Lu Rui-qi, Wu Ju-gang, Zhou Guo-cai, Jiang Hai-guang, Yu Ji-wei, Jiang Bo-jian
Department of General Surgery, Shanghai Jiaotong University School of Medicine, Shanghai, China.
Zhonghua Wei Chang Wai Ke Za Zhi. 2012 Feb;15(2):174-9.
To sort CD133(+) subset cells in human gastric cancer (GC) and to identify their tumor initiating cell-like properties.
The tissues of GC and normal tissues adjacent to GC were obtained from 50 patients. Samples were stained for CD133 by immunohistochemistry. Likewise, assessments of CD133 were undertaken by Western blot. Flow cytometry was used to determine the proportion of CD133(+) cells in four GC cell lines therein the KATO-III was sorted by magnetic activated cell sorting (MACS) method. The growing characteristics and the tumorigenic ability of CD133(+) cells were evaluated in vitro and in vivo. Meanwhile, the growth of single cells in suspension culture was observed and expression of stem cell-specific marker were determined using reverse transcription-polymerase chain reaction (RT-PCR).
The expression of CD133 was demonstrated on the cell membranes in the mucosa and submucosa of primary GC, which were higher than those in the normal gastric tissues adjacent to cancer (P<0.05). Four GC cell lines including KATO-III, SGC-7901, AGS and MKN-45 were found to contain (28 ± 2)%, (17 ± 2)%, (6 ± 2)%, and (4 ± 2)% of CD133(+) cells respectively. In addition, the purity of CD133(+) cells isolated from KATO-III by MACS was (91 ± 3)% and up to(95 ± 2)% after 1-week culture. CCK-8 detection showed that population doubling time of the CD133(+) cells was (21 ± 3)h, significantly shorter than that of the CD133(-) cells[(40 ± 8)h, P<0.05]. Notably, there was a remarkable difference of tumor formation rate between CD133(+) cells (100%), non-sorted cells (80%), and CD133(-) cells(0). The average mass and volume of tumor in group of CD133(+) cells was larger and heavier than those in non-sorted cells (P<0.05, P<0.05). Furthermore, the single cell proliferated well, formed the big sphere and semi-quantitative RT-PCR showed expression of stem cell markers such as Oct-4, Nanog, Sox-2, Musashi-1 and EGFR.
CD133 protein expression in primary lesions is higher than those in the normal gastric tissues. CD133(+) subset cells can be isolated, purified, and amplified in human GC, and possess some properties including the ability of self-renewal, proliferation, and higher tumorigenic ability in vivo and can express some stem cell markers.
分选人胃癌（GC）中的CD133(+)亚群细胞，并鉴定其肿瘤起始细胞样特性。
从50例患者获取GC组织及癌旁正常组织。样本采用免疫组织化学法进行CD133染色。同样，通过蛋白质免疫印迹法对CD133进行评估。使用流式细胞术测定四种GC细胞系中CD133(+)细胞的比例，其中KATO-III细胞系采用磁珠分选法（MACS）进行分选。在体外和体内评估CD133(+)细胞的生长特性和致瘤能力。同时，观察悬浮培养中单个细胞的生长情况，并采用逆转录-聚合酶链反应（RT-PCR）检测干细胞特异性标志物的表达。
原发性GC黏膜及黏膜下层细胞膜上可检测到CD133表达，其表达水平高于癌旁正常胃组织（P<0.05）。四种GC细胞系KATO-III、SGC-7901、AGS和MKN-45中CD133(+)细胞比例分别为(28±2)%、(17±2)%、(6±2)%和(4±2)%。此外，通过MACS从KATO-III细胞系中分离得到的CD133(+)细胞纯度为(91±3)%，培养1周后可达(95±2)%。CCK-8检测显示，CD133(+)细胞群体倍增时间为(21±3)小时，显著短于CD133(-)细胞[(40±8)小时，P<0.05]。值得注意的是，CD133(+)细胞（100%）、未分选细胞（80%）和CD133(-)细胞（0）的肿瘤形成率存在显著差异。CD133(+)细胞组肿瘤的平均质量和体积均大于未分选细胞组（P<0.05，P<0.05）。此外，单个细胞增殖良好，形成大的球体，半定量RT-PCR显示干细胞标志物Oct-4、Nanog、Sox-2、Musashi-1和EGFR表达。
原发性病变中CD133蛋白表达高于正常胃组织。CD133(+)亚群细胞可在人GC中分离、纯化和扩增，具有自我更新、增殖能力，在体内具有更高的致瘤能力，并可表达一些干细胞标志物。
