【作者】 潘金兰；

【导师】 薛永权； 陈苏宁；

【作者基本信息】 苏州大学 ， 内科学， 2011， 博士

【摘要】 目的和意义白血病细胞株有着良好的稳定性,即在体外长期传代也能较好地保持其在体内的特征,因而被认为是体内白血病细胞在体外的优秀模型,从而被大量应用于白血病的发病机制、治疗药物和生物学技术等方面的研究中。目前已建立了大于一千株白血病细胞株,但其中髓、单核系细胞株较少,且相当一部分未能进行全面的生物学特性鉴定,因而难以被各种研究所应用。本研究先后通过对两例髓、单核系白血病(一例为AML-M4,另一例为CML急性髓、单核系变)患者的原代白血病细胞的体外培养,建立了两株伴有特异性染色体异常的白血病细胞株(JIH-3和JIH-6),并对其生物学特性进行了较为全面的鉴定,从而为白血病的研究又提供了有价值的工具。一、JIH-3细胞株的建立及其生物学特性的鉴定方法在本科室现有的建立白血病细胞株的经验基础上,再结合国外文献发现,初诊、预后良好的患者,其白血病细胞难以建株,而复发以及难治性患者的白血病细胞建株成功率较高。因此我们有意识地选取难治和复发患者的白血病细胞进行体外培养,从十多例白血病患者中成功建立了一株白血病:JIH-3。该细胞株建自一例急性髓、单核系(M4)患者的外周血白血病细胞。该部分内容共分为四部分。第一部分我们首先对成功建成JIH-3细胞株的AML-M4患者进行了深入的遗传学研究:以常规R显带法进行核型分析;FISH检测CBFβ基因重排;7号染色体长臂和短臂涂抹探针检测7号染色体短臂的缺失;多重逆转录-聚合酶链反应(RT-PCR)检测有无特异性重排。第二部分中,我们对JIH-3的建株过程进行了较为详细的阐述,并对其基本生物学特性进行了鉴定:倒置显微镜下观察活细胞的生长特征;瑞氏染色后于镜下观察细胞形态;α-醋酸萘酚酯酶染色+氟化钠抑制试验、过氧化物酶(POX)染色观察其细胞化学染色特点;电子显微镜下观察细胞超微结构;绘制生长曲线,计算倍增时间;半固体甲基纤维素中集落培养检测JIH-3细胞的集落生成能力;流式细胞仪(FCM)检测JIH-3细胞的免疫表型和细胞周期分布;荧光定量PCR检测EB病毒基因组DNA;PCR检测支原体;R显带核型分析动态监测建株过程中JIH-3细胞的染色体改变;FISH检测CBFβ基因重排、PML基因重排;应用4号和7号着丝粒探针检测JIH-3核型中衍生染色体的着丝粒性质;应用7号染色体长臂和短臂涂抹探针检测7号染色体短臂的缺失;应用M-FISH检测JIH-3细胞是否存在其它核型分析未能发现的隐匿性重排;应用多重RT-PCR检测常见29种融合基因;PCR扩增JIH-3细胞常见基因的热点外显子,包括KIT、P53、FLT3、JAK2、NPM1、CEBPA、RUNX1和WT1,测序分析其有无突变;短串联重复序列(STR)-PCR检测JIH-3细胞和原代白血病细胞是否来自同一个体。第三部分中,我们对JIH-3细胞在裸小鼠体内的致瘤性进行了研究。将JIH-3细胞接种至4只四周龄NC裸小鼠皮下,观察肿瘤生长情况;取肿瘤组织行常规病理检测;分离瘤组织中单个核细胞(MNC)进行R显带核型分析;流式细胞仪(FCM)检测瘤组织中的单个核细胞的免疫表型。第四部分中我们对JIH-3细胞株和原代白血病细胞进行了芯片比较基因组杂交(aCGH)研究。将JIH-3细胞和原代白血病细胞提取DNA后,送至上海康成生物公司,进行aCGH研究。结果(一)患者整个病程中共作3次核型分析,初诊时骨髓细胞核型分析揭示为46,XY正常男性核型,第一次和第二次复发时骨髓细胞和外周血细胞核型分别为46,XY,del(7)(p1?3p2?2)[10]和46,XY,del(7)(p1?3p2?2)[10]/ 46,XY[2]。7号染色体长臂和短臂涂抹分析证实了7号短臂的缺失。间期FISH未发现CBFβ基因重排,多重RT-PCR也为检测到包括MYH11/CBFβ融合基因在内的29种白血病融合基因。流式细胞仪检测为髓系、单核系表达,并排除了M4eo的诊断。(二) JIH-3细胞可在无任何细胞因子的条件下持续增殖,其适宜培养条件是含15%胎牛血清的IMDM培养基。该细胞在培养基中呈单细胞样悬浮生长,目前已于体外持续培养1年半余,经液氮冻存、复苏后仍能持续增殖,其倍增时间为102.5个小时。瑞氏染色下JIH-3细胞胞体中等大小或偏大,胞浆量多丰富,少部分可见伪足样突起,染灰蓝色,部分细胞胞浆中可见少量细小紫红色颗粒;胞核圆形,类圆形,易见扭曲、折叠,核染色质细致疏松,部分可见核仁。电镜下可见丰富的线粒体、内质网、溶酶体等细胞器及空泡结构,胞核不规则,部分细胞可见核凹陷、折叠,多可见明显的核仁。POX染色18%为阳性,82%为阴性。α-醋酸萘酚酯酶染色为阴性。免疫表型检测显示JIH-3细胞主要表达髓系、单核系抗原,也有部分T淋系、B淋系、自然杀伤细胞系抗原和干祖细胞标记。细胞周期分析结果显示S期细胞所占比例为17.065%, G1期细胞所占比例为78.4%,G2期细胞所占比例为4.5%。体外培养至2009年9月18日时,核型出现了变化,为46,XY,del(7p)[6]/45,XY,dic(4;7) (p11;p11),del (15q)[4]两个克隆,直至2009年12月22日,核型均为45,XY,dic(4;7)(p11;p11), del(15q),并一直稳定生长至2010年8月18日。新的克隆由着丝粒FISH、涂抹FISH和M-FISH加以证实,M-FISH也未发现任何核型未检出的隐匿性异常。无CBFβ和PML基因重排。JIH-3细胞于1.4 %甲基纤维素培养基内生长14天后,其集落生成率为8.3%。荧光定量PCR未检测到EB病毒感染,培养细胞上清中也未检测到支原体污染。多重PCR未检出任何融合基因阳性。测序分析未发现8种常见基因的突变。STR-PCR结果显示JIH-3细胞和原代白血病细胞来自同一个体。(三)注射JIH-3细胞的4只裸鼠于第60天时发现注射部位均有肿块形成,85天时肿块大小约在0.4×0.3～1.5×1.0cm2之间。病理检测显示瘤体组织绝大部分由白血病细胞组成,可见血管生成和少量坏死组织。从肿瘤组织分离的MNC经R显带核型分析后显示为45,XY,dic(4;7)(p11;p11),del(15q)。FCM检测结果,小鼠内取出的致瘤肿瘤细胞的表面抗原表达与患者初诊时骨髓以及体外培养细胞的表面抗原表达的结果基本相一致,为髓系、单核系、干系以及NK系表达。(四)患者原代细胞经aCGH检测后,不仅证实了核型分析检出的del(7p),并对其断裂位点进行了调整,由原来的7p1?3p2?2修正为7p14.1p21.1和7p22.3,片段大小分别为18.8Mb和601Kb,另外,aCGH还检出了核型分析无法知晓的小片段的缺失和扩增。