多发性骨髓瘤细胞株PRIM8226侧群细胞分选及其生物学特性的研究

The separation of side population cells in multiple myeloma cell line PRIM8226 and their biological characteristics

目的 检测和分选多发性骨髓瘤（MM）细胞株PRIM8226中的侧群细胞（SP细胞）并鉴定其生物学特性.方法 以Hoechst33342/碘化丙啶（PI）荧光染料双染,维拉帕米拮抗对照,应用流式细胞术荧光激活分选法检测并分选MM细胞株PRIM8226 SP细胞,并通过细胞生长曲线、细胞周期、免疫表型、集落形成实验、RT-PCR检测干细胞特异标志物mRNA表达量、裸鼠体内成瘤实验等对SP细胞的生物学特性进行初步探讨.结果 MM细胞株PRIM8226 SP细胞含量为（1.78±0.89）％,采用流式细胞术成功分选SP细胞.生长曲线显示：SP细胞分选初期生长较主群细胞（MP细胞）缓慢,进入稳定增长期后增殖能力与MP细胞差异无统计学意义（P＞0.05）.细胞周期分析显示：SP细胞与MP细胞相比,周期多处于Go/G1期[（44.34±3.09）％、（28.49±1.97）％,P＜0.05],较少的细胞处于S期[（38.83±3.69）％、（51.49±4.62）％,P＜ 0.05].在免疫表型研究中,观察到SP和MP细胞的CD138、CD38表达分别为（78.5±8.5）％、（82.0±4.0）％和（72.3±15.7）％、（84.3±11.9）％,差异均无统计学意义（均P＞0.05）.MM细胞株PRIM8226 SP细胞的单细胞克隆直径、克隆形成数、克隆形成率均高于MP细胞[0.280±0.016和0.118±0.019、1 722±127和358±14、（86.1±3.46）％和（17.9±1.88）％,P＜0.05].RT-PCR显示SP细胞干细胞标志性基因的表达高于MP细胞：c-myc[（29.90±3.73）％、（16.84±2.35）％]、KIF4[（29.97±2.89）％、（19.06±1.23）％]、SOX2[（40.00±4.58）％、（16.62±2.09）％]、OCT4[（32.96±1.56）％、（23.27±0.92）％]（均P＜0.05）.裸鼠体内成瘤实验显示SP细胞成瘤能力显著高于MP细胞（最低成瘤数量分别为5×103、5×105个）.结论 MM细胞株PRIM8226的SP细胞在静止期细胞比例、集落形成能力、干细胞标记c-myc、KIF4、SOX2、OCT4基因表达量、体内成瘤能力上与MP细胞差异均有统计学意义,而SP细胞和MP细胞在体外长期增殖能力、CD38和CD138的表达上差异均无统计学意义.

Objective To detect and separate the side population cells（SP） from multiple myeloma （MM） cell lines PRIM8226,and to study their biological characteristics.Methods Fluorescence activated cell sorter （FACS） and Hoechst33342/PI dye were used to sort SP cells of PRIM8226.The multiplication capacity was tested by the growth curve and MTT test,SP cells proportion and the cell cycle were analyzed by flow cytometry,the colony-formtion ability was compared in terms of colony forming experiment,the expression of c-myc,KIF4,SOX2,OCT4 was tested by RT-PCR.The oncogenicity of the cells was analyzed by nude mouse transplantation tumor experiment in vivo.Results The ratio of SP cells in PRIM8226 was （1.78±0.89） %.More SP cells in the G0 / G1 period,（44.34±3.09） % vs （28.49±1.97） %,P 〈 0.05,and fewer cells in S phase than MP cells,（38.83±3.69） % vs （51.49±4.62） %,P 〈 0.05.There were no difference in the expression of CD38 and CD138 between SP cells and MP cells,respectively,（78.5±8.5） % vs （82.0±4.0） % and （72.3±15.7） % vs （84.3±11.9） %,P 〉 0.05.Colony formation assay showed that the colony forming efficiency of the SP cells was higher than the MP cells,single cell clone diameter,the number of clone forming,the clone formtion rate were significantly higher than MP cells,0.280±0.016 vs 0.118±0.019,1 722±127 vs 358±14,（86.1±3.46） % vs （17.9±1.88） %,P 〈 0.05.The mRNA expression levels of c-myc,KIF4,SOX2,OCT4 in SP cells were higher than those in MP cells,c-myc （29.90±3.73） % vs （16.84±2.35） %,KIF4 （29.97±2.89） % vs （19.06±1.23） %,SOX2 （40.00±4.58） % vs （16.62±2.09） %,OCT4 （32.96±0.92） % vs （23.27±0.92） %,all P 〈 0.05.Nude mouse transplantation tumor experiment in vivo showed the oncogenicity of the SP cells was more stronger than MP cells （5×103 vs 5×l05）.Conclusion There are notably difference in the cell cycle,colony formation assay,the mRNA expression levels and oncogenicity,but no difference in the expression of CD38 and CD138,the proliferation ability between SP cells and MP cells.