Effects Of ERK Signal On The Proliferation And Apoptosis Of HaCaT Cells

Objective:To study the effects of ERK signal pathway on proliferation and apoptosis of HaCaT cells.Methods:1. Culture of HaCaT and HEK293: Cells were cultured with DMEM that contained 10% FBS in constant 37℃ temperature saturated humidity incubaton with 5%CO₂. Cells were digested by 0.25% tripsin when fused about 80-90%, then to blow it to simple cell and passage with 1:3 rate. It is usually passaged after three or four days. The density of cells was 5 ×10⁴ cells/ ml at experiments.2. Disposation of cells1) Construction of proliferation model. EGF was added when cells fused about 70-80% and its final concentration were 1, 10 and 100 μg/L, then cells were cultured for 24 hour. MEK specific blocking agent — PD98059 was added 5h before EGF, its final concentration were 6.25,12.5, 25, 50 and 100μM.2) Construction of apoptosis model. Cells were washed by PBS for two times when cells fused about 80%, then irradiated it with ultra violit. The irradiation dose of low dose group was UVA2J/cm² and UVB10mJ/cm², high dose group was UVA6J/cm²,UVB 30mJ/cm². The control group did not irradiated.3. The method of proliferation detection: the doses of 37kBq ³H-TdR were added, and then cells were cultured for 6h. To detect the incorporation of ³H-TdR according the operating procedure of Wallac. The results were demonstrated by Counts/min...