急性单核细胞白血病细胞系THP-1侧群细胞的分选、鉴定与富集  

Sorting, identification and enrichment of side population cells in acute monocytic leukemia THP-1 Cells

作　　者：王颖超[1] 马丽娜[1] 殷楚云[1] 冯磊[1] 魏永纬[1] 盛光耀[1] 

机构地区：[1]郑州大学第一附属医院儿科,450052

出　　处：《中华实用儿科临床杂志》2013年第9期699-703,共5页Chinese Journal of Applied Clinical Pediatrics

摘　　要：目的探讨人急性单核细胞白血病细胞系THP-1中是否存在侧群（SP）细胞，鉴定其是否具有白血病干细胞（LSCs）的生物学特性，并研究从THP-1细胞系中富集sP细胞的方法。方法THP-1细胞用Hoechst33342荧光染料染色后，分别应用荧光显微镜和流式细胞术（FCM）检测是否存在sP细胞及sP细胞的形态和比例。分选sP和非侧群（NSP）细胞，FCM检测其表面抗原表达及细胞周期；实时荧光定量PCR检测耐药基因ABCG2、ABCBl和凋亡基因Bcl-2、Bax在两亚群细胞中的表达差异。将THP-1细胞用不同水平阿糖胞苷（Ara—C）处理24h，染色后检测sP细胞比例的变化。结果1．荧光显微镜观察到在THP-1细胞中存在拒染的sP细胞，形态与NSP细胞无明显区别。FCM检测结果显示，试验管sP细胞比例为（1．81-4-0．99）％，对照管可被维拉帕米完全拮抗。2．FCM分选sP细胞的纯度达到（96．71±0．90）％；SP细胞大部分处于静止期，G0／G1期细胞比例达（84．04±4．98）％，而NSP细胞则处于增殖期，S／G2／M期细胞比例为（56．38±1．48）％；CD34＋、CD34＋CD％一细胞在sP亚群中的比例要高于NSP亚群（P〈0．05）；SP细胞ABCG2及ABCBl表达水平显著高于NSP细胞（P均〈0．05）。SP细胞Bcl-2表达水平（0．99±0．08）显著高于NSP细胞（0．29±0．13）（P〈0．05），而Bax表达值（0．68±0．05）与NSP细胞（0．85±0．09）表达水平差异无统计学意义（P〉0．05），但Bcl-2／Bax比值显著高于NSP细胞亚群（P〈0．05）。3．经与Ara-C共培养24h后，sP细胞的比例显著提高，且随着Ara．C水平增大，sP细胞比例也越高。结论人急性单核细胞白血病细胞系THP-1中存在少量sP细胞，较之NSP细胞更具有干细胞的生物学活性。与一定水平Ara—C共培养可有效富集THP-1细胞系中的sP细胞，可作为LSCs研究的切入点，对进一步研究白血病有重要意义。Objective To assay and determine whether the human acute monocytic leukemia cell line THP-1 contains side populations （SP） cells ,and to increase the proportion of SP cells using cytarabine （Ara-C）. Methods Fluorescent microscope and flow cytometry （FCM） were employed for detecting the percentage of SP cells in THP-1 cells. Then,SP and non-SP （NSP） subpopulations were collected and identified. Finally, THP-1 cells were incubated with different concentrations of Ara-C for 24 hours and detected the proportion of SP cells, respectively. Results The results demonstrated that the percentage of SP cells was （ 1.810±0.99） % in THP-1 cells. A majority of the SP cells remained in the G0/G1 phase, and the expressions of CD34＋ and CD34＋ CD38 - and the proliferative ability of the SP cells were higher than those of NSP cells （ P 〈 0.05 ）. The mRNA expression of multidrug resistance genes （ ABCG2, ABCB1 ）, apoptosis regulation genes （ Bcl-2 ） and the Bcl-2/Bax ratio of SP ceils were higher than those of NSP cells. SP cells have been shown to be more tumorigenic than NSP cells. After co-culture with Ara-C, the proportion of SP cells increased significantly and presented in a concentration-dependent manor. Conclusions All of these findings suggest that the THP-1 cell line contains SP cells and the SP cells possess some intrinsic stem cell properties. The proportion of SP cells can be increased when co-cultured with Ara C,and this technique is a useful and important application for the study of LSCs.

关 键 词：THP-1细胞 白血病干细胞 侧群细胞 阿糖胞苷 

分 类 号：R733.7[医药卫生—肿瘤]