自组装短肽水凝胶RADA16-I在卵巢癌细胞培养中的应用研究

Application of self-assembling peptide RADA16-I nanofiber hydrogel in culture of ovarian carcinoma cells

目的构建纳米自组装短肽RADA16-I三维细胞培养模型,探讨卵巢癌细胞A2780三维培养的可能性。方法采用透射电镜观察细胞支架材料结构特征;MTT比色法观察细胞粘附情况;通过荧光显微镜和DNA含量测定检测细胞生长情况;免疫组化法检测细胞粘附分子(整合素β1、E-钙黏蛋白、N-钙黏蛋白)表达情况;ELISA检测细胞相关因子(VEGFA、EGF、FGF2、IGF1)表达水平;活性氧检测法检测支架材料中细胞氧活性情况。结果与Matrigel和I型胶原(Collagen I)相比,纳米自组装短肽RADA16-I具有较长纤维,且形成的纤维网架结构较为密集;A2780对短肽RADA16-I的粘附力相似于Matrigel、Collagen I(P>0.05);三维培养第3天A2780开始聚集成团且活性良好,在RADA16-I中细胞增殖相对Matrigel与Collagen I较慢,但保持一定增殖速率;在RADA16-I三维培养体系中,细胞氧活性良好,且细胞粘附分子(整合素β1、E-钙黏蛋白、N-钙黏蛋白)和细胞相关因子(VEGFA、EGF、FGF2、IGF1)均有表达。结论纳米自组装短肽RADA16-I可成为体外A2780研究的三维实验模型。

Objective To establish the three dimensional culture model for ovarian cancer cell line A2780 with self-assembling peptide RADA16-I nanofiber hydrogel as scaffolds.Methods The structures of matrices were characterized with transmission electron microscopy(TEM).The adhesion potential of cells onto different matrices was measured by MTT assay.Cell state in different 3D cell matrices was observed by immunochemical staining and evaluated by DNA-content measurement.Expression of cell adhesion molecules on different scaffolds was detected by immunohistochemistry.The transcripts of ovarian cancer cell in different culture systems were detected through ELISA assay.Cellular oxygen activity on various matrices was detected by the reactive oxygen species assay kit.Results Longer fibrils and more intensive nanofiber networks can be formed in self-assembling peptide hydrogel than in the other two kinds of materials.However,the A2780 cells’adhesion onto the three matrices was shown to be similar(P>0.05).On day 3,the cells began to aggregate and maintained good viability in these 3D cell matrices.Moreover,cells in the RADA16-I matrice kept proliferation at a certain proliferating rate,though the rate was slower than those in Matrigel and collagen I during the culture process.Expression of cellular oxygen activity,cell adhesion molecules(integrinβ1,E-cadherin,N-cadherin)and cell growth factors(VEGFA,EGF,FGF2,IGF1)were all detectable in RADA16-I.Conclusion The self-assembling peptide hydrogel can be explored to construct a relatively ideal culture system for ovarian cancer cell line A2780 in vitro.