| ObjectiveThis study was to investigate the apoptosis of human Leukemia HL-60 cells induced by crocin and its possible mechanisms.MethodCrocin solution which was prepared under sterile conditions were divided into six groups A-F, which propotional crocin final concentrations were 0,0.625,1.25,2.5,5.0 and 10 mg/ml. The growth curve of HL-60 cells was measured by blood cell count plate artificial count method after the role of different concentrations of crocin. Using fluorescence microscopy to examine cell morphology. After acridine orange/ethidium bromide (AO/EB) mixed dye staining,apoptotic cells were counted under fluorescence microscope,then calculating the apoptotic rate.Cell proliferation was measure through MTT method,calculating the inhibition rate of HL-60 cells under the role of different concentrations of crocin at different times.HL-60 cell cycle distribution was measured by flow cytometry after 48h under the role of Crocin, of crocin role of 24,48 h after bcl-2, bax gene expression.were detected by RT-PCR after 24h,48h under the role of crocin.Resultâ‘ .Determination result of cell growth curve showed that, when Crocin concentration among 0.625-10mg/ml, it could inhibit the proliferation of HL-60 cells, with the increase of drug concentration and the role of time, HL-60 cells decreased.The inhibition of the HL-60 cell was the most obvious under 10 mg/ml concentration for 48h.MTT assay showed that after crocin roled in HL-60 cells for 24h, the inhibition rate gradually increased when the drug concentration increased,the difference among groups were significant(F=2.84,P<0.05).The inhibition rate was highest when the crocin concentration was 10 mg/ml. After crocin roled in HL-60 cells for 48h, the inhibition rate gradually increased when the drug concentration increased,the difference among groups were significant(F=4.71,P<0.05).When the crocin concentration was 10 mg/ml,there was significant difference between the role of 48h and 24h(t=3.59,P<0.05).â‘¡.Cell morpholology observed under fluorescence microscopy showed that after crocin roled in HL-60 cells for 24h,apoptotic cells appeared occasionally in the control group.When the drug concentration increased from 0.625mg/ml to 5mg/ml,the number of apoptotic cells markedlly incread.The apoptotic cell morpholology was the most evident at the concentration of 5mg/ml.There were a lot of necrosis and disintegration cells inseased of apoptotic cells at the concentration of 10mg/ml. After crocin roled in HL-60 cells for 48h,the apoptpsis rate of HL-60 gradually increased when the drug concentration increased from 0.625mg/ml to 5mg/ml, the apoptpsis rate decreased at the concentration of lOmg/ml.When the drug concentration was too high it showed the role of necrosis.â‘¢.Cell cycle detected by flow cytometry showed that, after crocin roled in HL-60 cells for 48h, in 0-5mg/ml concentration range, the proportion of G0/G1 phase cell have significant differences between groups (F=185, P<0.05), there were significant differences in comparison group(P<0.05). In 0.625-5mg/ml concentration range,with the increase of drug concentration,the proportion of G0/G1 phase cell number gradually increased,proportion of G0/G1 phase cells was the highest at the concentration of 5mg/ml.which significant increase than other groups (P<0.01).But When the concentration reached 10mg/ml,proportion of G0/G1 phase cell number significant decreased compared with the concentration of 5mg/ml group (P<0.05), and there were not statistically significant compared with the A-D groups (P>0.05).â‘£.RT-PCR results showed that when reaction time was 24h,48h, with the increase of the concentrations of crocin,bcl-2 gene band intensity decreased gradually. Grayscale scan ratio results show that in 0-10mg/ml concentration range, the difference between two groups was significant (P<0.05),there was not significant differfence between A group and B group(P>0.05). With the increase of drug concentration, bcl-2 gene expression decreased, the expression was weakest at the concentration of 10mg/ml.When reaction time was 24h,48h,with the increase of the concentrations of crocin, bax gene band intensity increased gradually. Grayscale scan ratio results show that in 0-10mg/ml concentration range, the difference between two groups was significant (P <0.05), there was not significant difference between A group and B group (P>0.05).With increasing drug concentration,bax gene expression increased,the expression was strongest at the concentration of 10mg/ml.Conclucion1.The growth of HL-60 cells was inhibited remarkably in the dosage dependence and time way.The apoptosis rate was gradually increased within the concentration 0.625-5mg/ml.When the crocin concentration was 10mg/ml,the percentage of apoptosic HL-60 cells was not increased,but decreased,while the cells necrosed. 2.Flow cytometry profiles revealed that cells were blocked at G0/G1 phase,the cell proliferation was inhibited obviously at 5mg/ml.The rate of G0/G1 cells decreased at 10mg/ml.When the drug concentration was too high, the most role was cytotoxicity.3.Crocin inhibite the expression of Bcl-2 gene,it reflected inhibition role within 1.25-10mg/ml.The inhibition was strongest at the concentration of 10mg/ml. Crocin inhibite the expression of Bax gene,it reflected inhibition role within 1.25-10mg/ml.The inhibition was strongest at the concentration of 10mg/ml for 48h.4.Crocin can promote apoptosis,inhibit cell growth,the mechan ism was for the inhibi tion of Bcl-2 gene expression and the promotion of the Bax gene expression,ultimately leading to the cell cycle arrest at G0/G1 phase.
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