| Objective:To research whether the radiofrequency ablation could trigger quick growth of residual tumor cells around the focus of ablation, the molecular biological mechanism of killing the tumor cells by radiofrequency ablation, and the inhibited effect of artesunate for the residual cells after radiofrequency ablation.Methods:In the research of the influence of radiofrequency ablation on the growing status of the tumor cells around the focus of ablation, trypan blue staining was first applied to test the killing effect of radiofrequency ablation to HCT116cells with variant applying temperatures and time, and the status of the residual tumor cells at variant time points after ablated with variant temperatures; observation of the status of the residual tumor cells at variant time points after ablated with variant temperatures by optical microscope is also included. Then flow cytometry was used to detect the apoptosis rates of the residual tumor cells after ablated with variant temperatures.During the experimental research of application of radiofrequency ablation combined with artesunate, the best range of applying temperature of radiofrequency ablation was sifted out. And then the influence of artesunate on the proliferation of HCT116cells was tested by MTT; then3gradients of temperature and one best concentration of artesunate were combined, forming3combining experimental groups, added with the single-RFA group, the single-ART group and the blank control group without any interference. Experimental groups were incubated in the medium (0.4%FBS inside)with ART for24hours.The single-ART group was incubated in the medium (0.4%FBS inside)with ART for24hours as well. The single-RFA group was incubated in the medium (0.4%FBS inside) for24hours as soon as the radiofrequency ablation was finished. The blank group was incubated in the medium (0.4%FBS inside) for24hours directly. The status of the residual tumor cells at variant time points after ablated with variant temperatures was evaluated by optical microscope and trypan blue staining. The apoptosis rates of the residual tumor cells after ablated with variant temperatures were detected by flow cytometry later.Results:The research results of the influence of radiofrequency ablation on the growing status of the tumor cells around the focus of ablation:after the cells attached to the bottom of the plate were applied with radiofrequency ablation, part of the cells in tho centre of the plate touched by the electrode pin fell off, which formed an area with diameters ranging from1mm to3mm. The cells after applying with RFA were incubated for0hour,2hours,6hours,12hours, and24hours. Observed by the optical microscope, the cells near the focus of RFA suffered the coagulative necrosis which keeps the shapes proper to the cells. These cells atrophied and were smaller than surrounding normal tumor cells. The cells with coagulative necrosis were surrounded by a loop area formed with bubble-like cells that would gradually fall off during the following incubation. The cells within0.5cm outside of the loop formed by the bubble-like cells are ablation focus’surrounding tumor cells. Stained with trypan blue, the blue-stained area that was composed of dead cells surrounded the area where cells fell off. As applying temperature rose and applying time extended, the blue-stained area expanded, and the cells in a loop around the blue-stained area began to fell off. Under the radiofrequency ablation of55℃, there was an ablation focus with an average diameter of0.25cm in the plate; under70℃, there was an ablation focus with an average diameter of0.5cm in the plate; under85℃, there was an ablation focus with an average diameter of lcm in the plate; under100℃, most of the bottom of the plate were stained blue.24hours after the RFA of85℃,70℃, and55℃, the apoptosis rates of residual tumor cells around the ablation focus are18.92%±15.83%,10.89%±1.90%, and11.90%±3.25%, separately, which are all higher than the blank group (2.45%±0.23%).The research results of application of radiofrequency ablation combined with artesunate:After ART applying to IICT116cells for24hours and48hours, the IC50were98.887μg/ml and28.802μg/ml, separately. Low dosage of ART had mild inhibiting effect on IICT116cells; as the concentration of ART increased, the cell growth was evidently inhibited. Different concentrations of artcsunate showed a dose-dependent and time-dependent manner during their applying time. Incubated in the medium(0.4%FBS inside) with artesunate of100μg/ml of24hours after RFA of55℃,70℃, and85℃, most cells were round and atrophied except the eells with the coagulative necros is, some eel1s atrophied and the shape became irregular. Part of the cells within the ablation area fell off and floated. The bottoms of the combined group’s plates were almost stained blue according to the trypan blue staining experiment. A loop with cells falling off could be seen and became clearer as the applying temperature increased, compared with the single-ART group and the blank group. The apoptosis rates of the cells from every group were tested and we got a remarkable synergistic effect (P<0.05) when the RFA of85℃and the artesunate of100μg/ml was applied together which was obviously better than the single application of artesunate.Conclusion:Radiofrequency ablation could not stimulate residual tumor cell into rapid growth in vitro and may also results in progressive injury to tumor cells. Applying artesunate of100μg/ml after the RFA of85℃can produce a synergistic effect. |
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