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1、眼脉络膜黑色素瘤OCM-1细胞的机制【摘要】【目的】研究-70℃反复冻融体外杀伤人眼脉络膜黑色素瘤细胞系OCM-1的机制。【方法】OCM-1细胞反复冻融后,用噻唑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】噻唑蓝比色法检测及克隆形成实验显示,冻融可以直接杀伤OCM-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P1
2、6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
3、理。【关键词】反复冻融;脉络膜黑色素瘤;细胞凋亡;CD40;线粒体膜电位Abstract:【Objective】Toinvestigatethemechanismofrepeating-70℃freezethawingonhumanchoroidalmelanomacelllineOCM-1.【Methods】OCM-1cellswerefrozenbyrepeating-70℃freezethawingforvariousfrequency,thenthedeathrateofcellswereexaminedbyMTTessay.The
4、cellviabilitywasmeasuredbycloneessay.ThemorphologicalchangesofthecellswereobservedusingelectronmicroscopeandtheP16immunocytochemistrystainingwasperformed.ThecellapoptosisrateoftheOCM-1cellswasexaminedbylaser-scanningconfocalmicroscopy(LSCM).ThecellCD40positiveratesandmitoc
5、hondrialmembranepotential(MMP)wereobservedbyflowcytometry(FCM).【Results】GrowthofOCM-1cellswasinhibitedbyrepeating-70℃freezethawing.Andthatshowedfreezefrequencydependenteffects(P<0.01).Differentmorphologyofcellsafter-70℃freezethawingcouldbeseenbyelectronmicroscopy.Theclassi
6、cmorphologiccharacteristicsoficecrystalandapoptosiswereshown.TheP16immunocytochemistrystainingshowedpositivereactioninthecontrolgroupandnegativereactioninthegroupofrepeating-70℃freezethawing,thenthepositivereactionappearanceintheculturedcells.Differentcellapoptosisratesoft
7、heOCM-1cellswereshowedbyLSCM.FlowcytometryshowedapositivecorrelationbetweenthecellCD40positiveratesandlethaleffect,butshowedainversecorrelationtheinversecorrelationbetweentheMMPandthecellapoptosisrate.【Conclusion】FreezethawingcankillOCM-1cellsandalsoinhibitsthegrowthofsurv
8、ivals.Repeating-70℃freezethawinginducesagooddealoftumorcellstoapoptosis,whichistheimmedia
