摘要
目的:研究反复-70℃冻融对人眼脉络膜黑色素瘤OCM-1细胞中肿瘤干细胞的杀伤作用。方法:复苏OCM-1细胞,使用标准DMEM/F12培养基培养后,改用无血清化学限定培养基(serum-freemedium,SFM)培养,并分离纯化肿瘤干细胞(choroidalmelanomastemcells,CMSCs)。分别对OCM-1和纯化的CMSCs反复-70℃冻融后,MTT比较杀伤比率的差异。光镜及共聚焦显微镜观察细胞形态学改变。结果:在标准DMEM/F12培养基中贴壁生长的OCM-1细胞,使用SFM培养基培养后,贴壁细胞明显减少,第6代的CMSCs细胞,均为悬浮团状集落生长,呈现典型的干细胞生长方式,CD133阳性比率达到79.8%,Musashi-1强阳性表达。MTT比色法显示,反复-70℃冻融对OCM-1和CMSCs的杀伤比率存在显著差异。光镜及共聚焦显微镜观察发现,冻融后OCM-1细胞出现大量的凋亡细胞,而CMSCs则以坏死为主,凋亡细胞少见。结论:反复-70℃冻融对分化良好的OCM-1细胞主要是诱导其凋亡。而OCM-1中存在的肿瘤干细胞CMSCs对冷冻更加敏感,冻融后大量死亡,这是肿瘤在冷冻治疗后发生退变消失的根本原因。
AIM： To investigate the effects of repeating -70℃ freeze thawing on tumor stem cells in human choroidal melanoma cell line OCM-1 （CMSCs）. METHODS： OCM-1 cells were continuously cultured in standard DMEM/F12 medium. Serum-free medium （SFM） was used to culture cells and purify the CMSCs. The cell CD133 positive rates were observed by flow cytometry （FCM）. The cell Musashi-1 immunocytochemistry of the CMSCs were examined by laser-scanning confocal microscopy （LSCM）. After OCM-1 cells and CMSCs were frozen by repeating -70℃ freeze thawing for various durations, the kill and wound condition of cells were examined by M1-F assay. The morphological changes of the cells were observed by confocal microscopy and the HE staining staining. RESULTS： Adherent OCM-1 cells in standard DMEM/F12 medium turned to spheroid cells in SFM, similar to the growth pattern of stem cells. The CD133 positive rate in the cells of the sixth generation was 79.8%, and Musashi-1 expressed highly. M1-F assay showed significant deviation in the lethal effect of OCM-1 and CMSCs. After repeating -70℃ freeze thawing, considerable apoptosis cells necrosis cells appeared in OCM-land CMSOs, respectively. OONCLUSION： There are tumor stem cells existing in hu- man choroidal melanoma OCM-1 cells. Repeating-70℃ freeze thawing could straightly kill CMSOs and induced OCM-lcells to apoptosis. Cell growth ability of the reliquus OCM-1 cells was inhibited, and cells died at last because considerable CMSOs had been killed. This is the important mechanism of the tumor cataplasis after cryotherapy.
出处
《国际眼科杂志》
CAS
2006年第6期1278-1282,共5页
International Eye Science
基金
中国国家自然科学基金资助项目(No.39870801
30672276)
中国广东省社会发展攻关项目(No.2003A3020302)~~
关键词
脉络膜黑瘤
肿瘤干细胞
反复冻融
choroidal melanoma
tumor stem cell
repeating freeze thawing
