检验医学与临床
檢驗醫學與臨床
검험의학여림상
JOURNAL OF LABORATORY MEDICINE AND CLINICAL SCIENCES
2014年
z1期
290-293
,共4页
结直肠癌%肿瘤干细胞%干细胞标志物%无血清培养%CD133+
結直腸癌%腫瘤榦細胞%榦細胞標誌物%無血清培養%CD133+
결직장암%종류간세포%간세포표지물%무혈청배양%CD133+
colorectal carcinoma cell lines%cancer stem/tumor-initiating cells%stem cell related marker%serum-free culture%CD133
目的：探讨结直肠肿瘤干细胞在体外分化过程中细胞形态、干细胞及转移相关标志物表达和转移侵袭能力的变化，为进一步研究结直肠肿瘤干细胞分化走向及转移侵袭能力改变提供实验依据。方法取来源于人结直肠癌的细胞系 HCT116，无血清培养分离出 CD133＋细胞，加血清诱导分化，相差显微镜下观察其形态变化；在未分化状态下无血清培养第7天和14天与血清诱导分化后收集细胞，利用流式细胞仪检测干细胞标志物 CD133＋其表达量，采用激光共聚焦、RT-PCR 和免疫荧光检测 CD133＋、N-cadherin 及 Vimentin 表面标记分子的表达，Tr-answell 小室检验 CD133＋细胞的转移侵袭能力。结果（1）细胞形态：无血清培养分离的 CD133＋细胞，在生长过程中聚集成规则的细胞球，血清诱导后即贴壁生长，贴壁形态与同来源细胞形态一致，且再次无血清悬浮培养后聚集成球稳定生长。（2）标志物变化：结直肠肿瘤干细胞未分化时 CD133＋、N-cadherin 及 Vimentin 表面标记分子均高表达，流式细胞仪检测未分化细胞 CD133第7天表达率为（20．4±0．52）％，第14天表达率为（78．5±2．80）％，分化后表达率为（0．50±0．17）％。（3）转移侵袭能力：CD133＋细胞穿过孔膜细胞数为104．67±14．64，显著高于血清诱导的分化细胞28．67±6．5（P ＜0．05）。结论细胞形态、标志物表达及转移能力的改变均表明高表达 CD133＋的 HCT116结直肠癌肿瘤干细胞可定向分化为同源的结直肠癌细胞，且 CD133＋细胞血清诱导分化后通过 MET而导致转移侵袭能力下调。
目的：探討結直腸腫瘤榦細胞在體外分化過程中細胞形態、榦細胞及轉移相關標誌物錶達和轉移侵襲能力的變化，為進一步研究結直腸腫瘤榦細胞分化走嚮及轉移侵襲能力改變提供實驗依據。方法取來源于人結直腸癌的細胞繫 HCT116，無血清培養分離齣 CD133＋細胞，加血清誘導分化，相差顯微鏡下觀察其形態變化；在未分化狀態下無血清培養第7天和14天與血清誘導分化後收集細胞，利用流式細胞儀檢測榦細胞標誌物 CD133＋其錶達量，採用激光共聚焦、RT-PCR 和免疫熒光檢測 CD133＋、N-cadherin 及 Vimentin 錶麵標記分子的錶達，Tr-answell 小室檢驗 CD133＋細胞的轉移侵襲能力。結果（1）細胞形態：無血清培養分離的 CD133＋細胞，在生長過程中聚集成規則的細胞毬，血清誘導後即貼壁生長，貼壁形態與同來源細胞形態一緻，且再次無血清懸浮培養後聚集成毬穩定生長。（2）標誌物變化：結直腸腫瘤榦細胞未分化時 CD133＋、N-cadherin 及 Vimentin 錶麵標記分子均高錶達，流式細胞儀檢測未分化細胞 CD133第7天錶達率為（20．4±0．52）％，第14天錶達率為（78．5±2．80）％，分化後錶達率為（0．50±0．17）％。（3）轉移侵襲能力：CD133＋細胞穿過孔膜細胞數為104．67±14．64，顯著高于血清誘導的分化細胞28．67±6．5（P ＜0．05）。結論細胞形態、標誌物錶達及轉移能力的改變均錶明高錶達 CD133＋的 HCT116結直腸癌腫瘤榦細胞可定嚮分化為同源的結直腸癌細胞，且 CD133＋細胞血清誘導分化後通過 MET而導緻轉移侵襲能力下調。
목적：탐토결직장종류간세포재체외분화과정중세포형태、간세포급전이상관표지물표체화전이침습능력적변화，위진일보연구결직장종류간세포분화주향급전이침습능력개변제공실험의거。방법취래원우인결직장암적세포계 HCT116，무혈청배양분리출 CD133＋세포，가혈청유도분화，상차현미경하관찰기형태변화；재미분화상태하무혈청배양제7천화14천여혈청유도분화후수집세포，이용류식세포의검측간세포표지물 CD133＋기표체량，채용격광공취초、RT-PCR 화면역형광검측 CD133＋、N-cadherin 급 Vimentin 표면표기분자적표체，Tr-answell 소실검험 CD133＋세포적전이침습능력。결과（1）세포형태：무혈청배양분리적 CD133＋세포，재생장과정중취집성규칙적세포구，혈청유도후즉첩벽생장，첩벽형태여동래원세포형태일치，차재차무혈청현부배양후취집성구은정생장。（2）표지물변화：결직장종류간세포미분화시 CD133＋、N-cadherin 급 Vimentin 표면표기분자균고표체，류식세포의검측미분화세포 CD133제7천표체솔위（20．4±0．52）％，제14천표체솔위（78．5±2．80）％，분화후표체솔위（0．50±0．17）％。（3）전이침습능력：CD133＋세포천과공막세포수위104．67±14．64，현저고우혈청유도적분화세포28．67±6．5（P ＜0．05）。결론세포형태、표지물표체급전이능력적개변균표명고표체 CD133＋적 HCT116결직장암종류간세포가정향분화위동원적결직장암세포，차 CD133＋세포혈청유도분화후통과 MET이도치전이침습능력하조。
