中文摘要：

目的观察结直肠癌中Ki-67阴性（Ki-67^-/low）细胞在肿瘤增殖及自我更新（self-renew）中的作用,探讨将Ki-67-/low作为分离肿瘤干细胞标记物的应用。方法通过将人结直肠癌标本消化成为单细胞,感染Ki-67启动子驱动GFP表达（pKi-67-GFP）的慢病毒后,注射至NOD/SCID小鼠背部建立pKi-67-GFP结直肠癌移植瘤模型;用流式细胞仪从移植瘤内分离出GFP＋EpCAM＋细胞（Ki-67＋癌上皮细胞）和GFP^-/lowEpCAM＋细胞（Ki-67^-/low癌上皮细胞）,并采用免疫印迹验证Ki-67的表达;用流式细胞仪检测Ki-67-/low的细胞周期;采用PKH26标记细胞后,根据滞留细胞分析其在体内的增殖能力;采用荧光定量PCR（qPCR）研究Ki-67细胞中CD133、CD44和Lgr5等肿瘤干细胞相关基因的表达水平,并用成球实验研究Ki-67^-/low结直肠癌细胞的自我更新能力。结果从新鲜人结直肠癌移植瘤中分离出GFP＋EpCAM＋细胞和GFP^-/lowEpCAM＋细胞,Western blot检测显示GFP＋EpCAM＋细胞高表达Ki-67,GFP^-/lowEpCAM＋细胞不表达Ki-67;细胞周期检测显示,（82.25±3.16）%的Ki^-67-/low细胞在G0/G1期,（51.67±4.11）%的Ki-67^＋细胞在G0/G1期,体内PKH26标记滞留实验显示Ki-67-/low和Ki-67＋细胞分别含（81.53±5.85）%和（4.57±1.05）%PKH26阳性细胞,差异有统计学意义（P〈0.01）,提示Ki-67-/low细胞主要是G0/G1期细胞,在体内处于慢增殖状态;荧光定量PCR证明Ki-67^-/low细胞CD133、CD44和Lgr5的mRNA水平为Ki-67＋细胞的（3.47±0.79）、（6.18±1.65）和（4.67±1.15）倍,差异有统计学意义（P〈0.05）,体外成球实验,Ki-67^-/low及Ki-67＋细胞的成球数量分别为（83.00±9.54）和（8.00±2.66）（P〈0.01）,提示Ki-67^-/low细胞高表达CD133、CD44和Lgr5等肿瘤干细胞相关标记物,并具有显著的自我更新能力,富集肿瘤干细胞。结论 Ki-67阴性的结直肠癌细胞具有慢增殖的特点,较Ki-67阳性结直肠癌细胞具有更强的成球能力,在结直

英文摘要：

Objective To investigate the proliferation and self-renewal capacity of Ki-67^-/low cells in colorectal cancer,and preliminarily discuss its function as a cancer stem cell marker.Methods Tumor specimens were obtained from patients with colorectal cancer（CRC）and were digested to single cell suspension.Cells infected by Ki-67promoter-driven GFP（pKi-67-GFP）lentivirus were then implanted into the back of NOD/SCID mice to establish models of pKi-67-GFP xenograft tumors.GFP＋EpCAM＋cells（Ki-67^＋cancer cells）and GFP-/lowEpCAM＋cells（Ki-67^-/lowcancer cells）were purified by flow cytometry（FCM）and Ki-67 levels were evaluated by Western blot.Cell-cycle analysis of Ki-67^-/lowcancer cells was performed by FCM,and cell proliferationin vivo was analyzed by PKH26 label retaining experiments.The mRNA levels of CD133,CD44 and Lgr5that are related to cancer stem cells were measured by qPCR and self-renewal ability was evaluated by sphere formation assay.Results Western blot showed that the purified GFP＋EpCAM＋cells were positive for Ki-67,whereas GFP^-/lowEpCAM＋cells negatively or lowly expressed Ki-67.Cell-cycle analysis revealed（82.25±3.16）% Ki-67^-/lowcells at G0/G1 phase and（51.67±4.11）% Ki-67＋cells at G0/G1 phase.PKH26 label retaining experiments in vivo showed that Ki-67^-/low cells contained（81.53±5.85）% PKH26＋cells while only（4.57±1.05）% Ki-67＋cells were positive for PHK26（P0.05）,suggesting that Ki^-67-/low cells were much more quiescent in vivo.qPCR revealed that the mRNA levels of CD133,CD44 and Lgr5in Ki-67-/low cells were（3.47±0.79）,（6.18±1.65）and（4.67±1.15）times that of those of Ki-67＋cells（P0.01）.Sphere formation assay revealed that Ki^-67-/low could generate（83.00±9.54）spheres,whereas Ki-67＋cells could generate only（8.00±2.66）spheres（P0.01）.ConclusionKi^-67-/lowcolorectal cancer cells slowly proliferate and they have higher capacity of sphere formation and self-renewal than Ki-67＋cells,suggesting that Ki-67^-/lowcan b