JIH-3细胞的aCGH结果表明保留了原代白血病细胞的主要特征,即del(7p),并在此基础发生了一些变化。这些具有异常DNA拷贝数的区域内含有多种基因,原代细胞异常区域中涉及138个基因,其中119个基因位于7p。JIH-3细胞则涉及366个基因,其中302个基因位于7p。原代细胞中的大部分异常基因在JIH-3细胞中仍旧异常。结论(一)报道一例伴有del(7)(p13p22)异常核型的AML-M4病例,并对其进行了深入的遗传学研究。结果经多种方法证实该例患者为核型仅伴有del(7)(p13p22)异常的AML-M4,而非初诊时形态所示的M4eo,表明del(7)(p13p22)预后不良,并且只有依靠MICM等多种手段,才能尽可能减少误诊,提高诊断准确率。(二)从一例AML-M4患者外周血中成功建立一株白血病细胞株JIH-3。形态学和免疫表型呈现髓系和单核系特征。JIH-3伴有dic(4;7)(p11;p11),del(15q)异常,FISH检测未发现CBFβ、PML基因重排,涂抹分析证实了7号染色体短臂的缺失,M-FISH未发现隐匿性异常,测序分析未发现常见基因的突变。EB病毒和支原体污染被排除,JIH-3细胞集落形成能力较弱,增殖较缓慢。STR证实JIH-3细胞和原代白血病细胞来自同一个体。(三) JIH-3细胞在裸小鼠皮下有较强的致瘤性。(四) aCGH是有效地检测DNA拷贝数变化的方法。不仅证实并调整原代白血病细胞的del(7)(p1?3p2?2)为del(7)( p14.1p21.1)和del(7)(p22.3),同时证实JIH-3为del(7)(p11p22),另外还检出核型分析无法知晓的小片段的缺失和扩增。二、JIH-6细胞株的建立及其生物学特性的鉴定方法从一例慢性髓系白血病急性髓系变患者的外周血中成功建立了另一株髓系白血病细胞株JIH-6。该部分内容分为二部分。第一部分我们首先对成功建成JIH-6细胞株的CML急性髓系变患者进行了深入的研究:以常规R显带法进行核型分析;FISH检测BCR/ABL基因重排、AML1和EVI1基因重排;逆转录-聚合酶链反应(RT-PCR)检测BCR/ABL融合基因;PCR扩增并测序分析有无常见BCR/ABL融合基因突变,流式细胞仪检测其白血病细胞免疫表型。因患者只有初诊时顺利抽取骨髓,此后始终干抽,故无加速或急变时骨髓形态学资料,以上检查所用标本均为患者外周血。第二部分中我们对JIH-6的建株过程进行了较为详细的阐述,并对其基本生物学特性进行了鉴定:倒置显微镜下观察活细胞的生长特征;瑞氏染色后于镜下观察细胞形态;过氧化物酶(POX)染色观察其细胞化学染色特点;绘制生长曲线,计算倍增时间;流式细胞仪(FCM)检测JIH-6细胞的免疫表型和细胞周期分布;荧光定量PCR检测EB病毒基因组DNA;PCR检测支原体;R显带核型分析JIH-6细胞的染色体异常;FISH检测BCR/ABL基因重排、AML1和EVI1基因重排;逆转录-聚合酶链反应(RT-PCR)检测BCR/ABL融合基因;PCR扩增并测序分析有无常见BCR/ABL融合基因突变;荧光定量RT-PCR检测JIH-6、JIH-3、JIH-4、SHI-1和HL60细胞株中EVI1基因表达;短串联重复序列(STR)-PCR检测JIH-6细胞和原代白血病细胞是否来自同一个体。结果(一)因患者诊疗过程中骨髓多部位干抽,故在本院治疗的整个病程中仅二次常规染色体检查,分别为初诊和急变时,初诊时骨髓细胞染色体结果为46,XX,t(9;22)(q34;q11),急变时因骨髓干抽,故抽取外周血行染色体检查,结果揭示为46,XX, der(3)?inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11)。FISH证实了BCR/ABL融合基因阳性,位于3q26的断裂点分开的EVI1基因探针不仅证实了3号染色体倒位,还发现部分细胞中该基因部分序列有扩增;AML1/ETO和AML1断裂点分开探针均证实了t(4;21)易位导致位于21q22的AML1基因受累,且部分细胞中AML1基因有扩增。荧光定量RT-PCR检测其BCR/ABL拷贝数为12301/10000abl;对BCR/ABL融合基因直接测序发现存在两群细胞,一群细胞中ABL基因的第1439个核苷酸“T”突变为“G”,另一群细胞无ABL基因突变;流式细胞仪检测其外周血白血病细胞为髓系和单核系表达。(二) JIH-6细胞适宜培养条件是含15%胎牛血清的IMDM培养基,可在无任何细胞因子的条件下持续增殖。该细胞在培养基中呈单细胞样悬浮生长,目前已于体外持续培养8月余,经液氮冻存、复苏后仍能持续增殖,其倍增时间为118.2个小时。瑞氏染色下JIH-6胞体大小不等,胞浆量丰富,少部分可见伪足样突起,染灰蓝色,少数细胞胞浆中可见少量嗜天青颗粒;胞核,类圆形,部分可见凹陷、折叠,核染色质细致疏松,部分可见核仁。POX染色1%为阳性,99%为阴性。免疫表型检测显示JIH-6细胞主要表达髓系、单核系抗原,也有部分细胞表达T淋系、B淋系和干祖细胞标记。S期细胞所占比例为24.78%, G1期细胞所占比例为73.29%,G2期细胞所占比例为1.92。因细胞增殖较缓慢,故仅体外培养6个多月时(2010年1月5日),才对其核型进行了检测,发现其核型与原代细胞相比并未发生变化,仍为46,XX, der(3)?inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11),与JIH-3细胞株相比,核型较为稳定。荧光定量PCR未检测到EB病毒感染,培养细胞上清中也未检测到支原体感染。FISH检测证实了EVI1基因受累重排及扩增,AML1基因受累重排及扩增,BCR/ABL融合基因阳性。RT-PCR检测BCR/ABL融合基因阳性。对BCR/ABL融合基因的直接测序未发现有ABL基因的突变。荧光定量RT-PCR检测发现,与正常对照相比,JIH-6、JIH-3、JIH-4、SHI-1和HL60细胞株中EVI1基因表达均增高。STR-PCR结果显示JIH-3细胞和原代白血病细胞来自同一个体。结论(一)报道一例伴有46,XX, inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11)复杂异常核型的CML急性髓、单核系变的病例,并对其做了深入的研究。结果证实:3号染色体倒位,导致位于3q26的EVI1基因重排并发生了扩增;t(4;21)(q21;q22)易位导致位于21q22的AML1基因受累重排并发生扩增;FISH和PCR均证实BCR/ABL融合基因阳性;ABL基因的第1439个核苷酸“T”突变为“G”。这些均为预后不良指标。(二)从该例患者建立了JIH-6髓单核系白血病细胞株。JIH-6伴有特异性染色体异常inv(3)(q21q26), t(4;21)(q21;q22)和t(9;22)(q34;q11)。FISH和PCR均证实BCR/ABL融合基因阳性,另外,FISH还证实EVI1和AML1基因受累重排并有扩增。BCR/ABL融合基因直接测序未发现有ABL基因的突变。EVI1基因表达增高。EB病毒和支原体污染被排除。STR-PCR证实JIH-6细胞株和原代白血病细胞来自同一个体。综上所述,JIH-3和JIH-6均为伴有特征性染色体异常的人急性髓、单核系白血病细胞株,具有清晰的生物学背景,为白血病研究又提供了两个新的重要的工具。更多还原

