Influence On Activity By Triptolide On COC₁/DDP In Vitro

Objecvtive:The purpose of this study is using triptotide in different concentration to interfere in COC₁/DDP,and observing the influence on activity of COC₁/DDP cell and the relation between quantity and effectiveness.In purpose to found the effective drug to treating DDP-fast ovary cancer patient.Methods:1 COC₁/DDP cell's serial subcultivation:Cultivated COC₁/DDP cell with COC₁/DDP complete nutritive medium.Adjusted the cell density to 5×10⁵/ml and put it in culture flask.Cultivated it in constant temperature incubator with the condition of 37℃,5%CO₂ and saturated humidity.Every 2～3 days when the cell full of culture flask,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 COC₁/DDP cell's growth curve:Picked the cell inlog phase into the 24 pore plate and cultivated it 5 days in constant temperature incubator with the condition of 37℃,5%CO₂ and saturated humidity.Used 3 pore cell every day to calculate the cell density with ttrypan blue,recorded the average.Drewed the COC₁/DDP cell's growth curve with the longitudinal axis for cell density and the lateral axis for times.From the COC₁/DDP cell's growth curve,it can be showing the growth cycle of COC₁/DDP cell and the best time to interfere in.3 The influence after the triptotide done with COC₁/DDP cell:Cells in action group was interfered with triptotide in different concentration(1ng/ml,3ng/ml,5ng/ml,10ng/ml,20ng/ml,50ng/ml,75ng/ml,100ng/ml),and it divided into 8 triptotide action groups according to different concentration.And the cells in blank group was interfered with COC₁/DDP complete nutritive medium.All the groups was 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.More high of the result of optical density,more active the cells were.the result can reflect the influence about the triptotide do with COC₁/DDP cell,and can simultaneously calculate the cell's inhibition ratio and the value of IC50.4 The chang of cell morphous and ultrastructural organization after the triptotide done with COC₁/DDP cell:From the inverted microscope and transmission electron microscope ,to observe the chang of cell morphous and ultrastructural organization in the blank group,low concentrantion TP group and high concentrantion TP group after 48h.Results:1 The growth cycle of COC₁/DDP cell cultivanted in vitro:From the growth curve of COC₁/DDP cell,it shows that cell actively proliferate in the first,second and third day,after the third day,cells go through into the platform,then the quantity of cell step down because of the limitation of growth space and the relatively short of nutrition.2 The influence after the triptotide done with COC₁/DDP cell:From the regult we can see that the effect was dependent on triptotide's concentration and working time.From 3ng/mlto100 ng/ml,it all worked which triptotide could inhibit the growth of COC₁/DDP cell efficiently.Its IC₅₀ after 48h was 51.76ng/ml,and IC50 after 72h was 10.78ng/ml.3 The chang of cell morphous and ultrastructural organization after the triptotide done with COC₁/DDP cell:3.1 The chang of COC₁/DDP cell morphous:From the inverted microscope,it seems that cells in blank group grown well;Cell clumps in low concentrantion TP group was reduced,cells became flat and the lucency descent.The quantity of cells reduced contrast to blank group;Cell morphous in high concentrantion TP group was multiformity,cytoplasm grana was increased and the quantity and cell concentration of cells reduced objectively contrast to low concentrantion TP group. 3.2 The chang of cell's ultrastructural organization after the triptotide done with COC₁/DDP cell:3.2.1 Blank group: To object the chang of COC₁/DDP cell's ultrastructural organization from transmission electron microscope,it shows that cells in blank group was orbicular-ovate,it has a big cell nucleus,the nuclear-cytoplasmic ratio was disproportion,and abnormity of cell nucleus.Chromoplasm was plentiful and less of cell organelle.3.2.2 In low concentrantion TP group: it can be seen that part of COC₁/DDP cell have large cell volume as polygon.Cell nucleus became larger and has content in it.,the chromoplasm became aggregated,pycnosis,fragmentation or dissolved.,the nuclear membrane invaginated,the quantity and morphous of mitochondria changed,and Golgi's apparatus dissolved.3.2.3 In high concentrantion TP group: it can be seen that COC₁/DDP cell's membrane was complete,the cell organelle increased.Most cell can be seen that chromoplasm in cell nucleus aggregated into apoptotic body beside the cell membrane.Conclusion:1 It has depressant effect By Triptolide on DDP-fast Overian Cancer in vitro,the working concentration was from 3ng/ml,and the depressant effect depend on drug concentration and working time.Its IC₅₀ after 48h was 51.76ng/ml,and IC₅₀ after 72h was 10.78ng/ml.2 Low and high concentrantion TP both have the depressant effect to COC₁/DDP cell,its mechanism may be which it could induce cell senium and apoptosis of COC₁/DDP cell.3 This study used the COC₁/DDP cell as experimental model in vitro,and it found that triptotide has depressant effect on DDP-fast Overian Cancer in vitro.it offered information for found effective drug to treat DDP-fast Overian Cancer,and also offered the experiment foundation in vitro for triptotide become the effective drug to treat DDP-fast Overian Cancer.