【作者】 梁佩燕；

【导师】 曾耀英；

【作者基本信息】 暨南大学 ， 免疫学， 2005， 硕士

【摘要】 目的: 建立放线菌酮（CHX）诱导HL-60细胞凋亡模型,并探讨该过程中的线粒体变化;同时,在该过程中分别阻断ERK和p38途径,研究细胞凋亡和线粒体变化,探讨CHX诱导HL-60细胞凋亡过程与这两个信号通路之间的关系。 方法: 利用PD和SB分别在CHX诱导HL-60细胞凋亡过程中阻断ERK和p38途径。通过AnnexinV-FITC/PI双染流式细胞术测定凋亡和坏死细胞比率;通过PI染色流式细胞术测定亚二倍体峰细胞比率;应用JC-1染色共聚焦显微镜和Horchest染色荧光显微镜进行观察与拍摄。通过JC-1染色流式细胞术,计算线粒体膜电势（（?）2/（?）1）,通过划分具有不同荧光强度的细胞群和反向射门,分析细胞凋亡过程中线粒体与细胞物理参数间的关系,并通过FL-2分析与比较不同时点低J—agregate的细胞比率。利用NAO染色流式细胞术检测实验组线粒体质量和H₂DCFDA/PI染色流式细胞术检测ROS含量。应用DiOC₆（3）/Annexin V-APC双染更多还原

【Abstract】 Aim:To establish the CHX-induced HL-60 cell apoptosis model and study on the change of mitochondria; to inhibit the ERK and p38 pathway respectively during the CHX-induced HL-60 cell apoptosis process in order to study on the influence of HL-60 cell apoptosis and change of mitochondria and the relationship between the cycloheximide-induced HL-60 cell apoptosis process and the the ERK and p38 pathway.Methods:To inhibit the ERK and p38 pathway respectively by PD and SB. Detecting the apoptotic and necrotic cell ratios by AnnexinV-FITC/PI double staining flow cytometry and the sub-diploid cell ratios by PI staining flow cytometry. To observe and take JC-1 staining picture and Horchest staining picture by confocal microscopyand fluorescene microscopy. To measure the J-aggregate （FL2） and J-monomer（FL1 ）by JC-1 flow cytometry and calculate the △Ψ_mby（FL1/FL2 ）, to analyze the relationship between the mitochondria and the physical parameter of the cell during the CHX-induced HL-60 cell apoptosis process by dividing different region of JC-1 staining cell according to the different fluorescene intensity, to divide and compare the low J-aggreagte cell of different group according to the FL-2. To measure the mitochondria mass by NAO flow cytometry and ROS producing ratios by H₂DCFDA/PI double staining flow cytometry. To measure the apoptotic cell ratios and △Ψ_m by DiOC₆（3）/Annexin V-APC double staining flow cytometry.Results:CHX can induce the HL-60 cell apoptosis. The apoptotic and necrotic cell ratios is lower than the CHX group when inhibiting the ERK pathway during the CHX-induced HL-60 cell apoptosis process; inhibiting p38 pathway during this process the apoptotic cell ratio has no significant difference and the necrotic cell ratios is higher compared with CHX group’s. The sub-diploid cell ratio is lower than the CHX group when inhibiting the ERK pathway during the CHX-induced HL-60 cell apoptosis process from 9h; The sub-diploid cell ratio is higher than the CHX group when inhibiting p38 pathway during this process from 9h. CHX can induce the Avm decrease. The △Ψ_mis higher than the CHX group when inhibiting the ERK pathway during the CHX-induced HL-60 cell apoptosis process; The △Ψ_m is lower than the CHX group when inhibiting p38 pathway during this process. The low mitochondria mass cell ratio of CHX group at 18h is higher than control group’s, The low mitochondria mass cell ratio is lower than the CHX group’s when inhibiting the ERK pathway during the CHX-induced HL-60 cell apoptosis process; the The ROS production cell ratio of CHX group at 18h is higher than control group’s.Conclusions:CHX can induce HL-60 cell apoptosis, the cell mitochondria depolarization, decrease of A W m and mitochondrial mass; Inhibiting the ERK pathway during the CHX-induced HL-60 cell apoptosis process can lessen the decrease of apoptotic and necrotic cell ratios, the cell mitochondria depolarization and the reduction of mitochondrial mass, indicating this process is closely related to the ERK pathway. Inhibiting the p38 pathway during the CHX-induced HL-60 cell apoptosis process can increase the necrotic cell ratio, and intensify the cell A Wm decrease, indicating that the p38 pathway is closely related to the necrosis of HL-60 cell.更多还原