【Abstract】 ObjectiveLeukemia cell lines sustained proliferation in vitro with preservation of most cellular features during long-term culture. They represent reliable model systems and provide powerful tools for the investigation of leukemogenesis, the analysis of drug effect and the research of biotechnology. To date, more than 1000 leukemia-lymphoma (LL) cell lines have been reported. The majority of them belong to lymphoid-originated cell lines, while myelomonocytic leukemias only account for a minor percentage. It is regretted that large panels of LL line cells are not available because they are not sufficiently characterized and are incompletely described in the literature. We establish two novel human myelomonocytic leukemia cell lines with specific chromosomal abnormality by culturing the primary leukemia cells from a acute myeloid leukemia(AML)-M4 and a chronic myeloid leukemia (CML) in myeloid blast crisis patients in vitro and to provide two novel well-characterized acute myelomonocytic leukemia cell lines for the research of leukemia.1. Establishment and characterization of an new AML-M4 cell line (JIH-3)MethodsMost of the described human myeloid leukemia cell lines are derived from aggressive or relapsed cases and have multiple cytogenetic aberrations. There are few cell lines derived from the patients with good prognosis. Based on the experience of the extablishment of SHI-1 and SHI-2 , the primary leukemia cell from over 10 relapsed leukemia patients were cultured in vitro. The acute myelomonocytic leukemia (AML-M4) cell line ,JIH-3 was established from the peripheral blood of an AML-M4 patient. There are four parts in this chapter.In the first part, we performed comprehensive genetic characterization on the AML-M4 patient. Conventional RHG banding was emploryed for karyotypic analysis. Fluorescence in situ hybridization (FISH) was used to identify the rearrangement of CBFβgene. Chromosome painting (CP) was performed using chromosome paint probes for the short arm and long arm of chromosome 7 to identify the deletion of the short arm of chromosome 7. The specific fusion gene transcript including MYH11/ CBFβwas studied by multiplex reverse transcriptase poiymerase chain reaction (Multiplex RT-PCR).In the second part, the process of establishment of the JIH-3 cell line was described and the basic biologic features of JIH-3 were characterized. The growth feature of the JIH-3 cell line in the 24-well plate was studied under the inverted microscope. The cell line was routinely stained with Wright’s staining method. Cytochemical studies were performed byα-naphthyl butyrate