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

Research on 3-dimentional culture of human choroidal melanoma cell line (OCM-1) in vitro and the mechanism of vasculogenic mimicry

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、Eph A2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7 d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1 d、4 d、7 d进行PI3 K和Eph A2免疫组织化学染色并观察其结果。结果OCM-1细胞三维培养4 d,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成半环状结构。7 d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1 d的细胞相比,培养4 d和7 d的PI3K和Eph A2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Eph A2和PI3 K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。

Objective To observe vasculogenic mimicry （VM） of human choroi- dai 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 v/tro 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 im- munohistochemical staining was performed to detect PI3K and EphA2 on day 1,4,7 and the results were observed. Results 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. Af- ter 7 days, a large number of tumor cells became long spindle, growing along the colla- gen 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 struc- ture. Moreover,the expression levels of PI3K and EphA2 on day 4 and 7 were signifi- cantly higher than those on day l （ 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.