摘要
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未缓解的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（J6；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型所取代.荧光原位杂交（FISH）和多色FISH证实了以上异常，并揭示-17导致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（CAG→CAT）。RT—PCR显示除了表达千细胞因子（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-π和p21。短串联重复序列PCR证明了SH-2细胞和患者白血病细胞的同源性。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。
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 obtain complete remission after chemotherapy and allogenie bone marrow transplantation were passed in a long term IMDM culture medium supplemented with 20% fetal calf sertim. Stromal cells were retained and rh-IL-3 was added in the culture system. A new human myeloid leukemia cell line SH-2 was successfully established with a cytogenetic 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. Various 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 decreased and replaced by the near-tetraploid cells with a karyotype of 73 - 102 （80） , XX, - Y, - Y, del （1q31） ×2, der（ 16）t（16;17） （q24;q12）×2, - 17, - 17, ＋ 19, ＋ 19. FISH and muhiple FISH delineated all the abnormalities and revealed a loss of one p53 allele due to monosomy 17. DNA direct sequencing detected 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. Conclusion SH-2 is a new myeloid leukemia cell line with a unique biology background, and will provide a useful tool for leukemia research.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2009年第7期458-463,共6页
Chinese Journal of Hematology
基金
基金项目：江苏省自然科学基金（BK2006.055）
卫生部基金（WKJ2005-2-025）