esterase, and peroxidase staining. The ultrastructure was assayed by the transmitted electronic microscope. The growth curve of the JIH-3 was obtained by incubating the cells in 24-well plate and the doubling time was calculated from the curve. The clonality was assayed by semi-solid methylcellulose clonogenic assay. The immunoprofile and the cell cycle distribution of JIH-3 were studied by flow cytometry (FCM). Quantitative fluorescent polymerase chain reaction was used to detected EBV genomic DNA. DNA-PCR was performed to determine the contamination of mycoplasma. RHG banding was employed for dynamic karyotypic analysis in the process of establishment of the cell line. FISH was used to identify the rearrangement of CBFβand PML gene. In order to assess the dicentric characterization of the derivative chromosome , FISH was performed using centromere-specific probes for chromosomes 4 and 7. CP was performed using CPs for 7p and 7q. Multiple FISH was used to exclude cryptic chromosomal abnormalities. The 29 specific fusion gene transcript was studied by Multiplex RT-PCR. The exons of KIT、P53、FLT3、JAK2、NPM1、CEBPA、RUNX1 and WT1 were amplified by PCR and sequenced, respectively. Cell line authentication was performed by short tandem repeating sequences-PCR (STR-PCR). In the third part, the tumorigenicity in mice was evaluated in four nude mice subcutaneous injected with the JIH-3 cell line. The tumor specimen was studied by the conventional pathologic assay. The mononuclear cells (MNC) of the tumor was assayed by RHG-banding and FCM for the immunoprofile.In the fourth part, array comparative genomic hybridization (aCGH) was carried out to determine the genomic copy number changes (loss or gain) in primary leukemia cell from the AML-M4 patient and JIH-3 cell line. Genomic DNA was extracted from primary leukemia cell and JIH-3 cell line. aCGH study was done by the limited Bio-tech company of KangChen.Results1. The cytogenetic analysis of the patient’bone marrow (BM) cells at presentation was normal male karyotype: 46, XY. The karyotype was 46,XY, del(7)(p1?3p2?2)[10] and 46, XY, del(7)(p1?3p2?2) [10] / 46,XY[2] at the first and second relapse ,respectively. The deletion of the short arm of chromosome 7 was confirmed by CP using CPs for 7p and 7q. The rearrangement of CBFβgene was exclude by FISH. It showed negative results of rearrangement of 29 fusion genes (including MYH11/ CBFβ) by Multiplex RT-PCR. The patient’s leukemia cells showed myeloid and monocytic antigens. The diagnosis of M4eo was excluded.2. The JIH-3 cell line could be cultured in IMDM medium with 15% fetal calf serum (FBS) without any cytokine, and has been maintained in continuous culture for more than one year. It grows as single cells in suspension in the 24-well plate. JIH-3 cells remain stable proliferation after repeated freezing and thawing and has doubling time