| Abstract:bjective:To investigate the apoptosis of acute promyelocytic leukemia (APL) resistant medicine cell MR2 cell lines induced by Radix Clematidis Saponins. We try to find a new treatment for patients with resistant acute Promyelocytic Leukemia to alleviate their symptoms, improve their quality of life, prevent recurrence and metastasis and prolong their life.Method:We extracted Radix Clematidis Saponins based on documents. MR2 cells were cultured with RPMI-1640 medium contained 10% fetal bovine serum in 37℃humidified atmosphere with 5%CO2 and passaged every 3-5 days. Cells in loganthmic phase cells were used for experiments. The cells on the concentration of 1 X 104/ml were suspended and seeded in 96 well microplates. There were eight treated ways with 0.01%DMSO as the blank control group and 1.0 umol/L As2O3 as the positive control group (n=5 parallel samples for every group, and repeat Experiment 3 times).The different treated concentration of 200 ug/ml,400ug/ml,600 ug/ml,800 ug/ml,1000ug/ml,1200 ug/ml Clematidis Saponins were used separately as the experimental groups. The proliferative inhibition rate of MR2 cell was examined using WST-1. Cells on the concentration of 1 X 106/ml were suspended and seeded in 6 well plates. There were 5 different treated ways with 0.01%DMSO as the blank control group and 1.0 umol/L As2O3 as the positive control group (n=5 parallel samples for every group, and repeat Experiment 3 times).The different treated concentration of 800 ug/ml,1000ug/ml,1200 ug/ml Clematidis Saponins were used separately as the experimental groups. After 72 hours treating, the appoptosis rate was examined using the FCM.At the same time, the changed morphology was examined using fluorescence microscope. Result:The effect of Radix Clematidis Saponins at different concentration of 200 ug/ml,400 ug/ml,600 ug/ml,800 ug/ml, 1000ug/ml, 1200 ug/ml on MR2 cells growth inhibition was obviously and it was accordance with the treating time and concentration of Radix Clematidis Saponins. Radix Clematidis Saponins at different concentration can inhibit MR2 cells growth. The inhabition rate increased gradually when medicine concentration increased and treating time prolonged. Radix Clematidis Saponins at height concentration had the same effect on MR2 cells as As2O3 group did. The apoptosis rate also increased when medicine concentration increased and treating time prolonged. In As2O3 group and the Saponins 1200ug/ml group there wasn't significant difference. Compared Saponins 1200ug/ml group with the Saponins 800ug/ml group and 1000ug/ml group, there were significant difference. Between Saponins 800ug/ml group and Saponins 1000ug/ml group, there was statistical difference. Annexin-V is a molecular weight of 35～36KD of the Ca2+dependent phospholipid binding protein which has a high affinity for phosphatiadylserine. In the apoptosis cells Annexin-V can bind with phosphatiadylserine on the outer membrane and produces green fluorescence signal. As a nucleic acid dye, PI can not come through the complete cell membrane.Bu it come through the membrance and bind to nucleus and produces red fluorescence signal in apoptosis cells and death cells. Under fluorescence microscopy, the volume of the treated cells were decreased, the nucleolus were highly condensed and even disrupts or formed apoptotic body which was same as the As2O3 group did.Discussion:1 Radix Clematids Saponins can inhabit cell growth and the effect is accordance with the treating time and concentration of Radix Clematidis Saponins.2 Radix Clematids Saponins can induce cell apoptosis and the effect is accordance with the treating time and concentration of Radix Clematidis. The apoptosis rate of MR2 cells treated by Radix Clematids Saponins at height concentration is same as AS2O3 group does.3 Radix Clematids Saponins will become a new economic,effective and low side effects of drugs which alone or in combination with other drugs treatment of acute promyelocytic leukemia and drug resistance in patients.
|
PDF Full Text Request