CAJ | 학술논문

目的：分离人卵巢癌细胞系SKOV3和原代卵巢癌细胞中的CD＋90细胞，并观察其肿瘤干细胞的生物学特性。方法从卵巢癌患者腹水中分离原代卵巢癌细胞，采用流式细胞术检测人卵巢癌细胞系SKOV3和原代卵巢癌细胞的CD133、CD90阳性率。流式分选得到CD＋90、CD－90细胞后，采用RT-PCR法检测其干细胞及上皮间质化（EMT）相关基因mRNA相对表达，Transwell小室侵袭试验观察细胞侵袭力，克隆形成试验观察细胞增殖分化能力，悬浮成球试验观察干细胞潜能，免疫缺陷小鼠体内有限稀释成瘤试验观察成瘤时间和成瘤率。结果人卵巢癌细胞系SKOV3的CD133和CD90阳性率均低于原代卵巢癌细胞，P均＜0．05。在人卵巢癌细胞系SKOV3和原代卵巢癌细胞中，CD＋90细胞干细胞相关基因（CD133、OCT4）和EMT间质标志相关基因（N-cadherin、Vimentine、MMP9）相对表达、穿到膜背面的细胞数、细胞克隆数、悬浮成球数均高于CD－90细胞，EMT上皮标志相关基因E-cadherin相对表达均低于CD－90细胞，P均＜0．05。随着接种细胞数目的增加，CD＋90、CD－90细胞的成瘤率和成瘤时间均升高，CD＋90细胞升高更明显。结论卵巢癌细胞中，CD＋90细胞高表达间质属性基因和干细胞相关基因，具备更高的侵袭力、增殖分化能力、体内成瘤能力和干细胞潜能，CD＋90细胞分离可能成为卵巢癌干细胞分离的新方法。
목적：분리인란소암세포계SKOV3화원대란소암세포중적CD＋90세포，병관찰기종류간세포적생물학특성。방법종란소암환자복수중분리원대란소암세포，채용류식세포술검측인란소암세포계SKOV3화원대란소암세포적CD133、CD90양성솔。류식분선득도CD＋90、CD－90세포후，채용RT-PCR법검측기간세포급상피간질화（EMT）상관기인mRNA상대표체，Transwell소실침습시험관찰세포침습력，극륭형성시험관찰세포증식분화능력，현부성구시험관찰간세포잠능，면역결함소서체내유한희석성류시험관찰성류시간화성류솔。결과인란소암세포계SKOV3적CD133화CD90양성솔균저우원대란소암세포，P균＜0．05。재인란소암세포계SKOV3화원대란소암세포중，CD＋90세포간세포상관기인（CD133、OCT4）화EMT간질표지상관기인（N-cadherin、Vimentine、MMP9）상대표체、천도막배면적세포수、세포극륭수、현부성구수균고우CD－90세포，EMT상피표지상관기인E-cadherin상대표체균저우CD－90세포，P균＜0．05。수착접충세포수목적증가，CD＋90、CD－90세포적성류솔화성류시간균승고，CD＋90세포승고경명현。결론란소암세포중，CD＋90세포고표체간질속성기인화간세포상관기인，구비경고적침습력、증식분화능력、체내성류능력화간세포잠능，CD＋90세포분리가능성위란소암간세포분리적신방법。
Objective To isolate and identity the CD+90 cells from SKOV3 cell line and primary ovarian cancer cells and to investigate the characteristics of cancer stem cells .Methods After successful isolation of ovarian cancer cells from ascites of patients with ovarian cancer .The flow cytometry was performed to detect the percentage of stem cell marker CD1+33 , CD+90 in SKOV3 and the primary cells .After the CD+90 and CD-90 cells were separated by means of fluorescence-based sorting, qRT-PCR was adopted to compare the difference of stem cell markers , transell assay was employed to analyze the ability of invasion , clone-formation test and sphere formation test were conducted to demonstrate the cell differentiation and proliferation capacity , limiting dilution transplantation assay was performed to check the tumorigenic ability in vivo . Results The percentage of CD+133 and CD+90 cells in SKOV3 cells were lower than those in primary cells (both P<0.05). In both SKOV3 and primary cells, the expression of CD133, CD44, ALDH1, OCT4, N-cadherin, Vimentine, MMP2, MMP9 in CD+90 cells were higher than those in CD-90 cells (all P<0.05).Compared with CD-90 cells, the invaded cell a-mount, colony formation number and suspended spheres formation increased in the CD +90 group, while the epithelial marker E-cadherin reduced , all P<0.05.The tumorigenic capacity and tumor mass escalated with the increase of implanted cell amounts and incubation time , which were more obvious in the CD+90 cells.Conclusions The CD+90 ovarian cancer cells re-presents a subpopulation characterized of enhanced mesenchymal and stem cell markers , obvious self-differentiation , marked self-renewal ability and evident in-vivo tumorigenic capacity .Therefore, CD+90 phenotype could be used as a marker to isolate ovarian cancer stem-like cells.