Effect Of Triptolide And Metoprolol On Mouse Melanoma B16 Cell Lines

Objective:The malignant degree and recurrence rate of melanoma are high,otherwise melanoma easily transfers to other place.Nowadays,its incidence continues to grow all around the world.There is no ideal treatment for it now.In order to find the effective drugs to cure melanoma,this paper discusses the effect of triptolide and metoprolol on B16 cell lines and the mechanism of action with the purpose of providing experimental basis for clinical therapy.Methods:1 Cell culture2 In order to understand the growth characteristics of the cells,to prepare for the follow-up test,the tetrazolium blue(MTT)assay were used to draw growth curve of B16 cells.3 The MTT method were used to detect the effects of two drugs on B16 cells at different time and different concentrations.4 The inverted microscope and HE staining were used to observe the changes of morphological characteristics of B16 cells before and after drug intervention.5 The application of FCM were used to detect cell cycle and apoptosis of B16 cells.6 The expression of vascular endothelial growth factor were detected by Immune Cell Technology.7 The statistics analysis by SPSS 16.0.Results:1 The growth curve of B16 cells in MTT wayAfter the cells were cultured in 96 well plates at different times.There OD numbers will be measured by the Microplate reader.The growth curve will be drew in the “s”way.2 MTT detection resultsWith the increase of drug concentration of the triptolide and metoprolol and the extension of time,the inhibitory effect of drugs on cells is very strong(P<0.05).Triptolide compared with metoprolol,the inhibition rate of 2.5ng/ml concentration triptolide group has no significant difference with the inhibition rate of 15.63ug/ml concentration metoprolol group after 24 hours(P>0.05);the inhibition rate of 5.0ng/ml concentration triptolide group has no significant difference with the inhibition rate of 31.25ug/ml concentration metoprolol group after 24 hours(P>0.05),there is significant difference compared with the rest of different concentration groups(P<0.05).The inhibition rate of 2.5ng/ml concentration triptolide group has no significant difference with the inhibition rate of 15.63ug/ml concentration metoprolol group after 48 hours(P>0.05);the inhibition rate of 5.0ng/ml concentration triptolide group has no significant difference with the inhibition rate of 31.25ug/ml concentration metoprolol group after 48 hours(P>0.05),there is significant difference compared with the rest of different concentration groups(P<0.05).There is significant differences between the two groups after 72 hours(P<0.05).Moreover,the group of 25ng/ml concentration of triptolide do maximal suppresion on B16 cells.3 Cell morphology under inverted microscope and the color of the mediumB16 cells grow well before drug intervention,B16 cells are polygonal or rhombic,good refraction,stick a wall to grow and single layer uniform distribution,there are a lot of filaments connections between cells,all of cell membrane are integrity,the nucleus can be seen.After drug intervention,the ability of cells to stick wall decreases,part of the cells are floating status and becoming small and round,intercellular connections disappeares,refraction decreases,and with the increase of concentration and the extension of time,there are greater changes in cell morphology,and compares with metoprolol,the effect of the triptolide on the cell morphology is stronger.The color of the medium showes a gradient color change from dark yellow to pink with the increasing of concentration and the action time of the drug extending in the triptolide group.Metoprolol group has no significant change in the color of the medium.4 Observation of B16 cells morphological characteristics with HE stainingBefore the drug intervention,the cytoplasm was pink,the nucleus were blue,the nucleus divided,the cell membrane were complete,the number of cell adherent was many,the cell connected closely,and cells distributed evenly.After drug intervention,the number of adherent cells decreases,intercellular connections disappeare,there are some isolated ones,clusters of cells and lots of shrinking and fractured nucleus,cell membrane are broken.Compared with metoprolol,the effect of the triptolide on the cell morphology is stronger.5 Effect of drugs on cell cycle and apoptosis of B16 cellsTwo drug groups compared with the control group,with the increase of drug concentration,the proportion of G0/G1 phase gradually increased(P<0.05),the cell ratio of S phase decreased(P<0.05).After 48 hours of the two drug intervention,it was not found significant sub G1 peak(apoptosis peak)before B16 cell DNA histogram G1 peak.6 Effect of drugs on the expression of VEGF protein of B16 cellsTwo drug groups compare with the control group,the expression of VEGF protein decreased(P<0.05),VEGF expression of triptolide treated groups decreased with drug concentration increased(P<0.05),the expression of VEGF of metoprolol groups had no significant decrease with the increase of drug concentration(P>0.05).Comparison of triptolide groups and metoprolol groups,VEGF protein expression of triptolide groups decreased more significantly.Conclusions:1 Triptolide and metoprolol can both inhibit proliferation of melanoma B16 cell,and have a certain concentration-dependent and time-dependent within a certain range.2 Triptolide and metoprolol can inhibit the cell cycle in G0/G1 phase and have a concentration-dependent manner within a certain range.This experiment does not detect apoptosis phenomenon caused by the two drugs,The reason may be because the intervened cells is during the early stage of apoptosis,it can not be ruled out the possibility of two drugs through apoptosis mechanism to inhibit B16 cells,which needs further explored.3 Triptolide and metoprolol can make B16 cells decrease the expression of VEGF and triptolide stronger than metoprolol and a concentration-dependent manner.It can be inferred that inhibition of VEGF expression may be one of the mechanisms of the anti-tumor effect of the two drugs.4 The effect of triptolide on proliferation of B16 cells is much stronger than metoprolol.