The Mechanism Of Ampelopsin Indmced Cell Apoptosis In Human Retinobl Astoma Cells Line

Objective By detecting ampelopsin on cultured human retinoblastoma cancer HXO-RB44 cells proliferation was observed morphological changes of cell apoptosis, mitochondrial transmembrane potential and intracellular reactive oxygen species(ROS)level changes.Discussion ampelopsin induced human retinoblastoma cancer HXO-RB44 cells apoptosis mechanism ampelopsin further elucidate the role of antitumor mechanism .Methods All the cultured HXO-RB44 cell the study used were in the logarithmic growth phase. 1.Observed the cell growth curve under of APS:Have seven different drug concentrations(50.00,33.33,22.22,14.84,9.90,6.60,4.40)μg/ml and blank control group,respectively,acting on the HXO-RB44 cells 0,24,48,72h,draw the growth curve chart. 2.Observed the cell proliferation of APS:Seven different drug concentrations of APS for HXO-RB44 cells 24h, the morphological changes observed by inverted microscope, MTT, colony formation in soft agar was observed APS on HXO-RB44 cells in vitro. 3.Morphology change on cell apoptosis:Select three different concentrations (50.00,14.84,4.40)μg/ml of the APS role in HXO-Rb44 cells after 24h, the apoptosis staining,using inverted fluorescence microscope and photographed.4.Apoptosis assay:Select the logarithmic growth phase cells, located three drug concentration groups and blank control group, after 24h,by flow cytometry to detect cell apoptosis.5.The changes of mitochondrial membrane potential and reactive oxygen species:Exponentially growing cells will be evenly suspended culture inoculated in 6-well plates,set three drug concentration groups and the control group after 24h,staining,detected the changes of mitochondrial membrane potential and reactive oxygen species by flow cytometry.Results 1.HXO-RB44 cell growth was inhibited after APS,with increased drug concentration in a dose dependent manner; most obvious inhibition for 24 hours.2.Morphological changes were dramatical under optical microscopy.The ampelopsin could obviously inhibit the growth of cultured HXO-RB44 cells,the IC50 in MTT assay is 14.71μg/ml;The repressive ratio in cell colony assay is 100% when medicine density is over 9.9μg/ml,the repressive ratio in cellcolony assay is 52.2% when medicine density is 4.4μg/ml.3.AO/EB fluorescent staining,there is a clear apoptotic cells,expressed as cell shrinkage,chromatin condensation,nuclear fragmentation and other morphological changes characteristic of apoptosis after the drug.4.APS can induce apoptosis significantly.At the 24 hours,the apoptosis rates of APS at concentration of 50.00μg/ml,14.84μg/ml,4.40μg/ml,0μg/ml are 88.13%,58.46%,14.83%,4.82 % ,there are significant difference between control group and experimental group.5.Different concentrations of APS role in HXO-RB44 cell, the level of mitochondrial transmembrane potential compared with the control group decreased significantly.6.The results showed that different concentrations of APS role in HXO-RB44 cell,intracellular reactive oxygen species levels and APS increased dose-dependent manner.Conclusions 1.Ampelopsin can inhibit the growth of HXO-RB44 in vitro. 2.Ampelopsin can induce apoptosis of HXO-RB44 in vitro.3.Ampelopsin induced HXO-RB44 cell apoptosis with a decline in mitochondrial membrane potential and an increase in the ROS generated.