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-free medium,SFM)培养,并分离纯化肿瘤干细胞(choroidal melanoma stem cells,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.

　　关键词：脉络膜黑瘤;肿瘤干细胞;反复冻融(6):1278-1282

　　Study on lethal effect of tumor stem cells in human choroidal melanoma OCM-1 cell line by repeating -70C freeze thawing in vitro

　　Bin Liu　 Yong-Ping Li　 Wen-Xin Zhang　 Zhan Peng

　　基金项目：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)

　　作者简介：刘斌,男,中山大学中山眼科中心2005级医学硕士.

　　作者简介：通讯作者:李永平[email protected].

　　作者单位：刘斌(510060,中国广东省广州市,中山大学眼科学国家重点实验室,中山大学中山眼科中心)

　　李永平(510060,中国广东省广州市,中山大学眼科学国家重点实验室,中山大学中山眼科中心)

　　张文忻(510060,中国广东省广州市,中山大学眼科学国家重点实验室,中山大学中山眼科中心)

　　彭展(510060,中国广东省广州市,中山大学眼科学国家重点实验室,中山大学中山眼科中心)

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