基于数字全息显微术的登革病毒感染C6/36细胞的3D形态学

Three-dimensional morphology of C6/36 cells infected by dengue virus:a study based on digital holographic microscopy

目的应用一种基于数字全息显微术的活细胞成像技术,监测登革病毒(DENV)感染C6/36细胞过程中3D形态学变化,分析病毒吸附和释放触发的系列细胞形态改变方向及强度,通过不同血清型间变化差异为深入研究相关感染机制提供线索。方法 6孔板中接种不同密度的C6/36细胞,以确定全息细胞成像仪下最佳成像密度;随后由28℃常规培养调节为37℃病毒培养条件,以分析培养环境改变对细胞状态的影响;4个血清型DENV感染C6/36细胞,在孵育2 h和培养24 h期间分别设计5个观察点;借助Holo Monitor M4全息细胞成像及分析系统记录相应三维全息图及相关形态学参数并应用软件进行统计学分析。结果 4×10~5接种量下,C6/36细胞全息图显示单个细胞三维形态表现统一,细胞面积、厚度和体积离散程度均最小(P<0.05),确定其为最佳成像密度;转移至37℃、5%CO_2培养24 h后细胞厚度、面积和体积改变无显著性差异(P>0.05);孵育和培养期间4个血清型DENV组细胞面积和体积增大表现一致,但孵育期间DENV1-4均表现细胞厚度减小不同,培养期间DENV1、2细胞厚度减小而DENV3、4出现截然相反的厚度增大现象。同时,不同血清型间变化趋势及程度具有各自特异性。结论成功运用此技术实时监测登革病毒感染形态学变化过程;不同时期4个血清型间特异性细胞病变现象存在差异,对登革病毒感染机制研究具有一定的指导意义。

Objective To monitor the 3-dimensional(3D) morphological changes of C6/36 cells during dengue virus(DENV)infection using a live-cell imaging technique based on digital holographic microscopy and provide clues for better understanding the mechanisms of DENV infection. Methods C6/36 cells were seeded in 6-well plates to determine the optimal imaging density under a holographic cell imager,and the morphological changes of the cells were recorded in response to a culture temperature change from 28 ℃ to 37 ℃. C6/36 cells were infected with 4 DENV strains with different serotypes at 28 ℃and incubated at 37 ℃ for 24 h,and the 3D holograms and relevant morphological parameters were recorded at different time points using Holo Monitor M4 holographic cell imaging and analysis system. Results The holograms of C6/36 cells inoculated at the optimal density for imaging(4×10~5 per well) showed unified 3D morphologies of the single cells with minimal dispersions in the cell area,thickness and volume(P<0.05),which did not undergo obvious changes when the cells were incubated at 37 ℃ for 24 h(P>0.05). The cell area and volume of the cells infected with the 4 DENV strains all increased and the cell thickness was reduced during incubation. Among the 4 strains,DENV-1 and DENV-2 caused reduced cell thickness while DENV-3 and DENV-4 increased the cell thickness,and the pattern and degree of such changes differ among the 4 strains.Conclusions Digital holographic microscopy allows monitoring of the complex morphological changes of cells during DENV infection. The 4 DENV strains with different serotypes causes characteristic cell damages during infection.