Objective To explore the changes of cell morphology,expression of stem cell related markers and metastasis ability of colorectal cancer stem cells after differentiation in vitro.Methods Tumor stem cells of the line CD133+ were obtained from colorectal cancer cell line HCT116 cells ,serum-free culture isolate CD133 + cells,and serum levels of differentiation,under phase contrast microscope to observe the morphological changesCD133+ cell were isolated by serum-free culture and then cultured to differentiate in medium containing 10% fetal bovine serum. The morphology of the cells was observed under phase contrast microscope.Cells were collected respectively before differentiation,after differentiate 7,14 days .The cell surface markers such as CD133,N-cadherin and Vimentin were investigated by flow cytometry,RT-PCR and immunofluorescence .Cell metastasis assay was applied to examine cell metastatic ability in vitro.Results (1)HCT116 cells were formed cancer cell spheres in SFM,Sphere cells were able to produce a heterogeneous cell population that could produce spheres.Cancer cell spheres were induced to differenti-ate by serum supplemented medium,these differentiated cell were adherence growth and kept the same morphology with the parental cells.(2)These cancer cell spheres were founded expressing markers such as CD133+,N-cadherin and Vimentin was higher than differentiated cells.The expression rates of CD133 was 20% after seven days ,90%after forteen days,and 0.5% of the differentiated cells.(3)The migrated cells number of CD133+ cells were 104.67 ±14.64,which were significant higher than differentiated cells 28.67±6.5(P <0.05).Conclusion The changes of morphology,cell marker expression and metastasis ability in the high-expression CD133+ CRC cells are oriented dif-ferentiated into the same origin CRC cells,and the metastasis ability of differentiated cells is higher than the cell sphere.
