生物材料表面微结构对循环肿瘤细胞粘附、增殖及捕获率的影响

Effect of surface microstructure of biomaterials on the adhesion, proliferation and capture rate of circulating tumor cells

目的探讨不同材料界面微观结构对循环肿瘤细胞(CTC)粘附、增殖及捕获率的影响。方法采用真空辅助软压印、水热法制备微米结构、纳米结构、微纳结构TiO2基底,连同氟掺杂氧化锡(FTO)玻璃共4种基底,修饰上皮细胞黏附分子抗体(anti-EpCAM)后进行人乳腺癌细胞系MCF-7和人宫颈癌细胞Hela的捕获实验,扫描电镜观察捕获细胞的形貌特征,荧光显微镜分析细胞增殖情况及捕获率。结果在微纳结构中,微米柱与纳米线在促进细胞粘附和铺展上具有协同作用,当微米柱间距合适时,细胞呈扁平状完全摊开。在捕获细胞孵育24 h后,微纳结构表面的细胞呈现明显增殖,细胞密度最大,单位面积细胞数为170个,明显多于其他3种结构界面(P<0.01)。随着孵育时间(30~60min)的延长,4种结构表面的捕获细胞逐渐增多(P<0.01),60 min后细胞捕获率基本不再增加;孵育60 min时,微纳结构的细胞捕获率最高,光滑表面则最低(P<0.01),且4种结构对Hela的捕获率均显著低于MCF-7(P<0.01)。结论相对于光滑表面、微米和纳米结构表面,仿荷叶微纳结构表面最有利于CTC粘附、铺展和增殖,具有最高的细胞捕获率。

Objective To investigate the effect of different interfacial microstructure on the adhesion, proliferation and capture rate of circulating tumor cells (CTC). Methods Micro-structure, nano-structure and micro-nano hierarchical TiO2 structure were manufactured using vacuum assisted soft imprinting and hydrothermal methods. Smooth surface FTO glass was selected for comparison, and all the four kinds of substrates were modified with anti-EpCAM before MCF-7 (a breast-cancer cell line) and Hela capture experiment. The morphology of captured cells was observed with scanning electron microscope. The cell proliferation and capture rate were analyzed with a fluorescence microscope. Results In the micro-nano structure, the micron column and nanowires had synergistic effect on promoting cell adhesion and spreading. When the spacing of micron columns was appropriate, the cells were flat and completely spread out. After the captured cells were incubated for 24 h, the cells on the surface of the micro-nano structure showed obvious proliferation and the maximum cell density, and the number of cells per unit area was 170, which was significantly more than that of other 3 structural interfaces (P<0.01). With the extension of incubation time (30-60 min), the number of captured cells on the surface of the four structures were gradually increased (P<0.01), and the cell capture rate basically stopped increasing after 60 min. At the time of incubation for 60 min, the cell capture rate of the micro-nano structure was the highest and that of the smooth surface was the lowest (P<0.01). The Hela capture rates of the four structures were significantly lower than the MCF-7 capture rates of the four structures (P<0.01). Conclusion Compared with smooth surface, micro or nano structure, lotus leaf-like micro/nano structure is most conducive to CTC adhesion, spreading and proliferation, and thus has the highest cell capture rate.