The Study Of The Mechanism About Effecting On Activity By Triptolide On Cisplatin-resistant Human Ovarian Cancer In Vitro

Objective:This experimental is about further study the effecting on activity by triptolide on COC1/DDP in vitro. Preliminary study the effecting on activity by triptolide on COC1/DDP in vitro of the mechanism and resistance mechanism.Triptolide is as for the treatment of advanced or cisplatin-resistant ovarian cancer drug and provide experimental basis.Methods:1, COC1/DDP cell's serial subcultivation:Cultivated COC1/DDP cell with 10%FBS and 90%RPMI-1640 complete nutritive medium. Adjusted the cell density to 5×10-7ml and put it in culture flask. Cultivated it in constant temperature incubator with the condition of 37℃.5%CO2 and saturated humidity.When the cell is logarithmic growth phase, we blowed the liquid in culture flask to monoplast suspension and centrifugated in 1000r/min for 10min.Then discard the supernatant fluid and changed it with COC1/DDP complete nutritive medium, adjusted the cell density to 5×105/ml and the liquid volume to 7ml in culture flask.2, The influence after the DDP done with COC1/DDP cell:Cells in action group was interfered with DDP in different concentration (0.625.1.25,2.5,5,10,20,40)μg/ml, and it divided into 7 DDP action groups according to different concentration. And the cells in blank group were interfered with COC1/DDP complete nutritive medium. All the groups were interfered for 24h,48h and 72h respectively. Then used the microculture tetrazolium (MTT) assay to determine the result of optical density which can reflect the quantity and the activity of cell.The result can reflect the influence about the DDP do with COC1/DDP cell, and can simultaneously calculate the cell's inhibition ratio and the value of IC50.3,The influence after the triptolide done with COC1/DDP cell:Cells divided into 9 groups(negative control group, DDP group, low concentration TP group, low concentration TP and DDP group, moderate concentration TP group, moderate concentration TP and DDP group, high concentration TP group, high concentration TP and DDP group, blank group), All the groups was interfered for 24h,48h and 72h respectively. Then used MTT assay to determine the result of OD, and can simultaneously calculate the cell's inhibition ratio by triptolide.4,The change of cell morphous and ultrastructural organization after the triptolide done with COC1/DDP cell:Use the inverted microscope and transmission electron microscope to observe the change of cell morphous and ultrastructural organization in the blank group and moderate concentration TP(10ng/ml) group after 24h.5,The influence of cell apoptosis rate and cell cycle after the triptolide done with COC1/DDP cell:Choose the logarithmic growth phase cells, use flow cytometry to observe the change of cell apoptosis rate and cell cycle in the blank group, low concentration TP group (3ng/ml), moderate concentration TP(10ng/ml) and high concentration TP group (50ng/ml) after 24h.6,The influence of DNA change after the triptolide done with COC1/DDP cell: Choose the logarithmic growth phase cells, get out the DNA of the two group cells.Used 1.2%Agarose Gel Electrophoresis to analyze DNA chromosome break condition in blank group and moderate concentration TP (10ng/ml) after 24h.7,Caspsase-7 protein expression on COC1/DDP cells by triptolide:Cells divided into two groups (blank group and moderate concentration TP group), after done 24h, make these two group suspension cells cell block and wax block according to conventional. Use the immunohistochemical to detect the difference of caspsase-7 protein expression in two groups.Results:1,Determine the logarithmic phase:From the growth curve of COC1/DDP cell, after 3-4d is the logarithmic growth phase cells, determined to take the role of 3-4d cells as target cells.2,MTT assay results of DDP on the inhibition of cells:The result shows the inhibitory effect of DDP on the COC1/DDP cells in a dose and time dependent. The IC50 after 24h was 5.567μg/ml, after 48h was 2.866μ/ml and after 72h was 1.161μg/ml. So,0.5μg/mlDDP in the complete nutritive medium didn't have effect on COC1/DDP cell.3,MTT assay results of TP on the inhibition of cells: Triptolide inhibition COC1/DDP cells were time-dose relationship.Different concentrations of TP and 2.5μg/ml cisplatin in the combined effect of synergistic effect COC,/DDP cells (P<0.05).4. TP effected the morphological and ultrastructure of the COC1/DDP:4.1 Inverted microscope:it seems that cells in blank group grown well, light of the strong cells, the cells were round or irregular. In the TP group, cells were multiformity, cell bodies became smaller, cytoplasmic particles increased, cells became flat, medium cell debris can be seen in the background.HE staining COCj/DDP cell atypia, increased nuclear cytoplasm ratio, giant multi-core and other changes.4.2 Observed cell ultrastructure:in the blank group, cells are oval and oval nuclei, chromatin is rich in cells with normal morphology. In the TP group, cells were apoptotic changes, as follows:disappearance of cell surface microvillus, mitochondria increased, endoplasmic reticulum, cell membrane shrinkage, cytoplasm condensation and cavity sizes appear, nuclear chromatin condensation, crescent-shaped pool around the nuclear membrane, there apoptotic body formation.5,The results of flow cytometry:The role of different concentrations of TP done with COC1/DDP cells after 24h. The role of flow cytometry after cell apoptosis.Low concentration TP group,Moderate concentration TP groups High concentration TP group all can see apoptosis image, apoptosis rate is (2.69±0.23)%,(5.30±0.18)%and (16.67±0.22)%, S phase is (48.86±0.61)%,(45.96±0.42)%and (38.72±1.11)%, G1 phase is (35.48±0.44)%, (45.38±0.35)%and (55.34±0.67)%, G2 phase is (15.67±0.71) %, (8.66±0.28)%and (5.94±0.90)%. The result hint:TP can promote COC,/DDP cell apoptosis, cells arrest in G1 phase, with the TP concentration increased, the rate of apoptosis increased, they are dose-effect relationship(P<0.05). Apoptosis mechanism by affecting cell cycle and promotes cell DNA fracture.6,The TP effected the DNA of the COC1/DDP:Triptolide effected on COC1/DDP cells after 24h, showing different lengths of apoptosis was specific "ladder" change, but no such cells in the blank group performance. TP induced COC1/DDP cell apoptosis individually. 7,Immunohistochemical detected the expression of Caspase-7 protein about TP done with COC1/DDP cells after 24h. The cytoplasmic staining seen to brown, apoptotic protein Caspase-7 was strongly positive, in the control group, the cytoplasmic staining seen to nankin, apoptotic protein Caspase-7 was weakly positive.Conclusion:1,DDP can significantly inhibit COC1/DDP cells in vitro,and it is concentration and time dependence. In the COC1/DDP complete nutritive medium,0.5μg/ml concentraton DDP can not inhibit COC1/DDP cells.2,TP can inhibit COC1/DDP in a time-dose relationship.3,Different concentration TP combined with 2.5μg/ml DDP can inhibit COC1/DDP cell, DDP combined inhibitory effects of TP on a synergistic effect.4,TP can promote COC1/DDP cells apoptosis, apoptosis rate and TP dose are positive correlation, apoptosis cycle arrest at G1 phase.Observed molecular level through the promotion cells to DNA break.5,In the COC1/DDP resistant cell lines, Caspase-7 protein expression was lower, after the TP done with COC1/DDP cell, Caspase-7 protein expression was highly expressed, TP promote the mechanism of COC1/DDP cell apoptosis that the expression of Caspase-7 protein increased, whereas the COC1/DDP cells resistant mechanism is downregulation of Caspase-7 protein.