研究目的
To examine the influence of the process of transduction on cellular morphology and cells’ behavior by comparing the movement of human skin fibroblasts with and without genetic modification using fluorescent markers.
研究成果
The process of transduction changes the volume and area covered by human skin fibroblasts during crawling, leading to modified movement aspects such as decreased efficiency in distance covered and region penetration due to changes in morphology. Transduced cells are less polarized and develop extra podia, with differences observed between EGFP and DsRed2 groups. General movement behavior remains unchanged, but further experiments are needed to explore underlying causes like cytoskeleton stiffness and adhesion.
研究不足
The study is limited by the challenges in image segmentation, where some sequences were only partly correct or had to be omitted. The focus on movement and shape may not capture other behaviors like proliferation or death, and the use of bright field images with low contrast and artifacts could affect accuracy. Future work could involve confocal microscopy to estimate cell size in the Z-axis and investigate changes in cytoskeleton stiffness and adhesion.
1：Experimental Design and Method Selection:
The study involved culturing and monitoring three groups of human dermal fibroblasts (control, EGFP-transduced, DsRed2-transduced) to analyze their movement and shape using image processing methods, specifically a modified level-set method for segmentation and quantification of movement descriptors and morphological features.
2：Sample Selection and Data Sources:
Fibroblasts were isolated from human skin, transduced with lentiviral vectors for stable expression of EGFP or DsRed2, and cultured in standard medium. Image sequences were captured using a microscope from multiple wells in 24-well plates.
3：List of Experimental Equipment and Materials:
Equipment included a Leica microscope LAS AF DMI 6000B with temperature and CO2 chamber BL-X, monochromatic camera DMI 6000B, bright field objective 40X (HCPL APO
4：25), 24-well plates with glass bottom from Nest Biotechnology Company, LTD, and a BD FACSCanto II flow cytometer. Materials included high glucose DMEM, fetal calf serum, nonessential amino acids, lentiviral vectors, and fluorescent markers. Experimental Procedures and Operational Workflow:
Cells were seeded and rested, then monitored with the microscope to capture image sequences in bright field and fluorescence at 1 or 2-minute intervals for up to 4 hours. Images were segmented using the level-set method, and movement and shape features were quantified. Flow cytometry was used to analyze cell volume.
5：Data Analysis Methods:
Data were analyzed using t-tests for statistical significance, with calculations performed in Matlab and Excel for movement descriptors and shape features.
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