TPA对人食管癌Eca109细胞增殖和细胞核形态的影响(2)
2014-09-16 02:10
导读：1.2 Cell Growth Studies Human esophageal cancer cell line Eca109 was kindly provided by professor Bai jingxiu，Zhengzhou University. The cells were maintained in RPMI 1640 medium with 10% fetal bovin
1.2 Cell Growth Studies Human esophageal cancer cell line Eca109 was kindly provided by professor Bai jingxiu，Zhengzhou University. The cells were maintained in RPMI 1640 medium with 10% fetal bovine serum (FBS) in the presence of antibiotics，penicillin (100 units/ml)and streptomycin (100units/ml). All cells were cultured at 37℃ in a humidified atmosphere containing 5% CO2. The medium was replaced after 3 days. The cells were routinely subcultured twice a week.
Cells were plated in either flasks (25cm2) or 96-well plates. TPA was added 24 hours after seeding，when the cells were attached to the surface of the containers. An equivalent volume of ethanol was added to the controls and had no effect on growth. After removing the cells from the flasks by trypsinization they were counted using a hemocytometer，fixed with 70% ethanol and stained by H.E. When the cells were plated into a 96-well microplate，the culture medium containing different concentrations of TPA was replaced 24 hours later. After 3-5 days of incubation，cell viability was evaluated by MTT assay.
1.3 Nuclear morphometry measurements Measurements of nuclear morphometry including nuclear area (NA)，nuclear perimeter (NP)，maximal diameter (Dmax)，minimal diameter (Dmin)，nuclear plasma ratio (NPR)，nuclear axis ratio (NAR)，nuclear roundness factor (NRF)，were performed with computer-assisted image analysis system MPIAS-500 (QingPing Image Engineering Company，Tongji University). Two fields were selected at random from each case for the measurements. Each field was viewed under a ×100 oil-immension lens and displayed on the screen of a color video image processor. The images of 50 nuclei of the tumour cells per field were outlined with a drawing pen，and the data were processed by a computer in the system，which calculated the parameters. In the end all the mean values of each case were calculated.
1.4 Statistical Analysis The data were expressed as means±SD. Statistical analysis was performed by SPSS 10.0 software. The criterion for statistical significance was taken as P
