| Tongue squamous cell carcinoma is one of the most common malignant tumors in oral and maxillofacial region, which occur in male more than female. On the one hand, because of the special position and important function, there will be a great challenge for survival of patients once the tongue is damaged. On the other hand, TSCC pathogenesis is not clear, and it with insidious onset, high malignant, high lymph node metastasis rate, and easy to recurrence, all above has brought the huge challenge for the clinical diagnosis and treatment. In recent years, potassium channel, especially voltage-gated potassium channel is paid more attention by the tumor researchers. Voltage-gated potassium channel is one of Potassium channel, which exists widely in membrane of excitable and non-excitable cells. It involved in a variety of cell physiological function, with a certain effects on a variety of cell proliferation, cycle and apoptosis.4-aminopyridine is a potassium channel blocker, which can selectively blocking Kv channels. A number of studies shows that,4-AP can inhibit a variety of tumor cell proliferation, induce cell apoptosis, regulate and participate in cell cycle. Objective:This experiment adopts the CCK-8 to detect the Inhibition of human tongue squamous carcinoma Tca8113 cell proliferation caused by 4-AP. Influence of Tca8113 cell cycle and apoptosis that 4-AP caused was detected by flow cytometry. Through above methods to determine whether 4-AP can inhibit human tongue squamous cell carcinoma cell growth or proliferation, whether it can promote the cell apoptosis, and provide reliable theoretical basis to the prevention, diagnosis and treatment of human tongue squamous cell carcinoma. Methods:1. CCK-8 assay was used to detect the effect of 4-AP on the proliferation of human tongue squamous carcinoma Tca8113 cell:human tongue squamous carcinoma Tca8113 cell co-cultured with 4-AP at the concentration of 1,5,10,20,50, 100mmol/L for 12,24, 48hours, observate the cell morphology by using the inverted phase contrast microscope, detect the OD value of each group and calculate the inhibition of the cell proliferation rate caused by 4-AP at different concentration and time; 2. Detect the effect of 4-AP on cell cycle of human tongue squamous carcinoma Tca8113 cell by flow cytometry:Co-culture cells and 4-AP at the concentration of 5,10,20,50mmol/L for 24hours, then flow cytometer detect the cell cycle changes after 70% ethanol fixation cell for 12hous; 3. Detect the effect of 4-AP on cell apoptosis of human tongue squamous carcinoma Tca8113 cell by flow cytometry:Co-culture cells and 4-AP at the concentration of 5,10,20,50,100mmol/L for 24hours, Annexin V-FITC and PI double staining, detecte the apoptosis by flow cytometer; 4. All data were analyzed by SPSS 19.0 software, and they expressed by x±s. Experiment groups were compared with single factor analysis of variance (one-way ANOVA). There are significant differences once P<0.05. Results:1.The inverted phase contrast microscope shows that, with the increase of 4-AP concentration and culture time, cells occured a series of morphological changes, including a decrease in cell volume, cell shrinkaged and turned round, cell chromatin agglutination, many vacuoles intracellular and so on.2. 4-AP can cause an inhibitory effect on proliferation of human tongue squamous cell carcinoma Tca8113 cell. Single factor analysis of variance (One-way ANOVA) shows that, compared with the negative control group, the inhibition rate that 4-AP at concentration of 1,5,10,20,50 and 100mmol/L groups for 12hours, respectively, was 0.65%,1.48%,2.98%, 6.43%,23.57%,and 47.26%(P<0.01, the difference was significant); when the co-culture time increased to 24 hours, cell proliferation inhibition rates were 0.76%,8.40%,10.64%,13.30%,36.95% and 74.50%(P<0.01, the difference was significant); and when the drug intervention time increased to 48 hours, cell proliferation inhibition rate was 1.01%,6.69%,30.16%, 72.45%,79.33% and 80.15%(P<0.01, the difference was significant). Analysed the cell proliferation inhibition rates of 4-AP on Tca8113 cell at the same concentration in different times, the results shows that the cell proliferation inhibition rates in 1mmol/L concentration group, respectively, was 0.65%,0.76%and 1.01%, compared with the control group, P>0.05, there is no significant difference. Comparison of the concentration of 5,10, 20,50,100mmol/L, the rest of the experimental group, were P<0.01, which has significant difference.3. Flow cytometry analyses the Tca8113 cell cycle that 4-AP caused at the concentration of 5,10,20,50mmol/L. The result was Go/G] phase cell proportion rose form49.60% to 71.73% compared with control group, there is significant difference(P<0.01); S phase cells ratio is reduced from 40.97% to 17.03%, which was also compared with control group, with significant difference(P<0.01); G2/M phase cells ratio, respectively, is 10.63%,10.97%,12.27% and 10.97%, the difference was not statistically significant (.P>0.05); All result above indicated that 4-AP can block Tca8113 cells at the G0/G1 phase.4. Flow cytometry test the cell apoptosis rate of human tongue squamous carcinoma Tca8113 cell that 4-AP induced at concentration of 5,10,20,50, 100mmol/L for 24hours. The apoptosis rate of 4-AP concentration of 5,10,20,50mmol/L, respectively, is 6.79%,6.34%,7.28%,7.39%, and 7.08% compared with control group were, the difference was not statistically significant (P>0.05); the apoptosis rate of the concentration of 100mmol/L group is 35.01%, with significant difference (P<0.01). Conclusion:1.4-AP can obviously inhibit the proliferation of Tca8113 cells, and this effect is time and concentration dependence.2.4-AP can effectively regulate the cell cycle of human tongue squamous carcinoma Tca8113 cells, cells were arrested in G0/G1 phase.3.4-AP can induce or promote human tongue squamous cell carcinoma Tca8113 cell apoptosis. |
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