一株新的急性髓系白血病细胞系SH-2的建立及其鉴定

目的 采用自体骨髓基质细胞与原代白血病细胞加rhIL-3后共培养的方法建立1株新的人急性髓系白血病(AML)细胞系SH-2,并对其生物学特征进行全面鉴定.方法 采集1例经联合化疗和异基因外周血干细胞移植末缓解的AML-M2a型患者骨髓,分离单个核细胞,加入含20%胎牛血清的IMDM培养基内置37℃、5%CO2、饱和湿度培养箱中传代培养,培养过程中保留自体骨髓基质细胞,同时加入细胞因子rhIL-3,长期体外培养成功建立伴有-Y,der(16)t(16;17)(q24;q12),-17,+19和p53突变的AML细胞系SH-2,并通过细胞学、遗传学、免疫学、分子学和小鼠致瘤实验等多种方法对SH-2细胞的生物学特征进行全面鉴定.结果 SH-2细胞已在体外持续生长3年余而不需加用生长因子和基质细胞.其EB病毒、支原体检测均为阴性.SH-2细胞具有和原代白血病细胞相同的髓系细胞形态学特点,伴有自然杀伤相关抗原表达的AML免疫表型(CD13+、CD33+、CD56+、CD16/56+)和45,X,-Y,der(16)t(16;17)(q24;q12),-17,+19的亚二倍体核型,后者逐渐被伴有上述染色体异常的近四倍体核犁所取代.荧光原位杂交(FISH)和多色FISH证实了以上异常,并揭示-17导敛1个p53基因丢失.DNA序列分析揭示另1个p53等位基因第5号外显子的576编码子点突变(CAC→CAT).RT-PCR显示除了表达于细胞因子(SCF)外,其余细胞因子均不表达;不表达多药耐药基因而表达凋亡相关基因,如bcl-2、Fas、GST-1T和p21.短串联重复序列PCR证明了SH-2细胞和患者门血病细胞的同源性.SH-2细胞有一定的集落形成能力并能在裸鼠皮下及SCID小鼠内脏成瘤.结论 SH-2是一株新的具有明晰生物学背景的AML细胞系,为白血病研究提供了又一有用工具.

Establishment and characterization of a new human myeloid leukemia cell line SH-2

Objective To establish and characterize a novel human myeloid leukemia cell line SH-2. Methods Bone marrow mononuclear cells(BMMNC) isolated from a AML-M2 patient, who failed to ob-tain complete remission after chemotherapy and allogenic bone marrow transplantation were passed in a long term IMDM culture medium supplemented with 20% fetal calf serum. Stromal cells were retained and rh-IL-3was added in the culture system. A new human myeloid leukemia cell line SH-2 was successfully established with a cytogeuetic characteristics of a loss of Y chromosome(- Y), a derivative chromosome 16 resulting from unbalanced translocation between chromosome 16 and 17, monosome 17, trisomy 19 and p53 alteration. Vari-ous methods were employed to characterize SH-2 cell line. Results SH-2 cells has been maintained without cytokine and stromal cells for more than 3 years without EB virus and mycoplasma contamination. SH-2 cells had the basically same morphological, immunophenotypic and cytogenetic features as the patient' s leukemia cells did, such as myeloid morphology, an immunophenotype of CD13+ , CD33+ , CD56+ , CD16/56+ and a hypodiploid karyotype of 45, X, - Y, der(16) t(16;17) (q24;q12) , - 17, + 19, which were gradually de-creased and replaced by the near-tetraploid cells with a karyotype of 73 - 102 (80), XX, - Y, - Y, del (lq31) ×2, der(16)t(16;17) (q24;q12) ×2, - 17, - 17, + 19, + 19. FISH and multiple FISH delineated all the abnormalities and revealed a loss of one p53 allele due to monosomy 17. DNA direct sequencing detec-ted a point mutation of CAG to CAT at codon 576 of exon 5 in another p53 allele. RT-PCR showed that SH-2 cells expressed apoptosis-related genes (bcl-2, Fas, GST- π and p21) rather than MDR-related genes. Short tandem repeat PCR provided powerful evidence for the derivation of SH-2 cell line from the patient' s leukemia cells. SH-2 cells had certain colony formation and tumorigenic capacities in nude and SCID mice. Conclu-sion SH-2 is a new myeloid leukemia cell line with a unique biology background, and will provide a useful tool for leukemia research.