CAJ | 학술논문

目的探讨人胃癌细胞CD133+亚群的分选及其特性的鉴定.方法对50例胃癌原发灶和癌旁胃黏膜组织标本行免疫组织化学染色及Western blot检测CD133蛋白的表达.采用流式细胞仪检测不同分化胃癌细胞系CD133所占百分比,免疫磁珠法分选CD133+细胞亚群,悬浮培养并观察其生长特性,并检测CD133阳性细胞裸鼠皮下接种致瘤能力.单细胞克隆观察单个CD133+细胞生长特性,半定量反转录聚合酶链反应法鉴定相关干细胞标记的表达.结果 CD133蛋白多定位于胃癌原发灶黏膜及黏膜下层的肿瘤细胞膜表面,其相对表达量高于癌旁胃黏膜组织(P＜0.05).不同分化程度的人胃癌细胞系KATO-Ⅲ、SGC-7901、AGS及MKN-45中CD 133+亚群所占相对百分比为(28±2)％、(17±2)％、(6±2)％及(4±2)％.人胃癌细胞系KATO-Ⅲ分选后阳性组中CD133+亚群比例分别为(91±3)％；培养1周后,达到(95±2)％,并不断增殖形成细胞球.而且其增殖能力强于阴性细胞[群体倍增时间分别为(21±3)h和(40±8)h,P＜0.05].CD133阳性组和未分选组细胞裸鼠皮下接种时成瘤率分别为100％和80％；而CD133阴性组不成瘤,CD133阳性组移植瘤平均体积及重量均大于未分选组(P＜0.05,P＜0.05).单克隆形成实验示单个CD133+细胞可形成新的细胞克隆.半定量RT-PCR检测示其表达干细胞标记物Oct-4、Nanog、Sox-2、Musashi-1及EGFR.结论体外可成功分离、纯化和扩增人胃癌细胞CD133+亚群,其具有自我更新、增殖能力及较强的致瘤能力,并表达部分干细胞相关基因.
목적 탐토인위암세포CD133+아군적분선급기특성적감정.방법 대50례위암원발조화암방위점막조직표본행면역조직화학염색급Western blot검측CD133단백적표체.채용류식세포의검측불동분화위암세포계CD133소점백분비,면역자주법분선CD133+세포아군,현부배양병관찰기생장특성,병검측CD133양성세포라서피하접충치류능력.단세포극륭관찰단개CD133+세포생장특성,반정량반전록취합매련반응법감정상관간세포표기적표체.결과 CD133단백다정위우위암원발조점막급점막하층적종류세포막표면,기상대표체량고우암방위점막조직(P＜0.05).불동분화정도적인위암세포계KATO-Ⅲ、SGC-7901、AGS급MKN-45중CD 133+아군소점상대백분비위(28±2)％、(17±2)％、(6±2)％급(4±2)％.인위암세포계KATO-Ⅲ분선후양성조중CD133+아군비례분별위(91±3)％；배양1주후,체도(95±2)％,병불단증식형성세포구.이차기증식능력강우음성세포[군체배증시간분별위(21±3)h화(40±8)h,P＜0.05].CD133양성조화미분선조세포라서피하접충시성류솔분별위100％화80％；이CD133음성조불성류,CD133양성조이식류평균체적급중량균대우미분선조(P＜0.05,P＜0.05).단극륭형성실험시단개CD133+세포가형성신적세포극륭.반정량RT-PCR검측시기표체간세포표기물Oct-4、Nanog、Sox-2、Musashi-1급EGFR.결론 체외가성공분리、순화화확증인위암세포CD133+아군,기구유자아경신、증식능력급교강적치류능력,병표체부분간세포상관기인.
Objective To sort CD133+ subset cells in human gastric cancer (GC) andto identify their tumor initiating cell-like properties.Methods 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 determin 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).Results 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.Conclusions 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.