In Vitro wound healing assay revisited: aided by a long-term, time-lapse recording system

• Main Authors: Pei-Shuan Chiang, 江旆萱
• Other Authors: Shaw-Jenq Tsai
• Format: Others
• Language: en_US
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/61577690168058185073

碩士 === 國立成功大學 === 分子醫學研究所 === 91 === Wound healing is one of the most frequently used methods to study cell motility. A monolayer of cells is scratch-wounded and cells alongside the wound would proliferate and migrate to fill up the denuded area. The area change or the wound closure distance is considered to be a measurement of cell motility. However, the rate of wound closure may not be a true measurement of cell motility. By measuring the distance of wound closure, we found that high-density monolayers of T24 cells (a bladder cancer cell line) showed faster wound-closure rates than low-density ones, which, by conventional interpretation, implied that T24 cells at higher cell densities would have greater cell motilities. To clarify such an observation, we investigated the details of wound healing with our long-term, time-lapse recording system, which was able to record and depict the migration path of a single cell through the entire healing process. Only the first few rows of cells behind the wounded edge contributed to wound closure. These cells showed better moving directionality (toward the direction of wound closure) at higher cell-densities, explaining the greater wound-closure rate, whereas the average lengths of the migration paths are the same in high- and low-density experiments. The lengths of migration paths over a period of time are the better measurement of cell motility, whereas the wound-closure rate represents the combinational effect of cell motility and directionality. The effects of mitomycin C and β-Glycyrrhetinic acid on wound-closure rate and cell motility in wound healing were further investigated. It had been suggested that in order to minimize the effect of cell proliferation on wound healing, the proliferation activity should be inhibited or the assay time should be kept as short as possible. However, we found that inhibition of cell proliferation by mitomycin C treatment may affect cell motility in a short period of time. Gap junctional communication was thought to play a role in wound healing. By inhibition of gap junction with GCA, we found that the percentage of forward moving cells as well as the migration rate significantly decreased. In the second part of the study, we applied the long-term time-lapse recording system to the functional analyses of genes through transient transfections. A preliminary procedure was established. Using a construct to co-express green fluorescent protein and EMP2 in NIH3T3 cells, we established a procedure to evaluate potential effects of EMP2 on cell morphology, viability, apoptosis, membrane ruffling and cell motility. The procedure could be utilized as a rapid screening test for gene functions.