of about 102.5 hours. Most of the cells showed a regular cell shape, intermediate size, folded nucleus with prominent nucleoli, finely reticular chromatin, and blue-grey cytoplasm with azurophilic granules. Transmission electron microscopy examination showed that most of cells have numerous mitochondria, endoplasic reticulum, lysosome, vacuole and folded nucleus with prominent nucleoli. Eighteen percent of JIH-3 cells were positive for peroxide staining. The cell line was negative forα-naphthyl butyrate esterase and the NaF inhibition test. The results of immunoprofile showed that the JIH-3 cell line expressed myeloid and monocytic antigens. A few markers of T、B lymphocytic lineage、stem cell and natural killer (NK)-related antigens were also presented. Cell cycle analysis showed 17.065% of cells in S phase, 78.4% in G1 phase and 4.5% in G2 phase. The karyotype of the JIH-3 cells harvested on September 18 of 2009 revealed the presence of two clones: 46,XY,del(7p)[6]/ 45,XY,dic(4;7)(p11;p11),del(15q)[4] and the karyotype of JIH-3 cells harvested on December 22 of 2009 was 45,dic(4;7)(p11;p11),del(15q). The dicentric characterization of the derivative chromosome dic(4;7) was revealed by FISH using centromere-specific probes for chromosomes 4 and 7. The translocation between chromosomes 4 and chromosomes 7 were detected by M-FISH. The JIH-3 had no the rearrangemenst of CBFβand PML gene. The clonality of the JIH-3 cell line was confirmed by semi-solid methylcellulose clonogenic culture. EBV genomic DNA and mycoplasma contamination were not revealed. Any positive results of fusion genes were not revealed by Multiplex RT-PCR. JIH-3 had no gene mutation of KIT、P53、FLT3、JAK2、NPM1、CEBPA、RUNX1 and WT1. Cell line authentication by STR-PCR showed that the primary leukemia cells of the patient and the JIH-3 cell line originated from the same individual.3. The tumor masses were found in hypodermic tissue in all four nude mice injected with JIH-3 cells after 60 days. The sizes of the tumor masses range from 0.4×0.3～1.5×1.0cm~2. The results of the conventional pathologic assay showed the tumor masses were composed of the leukemia cells in which blood vessels and necrosis could be seen. Chromosome analysis was performed on mononuclear cells isolated from the tumor masses and showed the same chromosomal abnormalities as the JIH-3 cell line: 45,XY,dic(4;7)(p11;p11),del(15q). Their immunophenotypic profile corresponded to the JIH-3 cell line.4. Array CGH study was performed using DNA extracted from the primary leukemia cells. The aCGH analysis confirmed the deletion of the short arm of chromosome 7 identified by RHG-banding. However, the revised lost locus was 7p14.1p21.1 and 7p22.3 rather than 7p1?3p2?. The size was 18.8Mb and 601Kb, respectively. In addition to the large-scale chromosome aberrations, aCGH revealed cryptic genomic gains and losses