多发性骨髓瘤侧群细胞分选及其生物学特征分析  

Sorting of Side Population Cells from Multiple Myeloma Cell Lines and Analysis of Their Biological Characteristics

作　　者：张晓丽[1] 张丽娜[1] 黄红铭[1] 丁润生[1] 施维[2] 徐瑞容[1] 于晓棠[1] 姜胜华[1] 

机构地区：[1]江苏省南通大学附属医院血液科,江苏南通226001 [2]江苏省南通大学附属医院外科综合实验室,江苏南通226001

出　　处：《中国实验血液学杂志》2014年第3期747-752,共6页Journal of Experimental Hematology

基　　金：江苏省自然科学基金(BK2011388);江苏省六大人才高峰资助计划(2010-WS-037)

摘　　要：本研究旨在从人多发性骨髓瘤细胞株中分选出侧群(SP)细胞并对其生物学特性进行分析。细胞经Hoechst33342染色后,应用荧光显微镜观察细胞内Hoechst荧光染色情况,采用流式细胞荧光激活分选(FACS)技术,将细胞分选为SP细胞与主群(main population,MP)细胞,用细胞生长曲线法比较两群细胞的体外增殖能力、集落形成试验比较细胞集落形成能力、流式细胞术分析两群细胞CD138的表达情况,应用RT-PCR法检测ABCG2mRNA在两群细胞中的表达不平、硼替佐米对CCK-8法和集落形成试验检测对两群细胞增殖、活力和细胞集落形成能力的影响。结果表明,骨髓瘤细胞株中存在小部分比例的SP细胞,RPMI 8226细胞株中SP细胞高达(7.10±2.69)%,在荧光显微镜下也检测到SP细胞;SP细胞增殖活力、细胞集落形成能力明显高于MP细胞(P<0.05);该基因SP和MP细胞的CD138表达率无明显差异(P>0.05);SP细胞表达耐药基因ABCG2,而MP细胞几乎不表达该基因;SP细胞的硼替佐米抑制率明显低于MP细胞(P<0.05),但在40 nmol/L作用下,两者抑制率未显示差异有显著统计学意义(P>0.05);硼替佐米均能降低两群细胞的集落形成能力,但MP细胞降低更为明显。结论:骨髓瘤细胞系中存在少量SP细胞,具有肿瘤干细胞的相关生物学特性。This study was aimed to sort the side population （SP） cells from human multiple myeloma cell lines, then detect the biological characteristics of those SP cells. After Hoechst33342 staining, intracellular Hoechst33342 fluorescence staining differences of myeloma cell lines observed by the fluorescence microscopy. The fluorescenceactivated cell sorting （FACS） technology was used to isolate SP cells and main pupolation （MP） cells; proliferative capacity in vitro was determined by cell growth curve; the cell colony forming ability was compared by colony forming test. The CD138 expression was detected by flow cytometry. The expression of ABCG2 mRNA was detected by reverse transcription PCR; CCK-8 assay and colony forming test were used to evaluate the effect of bortezomib on the cell proliferation, vitality and colony forming ability of the two populations. The results showed that the myeloma cell lines had a small proportion of SP cells, especially, RPMI 8226 cells accounted for the highest proportion of SP cells （7.10 ± 2.69） %, which have also been confirmed under the fluorescence microscope; the proliferative activity and cell colony forming ability of SP cells were significantly higher than those of MP cells （ P 〈 0.05 ）. The expression levels of CD138 in SP and MP cells were not significantly different （ P 〉 0.05 ）. RT-PCR results showed that SP cells expressed the drugresistance gene ABCG2, but MP cells hardly express these genes. The inhibition rate of bortezomib on SP cells was significantly lower than that on MP cells （ P 〈 0.05 ）, however, the difference was not significant （ P 〉 0.05 ） at bortezomib 40 nmol/L. Bortezomib could reduce colony formation in the both two cell populations, but more severe reduction appeared in the MP cells. It is concluded that the myeloma cell line contain a small amount of SP cells with the cancer stem cell characteristics.

关 键 词：多发性骨髓瘤肿瘤干细胞 侧群细胞 主群细胞 

分 类 号：R733.3[医药卫生—肿瘤]