人结直肠癌干细胞休眠与增殖阶段细胞形态学的研究

目的初步研究人结直肠癌干细胞休眠与增殖阶段细胞形态的变化。方法流式细胞仪从新鲜人结直肠癌组织中分选EpCAMhigh／CD44＋／CD133＋细胞亚群，并通过裸鼠（NOD／SCID）成瘤实验鉴定其干细胞特性：采用三维培养，WST．1绘制人结直肠癌干细胞的生长曲线；流式细胞仪测定P27及Ki一67的表达水平，区分人结直肠癌干细胞休眠和增殖时相；细胞免疫荧光显示休眠期与增殖期人结直肠癌干细胞形态的变化。结果EpCAMhigh／CD44＋／CDl33‘细胞亚群在人结直肠癌组织中占1．6％．经NOD／SCID成瘤实验证实为人结直肠癌干细胞。人结直肠癌干细胞生长曲线呈“S”型；前3d生长缓慢，细胞为静止阶段（休眠期），P27表达水平逐渐增加，Ki．67表达偏低；从第4d开始，细胞进入增殖期，Ki-67表达逐渐增加，P27表达降低。细胞免疫荧光染色显示：休眠期人结直肠癌干细胞圆而大，伪足少；而增殖期细胞伪足增多，呈现增殖和侵袭的状态。结论肿瘤的复发、转移可能与肿瘤干细胞的生长状态发生改变有一定关系；人结直肠癌干细胞增殖期与休眠期相比。呈现出明显增殖和侵袭能力：这为治疗人结直肠癌及其他肿瘤的复发和转移提供了新的切人点。

Cell morphology in the dormancy and proliferation stage of colorectal cancer stem cells

Objective To study the cell morphology change in dormancy and proliferation stage of colorectal cancer stem ceils in order to provide reference to the treatment of colorectal cancer. Methods The subpopulation of EpCAM~/CD44＋/CD133 ~ was isolated from fresh colorectal cancer tissues. These cells were tested by xenograft assay in NOD/SCID nude mice. Coloreetal cancer stem cells underwent three-dimensional culture, and the growth curve of stem ceils was drawn by WST-1. The expression of P27 and Ki-67 was examined by flow cytometry to understand the phase of dormancy and proliferation of colorectal cancer stem cells. Then the morphological differences of eolorectal cancer stem cells between dormant and proliferation stages were recognized by immunofluorescence staining of actin. Results The percentage of EpCAM~sh/CD44~/CD133 ~ was 1.6%, and the subpopulation was confirmed to be colorectal cancer stem cells by means of the experiment of tumorigenicity in vivo. The growth curve of colorectal cancer stein cells was ＂S＂ type. Colorectal cancer stem cells grew slowly in the first three days. The expression of P27 was gradually up-regulated, and the level of Ki-67 was very low. These cells remained quiescence, which was the so-called dormancy. The expression of Ki-67 of colorectal cancer stem cells was at high level since the fourth day, and the P27 level was very low. According to the growth curve, this period belonged to the proliferative stage of colorectal cancer stem cells. On immunofluorescence staining, colorectal cancer stem cells with high level of P27 were round, large, and few pseudopodium, but no obvious death was found. These cells showed characteristics of dormancy. In contrast, the stem cells with high level of Ki-67 had much pseudopodium, showing proliferation and invasion. Conclusions Cancer recurrence and metastasis may be associated with the change of growth state of cancer stem cells. Colorectal cancer stem cells in the proliferation stage show greater proliferative and invasive ability as compared to the dormancy stage, which provides a new perspective for the treatment of colorectal cancer, and recurrence and metastasis of other tumors.