Experimental Study Of Arsenic Trioxide Combined With Radiofrequency Ablation In The Treatment Of Liver Cancer

Objectives:1.To study the effect of different concentration of arsenic trioxide on the survival rate of HepG2 cells;2.To study whether arsenic trioxide combined with radiofrequency ablation can enlarge the damage area of tumor radiofrequency ablation.Methods:1.The Hep G2 hepatoma cells were resuscitated and cultured.The HepG2 cells in the logarithmic growth stage were seeded in 96 well plate and placed in 37?and 5%CO₂ incubator.After 12 hours of culture,the cells were observed by light microscope.Then,the blank control group C?with the same amount of PBS?and the experimental group L?5?mol/L?,M?10?mol/L?,N?20?mol/L?and H?30?mol/L?were set,After continuous culture for 24 hours,the morphology and distribution of cells were observed under electron microscope.The OD value of each group was measured by MTT method.The survival rate of cells in the control group was calculated as 100%,The survival rate?S?=?experiment group od/control group OD?×100%,and the average survival rate of cells in each group was calculated and compared.2.Tumor bearing model of nude mice:24 5-week-old immunodeficient male nude mice?BALB/C??20g±2g?were injected with 0.1ml logarithmic growth HepG2 hepatoma cells?2×106 cells/ml?in the axillary midline of the right forelimb.They were fed on the laminar flow frame under SPF condition,following the principle of asepsis,disinfecting the bedding,feed and water,and the nude mice were fed freely.The tumor size was measured every 3 days.The tumor grew to1cm×1cm,and the experiment was started Intervention.3.Radiofrequency ablation and radiofrequency ablation combined with arsenic trioxide intervention:24 tumor bearing nude mice were randomly divided into four groups?n=6?.Radiofrequency ablation alone group R:radiofrequency ablation directly?70?±2??for 5 minutes;the other three groups?T1 group,T2 group,T3 group?were injected with arsenic trioxide?5mg/kg?intraperitoneally at the same time.Radiofrequency ablation was carried out after 1 hour,3 hours and 6 hours,respectively?70?±2??5 minutes.The tumor was removed after operation.4.Detection index:the tumor samples were fixed by tissue fixative,and immunohistochemistry?CD31 staining?was used to detect the microvessel density and tumor damage area.Results:1.The effect of different concentration of arsenic trioxide on HepG2cells under microscope?magnified 200 times?:After different concentrations of arsenic trioxides were applied to Hep G2 cells,the morphology and distribution of HepG2 cells changed 24 hours after the intervention of different concentrations of arsenic trioxides.In group C,the cell morphology was normal,the cell nucleus was clear,the cell growth was full,the distribution was uniform,and the cell growth was good;in group L,M,N,H,with the increase of the concentration of the drug,the cell distribution was uneven,the cell leaf disappeared,the cell growth was poor,the heteromorphism increased,the cell nucleus disappeared,and the cell apoptosis was vacuole like.2.The results of MTT showed that the OD value of L group,M group,N group and H group was significantly lower than that of C group?P<0.01?,S of L group,M group,N group and H group were 84%,77%,68%and 63%respectively,S of L group,M group,N group and H group were significantly lower than that of C group?P<0.01?,S of L group,M group,N group and H group were compared,L>M>N>H?P<0.05?.The results showed that arsenic trioxides could significantly reduce the survival rate of HepG2 cells,and the survival rate of HepG2 cells decreased in a dose-dependent manner.The cell survival rate of HepG2cells was significantly decreased when treated with 20?mol/L arsenic trioxides in different concentration gradients for 24 hours.3.Immunohistochemistry of paraffin section under microscope?magnification 200 times?:The number of microvessels in T1,T2 and T3groups was significantly reduced compared with that in R group,and the damaged area was increased.4.Microvessel density?MVD?:MVD was compared among groups,compared with R group,MVD of T1,T2 and T3 group decreased significantly?P<0.01?.The MVD of T1 group was significantly lower than that of T2 group and T3 group?P<0.01?.There was no significant difference in MVD between T2 group and T3 group?P>0.05?.The experimental results showed that arsenic trioxides could significantly reduce MVD in tumor,and the decrease of MVD in one hour after administration was more significant than that in three hours and six hours after administration,while the change of MVD in three hours after administration was not significant compared with that in six hours after administration.5.Tumor damage area:compared with R group,T1,T2,T3 group showed a significant increase in tumor lesion area?P<0.01?.The tumor damage area of T1,T2 and T3 combined group was compared,the tumor damage area of T1 group was significantly higher than that of T2 group and T3 group?P<0.01?.There was no significant difference between T2group and T3 group?P>0.05?.The experimental results showed that arsenic trioxides combined with radiofrequency ablation could significantly expand the tumor damage area after radiofrequency ablation.The tumor damage area at 1 hour after arsenic trioxides administration was significantly larger than that at 3 hours and 6 hours after arsenic trioxides administration,while the tumor damage area did not change significantly at 3 hours and 6 hours after arsenic trioxides administration.6.Correlation analysis of MVD and tumor lesion area:correlation coefficient r=-0.6,significance?double tail?P<0.01.The experimental results show that arsenic trioxides can reduce the microvessel density and increase the tumor damage area.Conclusions:1.Arsenic trioxide reduced the survival rate of HepG2 hepatocellular carcinoma cells in a dose-dependent manner;2.Arsenic trioxides can reduce the microvessel density in the tumor and increase the area of the tumor damaged by radiofrequency ablation.Single dosing for 1 hour has the most synergistic effect.