【作者】 王会营；

【导师】 曾耀英；

【作者基本信息】 暨南大学 ， 免疫学， 2006， 硕士

【摘要】 目的： 探讨ERK信号在HaCaT细胞增殖及凋亡中的作用。 方法： 1．HaCaT细胞和HEK293细胞的培养：培养基为含有10％FBS的DMEM培养基，培养于37℃、5％CO₂恒温饱和湿度培养箱。细胞80～90％融合时，0.25％胰蛋白酶消化，37℃孵育5min，加入2mL完全培养基，吹打成单个细胞，以1：3的比例接种，通常3～4天即可传代。实验时细胞接种密度为5×10⁴cells／mL。 2．细胞的处理： 1) 增殖模型的建立：培养板细胞达到70～80％融合时，加入EGF（终浓度分别为1、10和100μg／L），继续培养24h。MEK特异性阻断剂PD98059（6.25、12.5、25、50和100μM）在EGF加入前5h加入。 2) 凋亡模型的建立：细胞融合至80％以上时，PBS洗2次后每孔加入PBS 1mL，将细胞放置于紫外灯管下照射（低剂量组照射强度为UVA2J／cm²、UVB10mJ／cm²；高剂量组为UVA6J／cm²、UVB 30mJ／cm²），对照组不照射。 3．增殖的检测方法：在培养结束前每孔加入37kBq的³H-TdR，继续培养6h。 参照Wallac提供的操作程序检测³H-TdR的掺入量，结果以Counts／min（CPM）表示。更多还原

【Abstract】 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更多还原