白血病·淋巴瘤
白血病·淋巴瘤
백혈병·림파류
JOURNAL OF LEUKEMIA & LYMPHOMA
2014年
8期
460-464
,共5页
多发性骨髓瘤%肿瘤干细胞%侧群细胞
多髮性骨髓瘤%腫瘤榦細胞%側群細胞
다발성골수류%종류간세포%측군세포
Multiple myeloma%Cancer stem cells%Side population cells
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.
目的 檢測和分選多髮性骨髓瘤(MM)細胞株PRIM8226中的側群細胞(SP細胞)併鑒定其生物學特性.方法 以Hoechst33342/碘化丙啶(PI)熒光染料雙染,維拉帕米拮抗對照,應用流式細胞術熒光激活分選法檢測併分選MM細胞株PRIM8226 SP細胞,併通過細胞生長麯線、細胞週期、免疫錶型、集落形成實驗、RT-PCR檢測榦細胞特異標誌物mRNA錶達量、裸鼠體內成瘤實驗等對SP細胞的生物學特性進行初步探討.結果 MM細胞株PRIM8226 SP細胞含量為(1.78±0.89)％,採用流式細胞術成功分選SP細胞.生長麯線顯示:SP細胞分選初期生長較主群細胞(MP細胞)緩慢,進入穩定增長期後增殖能力與MP細胞差異無統計學意義(P＞0.05).細胞週期分析顯示:SP細胞與MP細胞相比,週期多處于Go/G1期[(44.34±3.09)％、(28.49±1.97)％,P＜0.05],較少的細胞處于S期[(38.83±3.69)％、(51.49±4.62)％,P＜ 0.05].在免疫錶型研究中,觀察到SP和MP細胞的CD138、CD38錶達分彆為(78.5±8.5)％、(82.0±4.0)％和(72.3±15.7)％、(84.3±11.9)％,差異均無統計學意義(均P＞0.05).MM細胞株PRIM8226 SP細胞的單細胞剋隆直徑、剋隆形成數、剋隆形成率均高于MP細胞[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顯示SP細胞榦細胞標誌性基因的錶達高于MP細胞: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).裸鼠體內成瘤實驗顯示SP細胞成瘤能力顯著高于MP細胞(最低成瘤數量分彆為5×103、5×105箇).結論 MM細胞株PRIM8226的SP細胞在靜止期細胞比例、集落形成能力、榦細胞標記c-myc、KIF4、SOX2、OCT4基因錶達量、體內成瘤能力上與MP細胞差異均有統計學意義,而SP細胞和MP細胞在體外長期增殖能力、CD38和CD138的錶達上差異均無統計學意義.
목적 검측화분선다발성골수류(MM)세포주PRIM8226중적측군세포(SP세포)병감정기생물학특성.방법 이Hoechst33342/전화병정(PI)형광염료쌍염,유랍파미길항대조,응용류식세포술형광격활분선법검측병분선MM세포주PRIM8226 SP세포,병통과세포생장곡선、세포주기、면역표형、집락형성실험、RT-PCR검측간세포특이표지물mRNA표체량、라서체내성류실험등대SP세포적생물학특성진행초보탐토.결과 MM세포주PRIM8226 SP세포함량위(1.78±0.89)％,채용류식세포술성공분선SP세포.생장곡선현시:SP세포분선초기생장교주군세포(MP세포)완만,진입은정증장기후증식능력여MP세포차이무통계학의의(P＞0.05).세포주기분석현시:SP세포여MP세포상비,주기다처우Go/G1기[(44.34±3.09)％、(28.49±1.97)％,P＜0.05],교소적세포처우S기[(38.83±3.69)％、(51.49±4.62)％,P＜ 0.05].재면역표형연구중,관찰도SP화MP세포적CD138、CD38표체분별위(78.5±8.5)％、(82.0±4.0)％화(72.3±15.7)％、(84.3±11.9)％,차이균무통계학의의(균P＞0.05).MM세포주PRIM8226 SP세포적단세포극륭직경、극륭형성수、극륭형성솔균고우MP세포[0.280±0.016화0.118±0.019、1 722±127화358±14、(86.1±3.46)％화(17.9±1.88)％,P＜0.05].RT-PCR현시SP세포간세포표지성기인적표체고우MP세포: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)％](균P＜0.05).라서체내성류실험현시SP세포성류능력현저고우MP세포(최저성류수량분별위5×103、5×105개).결론 MM세포주PRIM8226적SP세포재정지기세포비례、집락형성능력、간세포표기c-myc、KIF4、SOX2、OCT4기인표체량、체내성류능력상여MP세포차이균유통계학의의,이SP세포화MP세포재체외장기증식능력、CD38화CD138적표체상차이균무통계학의의.
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.
