以胶原水凝胶为支架构建腺病毒三维培养系统

In vitro establishment of a three-dimensional adenovirus culture system using collagen hydrogel as scaffolds

目的以胶原水凝胶为三维细胞培养支架,结合病毒学实验技术,构建能长时间体外三维培养腺病毒的模型,为病毒分离鉴定、致病机制研究、药物筛选等提供新的研究方法。方法扫描电镜(SEM)表征胶原水凝胶的结构特征,以胶原水凝胶为三维支架材料进行293T细胞培养,并感染腺病毒。用倒置显微镜观察3D培养293T细胞的形态特征;MTS法检测腺病毒感染前后细胞的增殖活性;透射电镜(TEM)观察三维培养293T细胞中的腺病毒颗粒,并用qPCR检测在胶原水凝胶三维培养293T细胞中腺病毒的增殖情况以及复制子的感染性。结果293T细胞在胶原水凝胶三维培养系统中能长时间良好生长。腺病毒感染持续时间长,有多个增殖峰。在此3D系统中增殖的腺病毒颗粒仍具有感染性。结论以胶原水凝胶为支架构建的腺病毒三维培养系统能长时间维持腺病毒的持续增殖,更接近体内感染情况,胶原水凝胶可作为腺病毒分离、培养和研究的三维培养支架材料。

Objective To construct a long-time three-dimensional(3D)culture model of adenovirus using collagen hydrogel as 3D cell culture scaffolds in vitro,and combined with virology experimental technology,so as to provide a novel research method for virus isolation and culture,pathogenesis study and antiviral drug screening.Methods The structural characteristics of collagen hydrogel were characterized by Scanning Electron Microscopy(SEM).293T cells were cultured with collagen hydrogel and infected with adenovirus.The morphological characteristics and proliferative activity of 3D-cultured 293T cells after adenovirus infection were observed by inverted microscope and the MTS cell proliferation assay,and the adenovirus particles in 3D cell culture system were observed by Transmission Electron Microscopy(TEM),and the proliferation of adenovirus and replicator infection in 3D cell culture system with collagen hydrogel were detected by qPCR.Results 293T cells could grow well for a long time in the three-dimensional culture system of collagen hydrogel.3D culture using collagen hydrogel can prolong the survival of adenovirus-infected 293T cells in vitro and has multiple proliferation peaks.Adenovirus replicators amplified in the 3D system are still infectious.Conclusion The 3D culture system of adenovirus using collagen hydrogel as scaffolds can maintain the continuous proliferation of adenovirus for a long time,which is closer to the infection in vivo.Collagen hydrogel can be used as 3D scaffolds for adenovirus isolation,culture and research.