深低温反复冻融杀伤人眼脉络膜黑色素瘤ocm1细胞的机制.docx

深低温反复冻融杀伤人眼脉络膜黑色素瘤0CM-1细胞的机制 作者：刘斌，李永平，张波，张文忻，彭展 【摘要】【目的】研究-70 °C反复冻融体外杀伤人眼脉络膜黑色 素瘤细胞系0CM-1的机制。【方法】0CM-1细胞反复冻融后，用嗟哩 蓝比色法及克隆形成实验检测细胞的杀伤情况和细胞生长活力，免疫 组织化学方法检测细胞P16表达的变化，电镜及共聚焦显微镜观察冻 融后细胞凋亡和坏死的形态学改变，并测定细胞的凋亡比率，流式细 胞仪检测冻融前后细胞CD40表达比率及细胞膜电位的变化。【结果】 嗟哩蓝比色法检测及克隆形成实验显示，冻融可以直接杀伤0CM-1细 胞，且残存的细胞生长能力也明显受抑制。免疫细胞化学染色见对照 组的细胞呈P16阳性，而冻融后的细胞均呈阴性，冻融后培养的细胞 部分呈阳性。电镜下观察到冰晶的结构特征及细胞的坏死和凋亡等变 化。激光共聚焦显微镜观察发现凋亡细胞随着冻融次数的增加而增 多。流式细胞仪测定显示冻融后细胞膜电位下降，冻融前后CD40阳 性细胞比率差异明显，且呈一定的量效关系。【结论】冻融不仅可以 明显杀伤OCM-1瘤细胞，还能抑制残存瘤细胞生长。反复冻融能诱导 大量的肿瘤细胞发生凋亡，此为杀伤肿瘤及诱导肿瘤退变的直接原 因，其机制与线粒体膜电位及P16表达下调有关。冻融后瘤细胞坏死 因子CD40表达增强，可能是增强机体的免疫应答，介导机体免疫杀 伤瘤细胞的机理。 【关键词】庾复冻融；脉络膜黑色素瘤；细胞凋亡；CD40；线 粒体膜电位 Abstract： L Objective] To investigate the mechanism of repeating -70 °C freeze thawing on human choroidal melanoma cell line OCM-1 ? 【Methods 】OCM-1 cells were frozen by repeating -70 °C freeze thawing for various frequency, then the death rate of cells were examined by MTT essay. The cell viability was measured by clone essay. The morphological changes of the cells were observed using electron microscope and the P16 immunocytochemistry staining was performed. The cel 1 apoptosis rate of the OCMT cells was examined by laser-scanning confocal microscopy (LSCM)? The cell CD40 positive rates and mitochondrial membra.ne potential (MMP) were observed by flow cytometry (FCM)?【Results】 Growth of OCM-1 cells was inhibited by repeating -70 °C freeze thawing. And that showed freeze frequency dependent effects (PIt； 0.01). Different morphology of cells after -70 °C freeze thawing could be seen by electron microscopy. The classic morphologic characteristics of ice crystal and apoptosis were shown. The P16 immunocytochemistry staining showed positive reaction in the control group and negative reaction in the group of repeating -70 °C freeze thawing, then the positive reaction appearanee in the cultured cells? Different cell apoptosis rates of the OCM-1 cells were showed by LSCM. Flow cytometry showed a positive correlation between the cell CD40 positive r