Research Between EMT And Its Relationship With The Radiation Resistance Of Radiation-resistant Human Esophageal Cancer Cell Lines TE13R120

Objective: To compare the expression levels of E-cadherin and vimentinbetween esophageal cancer cells TE13and its radiation-resistant cell linesTE13R120cell. Discuss the relationship of epithelial-mesenchymal transition(EMT) and the esophageal cancer cells’ radiation resistance.Methods: Separately routinely cultured human esophageal squamous cellcarcinoma TE13and the screened by irradiation of radiation-resistant cell linesTE13R120cells. We observe TE13cell and TE13R120cell’s morphologicalchanges with inverted phase contrast microscope. Compare the capacity ofinvasion and migration between TE13cell and TE13R120cell through cellscratch assay. Utilize Real-time quantitative fluorescent PCR method to detectthe epithelial phenotype markers E-cadherin and mesenchymal phenotypemarkers vimentin. Move forward a single step, test and verify the positiveexpression of E-cadherin and vimentin by Immunocytochemistry.Result:1Morphological difference: Observe logarithmic phase monolayer ofTE13cell and TE13R120cell’s morphological changes with inverted phasecontrast microscope, we found that TE13cells show a sheet growthphenomenon and there are no clear boundaries between cells. While inTE13R120cell, we observed its cell outline is clearer and cell connections arenot tight. TE13R120cells are slim, large or small, disorganized erratic,irregular and showing spindle or spindle-shaped.2Radiosensitivity difference between TE13cell and TE13R120cell:Obtain the radiobiology parameters of the two kinds of cells by cell colonyformation assay. The values of D0、Dq and N in TE13R120cell are larger thanthat in TE13cell, and the α/β ratio decreased, which prompt TE13R120 cells are more resistant to radiation compared with their parental cells.Survival fractions2(SF2) is an important indicator of cell radiosensitivitywhen they accepted2Gy rays. Greater the SF2value, the stronger theradiation resistance. TE13R120cells SF2value is higher than their parentalcells TE13cells, Further illustrated that TE13R120cells’ radiation resistanceincreased.3Radiation-resistant TE13R120cells cycle: Counting the number ofcells in24-well plates in Consecutive8days, then Draw out asemi-logarithmic cell growth curve of TE13cell and TE13R120cells. As canbe seen from the cell growth curve, TE13R120cells’ growth rate is slow thanTE13cells, TE13R120Cell population doubling time is（20.70±0.57）h，which is longer than the parental cells TE13cells’（17.67±0.99）h，（P=0.024）4Compare the capacity of invasion and migration between TE13cell andTE13R120cell: Culture the two kinds of cells in complete medium toapproaching covered in six-well plates, afterwards for cell scratch andchanged to serum-free medium to keep on culture cells. Observe the case ofcell invasion and migration in the scratch in an inverted microscope take aphotograph at6h、12h、24h after scratches. We found that at12h and24h,TE13R120cells in scratches are more than TE13cells. At24h the TE13cellmigration distance is （97.18±3.04）um; TE13R120cell migration distance is（203.52（40.42））um,(P=0.000).The difference was statistically significant,that is to say, compare with TE13cell, TE13R120cell invasion and migrationcapability enhanced.5TE13cell appeared Epithelial-mesenchymal transition phenomenonafter irradiation: After120Gy, TE13cells’ cell morphology ranging fromsmall polygons to a slender uniform, spindle-shaped variation. Use Real-timequantitative PCR method to detect the E-cad and Vimentin’s expression inTE13cell and TE13R120cell. With GAPDH as an internal, the epithelial cellphenotype marker E-cad’s expression is decreased in TE13R120cells thanTE13cells. The difference was statistically significant (P=0.000). The mesenchymal phenotype markers Vimentin’s expression is increased inTE13R120cells than TE13cells. The difference was statistically significant(P=0.030).6Immunocytochemistry method to detect E-cad and Vimentin’s positiveexpression of the cell membrane and cytoplasm between TE13cell andTE13R120cell: The expression of E-cad-positive cells in TE13cell werehigher than that in TE13R120cells, while Vimentin-positive cells lower.Conclusion:1Repeatedly irradiated by ionizing radiation to TE13cells esophagealscreened radiation resistant cell lines TE13R120cell. TE13R120cell’sdoubling time is longer than the TE13cells’, which also possess a stableradiation resistant phenotype.2Radiation resistant esophageal cancer cell TE13R120cell’s invasionand migration capacity is enhanced.3Repeated ionizing radiation induced TE13cells’ morphological andmolecular levels changed, in line with epithelial-mesenchymal transformationfeatures.