not visible by karyotype. In JIH-3, the range of deletion was from 7p11.1 to 7p22.3(53Mb). The regions of genomic copy number changes harbor genes that maybe play an important role in the development and progression of AML. 119 of 138 genes and 302 of 366 genes were in regions of genomic copy number changes on 7p in primary leukemia cells and in JIH-3, respectively.Conclusions1. To report an acute myelomonocytic leukemia (AML-M4) patient with del(7)(p13p22) in the first relapse. The comprehensive genetic studies were performed using RHG-banding, FISH, multiplex RT-PCR and CP. The results of these studies disclosed this case to be an acute myelomonocytic leukemia with del(7p).2. A human acute myelomonocytic leukemia cell line, JIH-3, was established from an AML-M4 patient. The morphology and immunoprofile of the cell line presented typical features of myelocytic and monocytic lineage. The results of RHG-banding and CP confirmed the existence of del(7p). No rearrangement of CBFβand PML gene were disclosed by FISH. No cryptic rearrangement and fusion genes were revealed by M-FISH and multiplex RT-PCR. No gene mutation of KIT、P53、FLT3、JAK2、NPM1、CEBPA、RUNX1 and WT1 were found by sequencing of the PCR products. EBV genomic DNA and mycoplasma contamination were not found. The clonality of the JIH-3 cell line was confirmed by semi-solid methylcellulose clonogeneic culture. Cell line authentication by STR-PCR showed that the primary leukemia cells of patient and JIH-3 cell line originate from the same individual.3. The JIH-3 cell line has high tumorigenicity in nude mice.4. Array CGH is a powerful technique for detection of genomic copy number changes. The deletion of the short arm of chromosome 7 was confirmed and the lost locus was revised to 7p14.1p21.1 and 7p22.3 in primary leukemia cells. The del(7)(p11.1 p22.3) was also confirmed in JIH-3 by aCGH. And cryptic genomic gains and losses not visible by karyotype were revealed by aCGH. 2. Establishment and characterization of an new CML-BC cell line (JIH-6) MethodsThe chronic myeloid leukemia(CML)- derived leukemia cell line, JIH-6 was established from the peripheral blood of a patient in myeloid blast crisis of CML. There are two parts in this chapter.In the first part, we performed comprehensive genetic characterization on the CML patient in myeloid blast crisis. Conventional RHG banding was emploryed for karyotypic analysis. Fluorescence in situ hybridization (FISH) was used to detect the rearrangement of BCR/ABL fusion gene, AML1 and EVI1 genes. the BCR/ABL fusion transcript was detected by reverse transcriptase poiymerase chain reaction (RT-PCR). The fusion transcript of BCR/ABL gene was amplified by PCR and sequenced to exclude the mutation. The immunoprofile were studied by flow cytometry (FCM). The specimens used in all studies were from peripheral blood.In the second part, the