CD105/CD133筛选鉴定SMMC-7721株干细胞表面标志物的实验研究

目的：观察人肝癌细胞株 SMMC-7721中膜抗原CD133、CD105的表达情况并对不同亚群的生物学性状进行体内外实验研究。方法以含10%胎牛血清的 DMEM 对SMMC-7721株细胞培养；采用流式细胞仪方法分选检测CD133、CD105在SMMC-7721中表达情况并分选出CD133+/CD105+、CD133+/CD105-、CD133-/CD105+、CD133-/CD105-4个亚群；CCK-8和 Transwell侵袭实验分别检测4个亚群和未分选细胞组的增殖及侵袭能力,软琼脂克隆实验检测5组细胞成球能力；裸鼠成瘤实验了解 CD133+/CD105+、CD133-/CD105-亚群和未分选组的成瘤能力。结果流式细胞仪分选的 CD133+/CD105+、 CD133+/CD105-、CD133-/CD105+、CD133-/CD105-4种细胞亚群的比例分别为1．61%、0．01%、97．88%和0．50%。 CD133+亚群的增殖和成球能力较 CD133-亚群及未分选细胞组强,而CD105+亚群侵袭能力较 CD105-亚群及未分选细胞组强。CD133+/CD105+组与CD133-/CD105-组及未分选细胞组相比成瘤所需的时间短、所需细胞数少、成瘤的体积大。结论 CD133在人肝癌细胞株 SMMC-7721中的表达与其增殖成球能力有关,CD105与其侵袭能力有关,CD133+/CD105+具有体内高度的成瘤能力。 CD133+/CD105+亚群在人原发性肝癌细胞株SMMC-7721中具有肿瘤干细胞特性。

CD105/CD133 as cell surface markers for screening and identification of stem cell in SMMC-7721

Objective The objective of this research is to compare the expression of CD105 and CD133 in mem-brane of human HCC cell line SMMC-7721 , different biological characters among the subpopulations in vitro and vi-vo. Methods SMMC-7721 cell line was cultured in DMEM containing 10%FBS. Flow cytometry was used to de-tect CD105, CD133 expression in SMMC-7721 cell in vitro. Four cell sub-populationsCD133 +/CD105 +,CD133 +/CD105 -,CD133 -/CD105 +, CD133 -/CD105 - were sorted by flow cytometry. Cell proliferation of these four sub-populations was detected by CCK-8 assay. Sphere formation ability of these four sub-populations was examined by soft agar test. Invasion ability of these four sub-populations was examined by Transwell cell invasion test. In vivo tumorigenicity assay was also performed on these four sub-populations using nude mice. Results The proportions of four sub-populations CD133 +/CD105 +,CD133 +/CD105 -,CD133 -/CD105 +,CD133 -/CD105 - were 1. 61%, 0. 01%, 97. 88% and 0. 50% sorted by flow cytometry, respectively. CD133 + subsets cells grew more quickly than those of CD105 + and unsorted cells. The numbers of colonies formed by CD133 + subsets cells were more than those of CD105 + and unsorted cells. Furthermore, the metastatic capacity of CD105 + subset cells observed by Tr-answell assay was higher than that of the CD133 + subsets cells and unsorted cells. In vivo tumor experiments showed CD133 +/CD105 +needed a shorter time and fewer cells,compared with unsorted group and CD105 -CD133 - groups were statistically significant ( P<0. 05 ) . Conclusion Our study finds that human live cancer cell line SMMC-7721 can be sorted into four sub-groups CD133 +/CD105 +, CD133 +/CD105 -, CD133 -/CD105 + and CD133 -/CD105 - successful by flow cytometry. It is found that CD133 +/CD105 + sub-group shows high proliferative poten-tial, high capacity for self-renewal by proliferation assay, clone forming assay and in vitro invasion assay. In vivo tumor experiment proves CD133 +/CD105 + sub-group shows a higher degree of tumorigenicity. Therefore, CD133 +/CD105 + subset cells are a subpopulation with stem properties in SMMC-7721 cells.