人脉络膜黑色素瘤细胞株OCM-1的三维培养及血管生成拟态机制的研究

摘要

Objective To observe vasculogenic mimicry (VM) of human choroidal melanoma cell line OCM-1 cultured in vitro and the expression of PI3K and EphA2 protein,as well as to explore the possible mechanisms.Methods OCM-1 cells were cultured in vitro and stained with periodic acid Schiff (PAS) on days 7,which aimed to observe the formation of PAS-positive cyclic structures,that is,VM formation.Then immunohistochemical staining was performed to detect PI3K and EphA2 on day 1,4,7 and the results were observed.Ressults On day 4 of 3-demintional culture,most of OCM-1 cells were polygonal and the cytoplasm was abundant;the nuclei were round and the nucleoli were visible.A small part of the tumor cells were long spindle.It was found that several long spindle cells were connected to each other to form hemicyclic structure.After 7 days,a large number of tumor cells became long spindle,growing along the collagen scaffold,and long protrusions appeared,forming a ring structure.PAS staining showed that the tumor cells were mostly arranged in a row,and tumor cells imitated the formation of body blood vessels,resulting in cell band and pipeline-like cell layers,with one layer of extracellular matrix (PAS-positive substance) making up the ring structure.Moreover,the expression levels of PI3K and EphA2 on day 4 and 7 were significantly higher than those on day 1 (all P ＜ 0.05),and their expression levels on day 7 were higher than those on day 4 (all P ＜ 0.05).Conclusion EphA2 and PI3K may play an important role in the VM formation in 3-dimentional culture of human choroidal melanoma cell line OCM-1.%目的 通过体外三维培养人脉络膜黑色素瘤细胞株OCM-1,观察其生成血管生成拟态(vasculogenic mimicry,VM)时,PI3K、EphA2蛋白的表达,探讨VM发生的可能机制.方法 体外三维培养OCM-1细胞,于三维培养7d进行过碘酸-雪夫反应(periodic acid schiff reaction,PAS)染色,观察PAS阳性的环状结构的形成(即其形成VM的能力),于1d、4d、7d进行PI3K和EphA2免疫组织化学染色并观察其结果.结果 OCM-1细胞三维培养4d,细胞大部分呈多角形,胞浆较丰富,核圆形,可见核仁,少部分肿瘤细胞长梭形,可见3～5个长梭形细胞彼此连接围咸丰环状结构.7d后,肿瘤细胞几乎均变成长梭形,沿着胶原支架生长,细胞伸出长的突起,彼此之间相互接触围成环状结构.PAS染色可见瘤细胞大多呈弥散排列,肿瘤细胞模仿机体血管生成而形成瘤细胞条索并围成管道,一层细胞外基质(PAS阳性物质)围成环状结构.与培养1d的细胞相比,培养4d和7d的PI3K和EphA2蛋白表达量均明显增加(均为P＜0.05),并随着时间的推移增加(7 d＞4 d＞1 d,P＜0.05).结论 EphA2和PI3K可能在三维培养人脉络膜黑色素瘤细胞株OCM-1的VM形成过程中具有重要作用.