process of establishment of the JIH-3 cell line was described and the basic biologic features of JIH-6 were characterized. The growth feature of the JIH-6 cell line in the 24-well plate was studied under the inverted microscope. The cell line was routinely stained with Wright’s staining method. Cytochemical studies were performed by peroxidase staining. The growth curve of the JIH-6 was obtained by incubating the cells in 24-well plate and the doubling time was calculated from the curve. The immunoprofile and the cell cycle distribution of JIH-6 were studied by flow cytometry (FCM). Quantitative fluorescent polymerase chain reaction was used to detect EBV genomic DNA. DNA-PCR was performed to determine the contamination of mycoplasma. RHG banding was employed for karyotypic analysis in the process of establishment of the cell line. FISH was used to identify the rearrangement of BCR/ABL fusion gene、AML1 and EVI1 gene. the BCR/ABL fusion transcript was detected by RT-PCR. The fusion transcript of BCR/ABL gene was amplified by PCR and sequenced to exclude the mutation. MDS1-EVI1/EVI1 gene expression was revealed by quantitative real-time RT-PCR in JIH-6、JIH-3、JIH-4、SHI-1、NB4 and HL-60 cell lines. Cell line authentication was performed by short tandem repeating sequences-PCR (STR-PCR).Results1. The cytogenetic analysis of the CML patient’bone marrow (BM) cells and peripheral blood showed 46, XX, t(9;22) at presentation and 46,XX, der(3)?inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11) in myeloid blast crisis, respectively. The BCR/ABL fusion gene was confirmed by FISH. The inversion of chromosome 3 was confirmed by FISH using dual-color EVI1 gene probe and EVI1 gene amplification was showed in some cells. The rearrangement of AML1 gene resulted from the translocation between chromosome 4 and 21 was cinfirmed by FISH using the probes of AML1/ETO and AML1, respectively. AML1 gene amplification was also showed in some cells. At the time of blast crisis, the BCR/ABL/ABL ratio was 12301/10000abl. A point mutation of T G of the ABL gene was found by sequencing of the PCR products. A leukemia marker study from peripheral blasts demonstrated that the blasts expressed antigens of myeloid and monocytic lineage. The diagnosis of CML in myelomonocytic blast crisis was made.2. The JIH-6 cell line could be cultured in IMDM medium with 15% fetal calf serum (FBS) without any cytokine, and has been maintained in continuous culture for more than eight months. It grows as single cells in suspension in the 24-well plate. JIH-6 cells remain stable proliferation after repeated freezing and thawing and has doubling time of about 118.2 hours. Most of the cells showed a regular cell shape, intermediate size, folded nucleus with prominent nucleoli, finely reticular chromatin, and blue-grey cytoplasm with azurophilic granules. JIH-6 cells were positive