The Study About The Apoptosis And It's Mechanism Of Acute Promyelocytic Leukemia Cell Lines NB₄ Cells Induced By Radix Clematidis Saponins

Objective:to investigate the apoptosis of Acute promyelocytic leukemia cell lines NB4 cells induced by Radix Clematidis Saponins and the expression of PML-RARa mRNA in NB4 cells induced by Radix Clematidis Saponins.Method:Radix Clematidis Saponins were extracted according to documents.0.01%DMSO were used as the negative control group,1.0umol/LAs2O3 as the positive control group, Radix Clematidis Saponins in different concentration as the experiment groups. NB4 cells were cultured with contained 10% fetal bovine serum in 37℃humidified atmosphere with 5%CO2 and passage every 3-5 days. NB4 cells were cultured separately in RPMI-1640 medium containing 0.01%DMSO, 1.0umol/LAs2O3, 10ug/ml Radix Clematidis Saponins,20ug/ml Radix Clematidis Saponins,40ug/ml Radix Clematidis Saponins,80ug/ml Radix Clematidis Saponins,160ug/ml Radix Clematidis Saponins.24,48,72 hours later, MTT was added and their A were measured. Cell growth inhibition rate and IC5o value were examined.NB4 cells were cultured separately in RPMI-1640 medium containing 0.01%DMSO,1.0umol/LAs2O3,30ug/ml Radix Clematidis Saponins.24,48,72 hours later, the NB4 cells were stained with Wright's-Giemsa liquid. The morphology changes were observed using optical microscope.NB4 cells were cultured separately in RPMI-1640 medium containing 0.01%DMSO,1.0umol/LAs2O3,30ug/ml Radix Clematidis Saponins.24,48 hours later, NB4 cells were mixed with Annexin V-FITC and Propidium iodide and incubated at room temperature for 20miri to detect the apoptosis rate.NB4 cells were cultured separately in RPMI-1640 medium containing 0.01%DMSO,1.0umol/LAs2O3,30ug/ml Radix Clematidis Saponins.24,48,72 hours later, NB4 cells were fixed by 75% ethanol and stained with PI liquid containing RNA enzyme to detect the distribution of cell cycle.NB4 cells were cultured separately in RPMI-1640 medium containing 0.01%DMSO,1.0umol/LAs2O3,30ug/ml Radix Clematidis Saponins.24,48,72 hours later, The mRNA of NB4 cells were extracted for real-time PCR to evaluate the influence of Radix Clematidis Saponins on the PML-RARa mRNA.Result:Radix Clematidis Saponins inhibited NB4 cells growth. The inhibition effect was increased with increasing Radix Clematidis Saponins concentrations and prolonged culture time. The difference has statistical meaning between any two groups. But the inhibition effect of Radix Clematidis Saponins was not as good as As2O3.The IC50 value was 247.91ug/ml in 24 hours group, 30ug/ml in 48 hours group,4.00ug/ml in 72 hours group.30ug/ml was chose as the best concentration.The morphological observation showed that cell volume decreased, nucleoli were highly condensed or apoptotic body formed, and the cytoplasm loosened to vacuolization as in the As2O3 group.The apoptosis effect was increased by increasing Radix Clematidis Saponins concentrations and prolonged culture time. But the apoptosis rate was lower than As2O3 group.The results of FCM showed that cell cycle was arrested at G2 stage and the best effect was found in 48 hours group.There was no difference of expression of PML-RARa mRNA between two groups or between different treated time of Radix Clematidis Saponins.Conclussion:Radix Clematidis Saponins can inhabit cell growth by inducing cell apoptosis.The optimal concentration and treament time of Radix Clematidis Saponins are 30ug/ml and 48 hours respectively.The possible mechanism of apoptosis is by cell cycle arresting.Radix Clematidis Saponins could not reduce the PML-RARa mRNA gene expression.