标本进行杂交,对比分析同一中期分裂相的G显带和FISH结果。结果转基因小鼠染色体检测到一对明显的荧光信号,推测其在14号同源染色体上,而对照组无信号。结论外源HBV基因以单位点形式稳定地整合到转基因小鼠的染色体上。