for peroxide staining. The results of immunoprofile showed that the JIH-6 cell line expressed myeloid and monocytic antigens. A few markers of T、B lymphocytic lineage and stem cell were also presented. Cell cycle analysis showed 24.78% of cells in S phase, 73.29% in G1 phase and 1.92% in G2 phase. JIH-6 showed a slow proliferation. And the karyotypic analysis only was performed on January 5 of 2010 and the result was 46,XX, inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11). Thus, the chromosome abnormalities of JIH-6 were the same as the patient’s leukemia cells. EBV genomic DNA and mycoplasma contamination were not revealed. The rearrangement and amplification of EVI1 gene were confirmed by FISH using dual-color EVI1 probe. The rearrangement and amplification of AML1 gene were also confirmed by FISH using AML1/ETO and AML1 dual-color probe. BCR/ABL fusion gene was detected by FISH and RT-PCR, respectively. The overexpression of EVI1 gene was revealed by quantitative real-time RT-PCR in JIH-6、JIH-3、JIH-4、SHI-1、NB4 and HL-60 cell lines.. Cell line authentication by STR-PCR showed that the primary leukemia cells of the patient and the JIH-6 cell line originated from the same individual.Conclusions1. To report an chronic myeloid leukemia (CML) patient in myelomonocytic blast crisis with inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11). The comprehensive genetic studies were performed using RHG-banding, FISH, RT-PCR and sequencing of PCR products. The results of these studies disclosed this case to be an CML in myelomonocytic blast crisis with a t(9;22) which resulted in BCR/ABL rearrangement, inv(3)(q21q26) which resulted in EVI1 gene rearrangement and amplification, and t(4;21) which resulted in AML1 gene rearrangement and amplification and a point mutation of ABL gene. Both are associated with her poor prognosis.2. A human CML with t(9;22) cell line, JIH-6, was established from an CML patient in myelomonocytic blast crisis. The morphology and immunoprofile of the cell line presented typical features of myelomonocytic lineage. The results of RHG-banding showed the existence of inv(3)(q21q26), t(4;21)(q21;q22),der(7), t(9;22)(q34;q11). The rearrangement of BCR/ABL were disclosed by FISH and PCR. The rearrangement and amplification of EVI1 and AML1 genes were revealed by FISH. No gene mutation of ABL were found by sequencing of the PCR products. EBV genomic DNA and mycoplasma contamination were not found. Cell line authentication by STR-PCR showed that the primary leukemia cells of patient and JIH-6 cell line originated from the same individual.In summary, JIH-3 and JIH-6 are two new myelomonocytic leukemia cell lines. the karyotype of JIH-3 is del(7p), and the JIH-6 is t(9;22), inv(3)(q21q26) and t(4;21)(q21;q22). They have clear biologic background and provides two new important tools for leukemia research.更多还原