| [Objective] To study the effect of Autophagy on hyperthermia-induced apoptosis in tongue squamous cell carcinoma Cal-27 cells,for by promoting or inhibiting cell autophagy role to improve the effect of thermal therapy to provide experimental basis.[Methods] (1) Cal-27 cells were cultivated with DMEM containing 10% fetal bovine serum sugar medium, with 37℃ and 5% CO2 incubator.(2) Experimental groups: control group(Cal-27 cells),hyperthermia-induced group(Cal-27 cells were treated with 42℃ 100min and 43℃ 100min), rapamycin treated group (Cal-27 cells were treated with 100nM rapamycin),3-MA treated group(Cal-27 cells were treated with 5mM 3-MA),rapamycin+hyperthermia-induced group(Cal-27 cells were treated with 100nM rapamycin+43℃ 100min),3-MA+hyperthermia-induced group(Cal-27 cells were treated with 5mM 3-MA+43℃ 100min). (3) The expression of Beclin-1 on hyperthermia-induced by immunocytochemical staining analysis. (4) Observe the differentiation of the autophagosome after normal training and hyperthermia-induced by electron microscopy. (5) Establish cell apoptosis model through the AnnexinV-FITC/PI marker flow cytometry instrument detecting apoptosis rate.(6)The expression of Bax、Beclin-1、LC3、Atg5 were analyzed through Western blot. (7) Statistical analysis was carried out with SPSS 17.0.[Results] (1) Chemical immunofluorescence experiments found that in the control group and the hyperthermia-induced group under the fluorescence microscope to observe autophagy related protein Beclin-1 light phenomenon, and at higher temperatures(42℃ 100min), light phenomenon Beclin-1 to slightly stronger with the control group. (2) The autophagosome in the control group cells were studied with electronic microscope.It was found the autophagosome within the monolayer film package waste using organelles, but fewer;When heated up 42℃ 100 min, the autophagosome significantly more than the control group, the monolayer membrane structure autophagosome was more in number, encircled the disuse organelles such as mitochondria, golgi apparatus, etc.,partially wrapped organelles had entered middle and later periods of the decomposition,other organelles in the cell showed good morphology;When heating 43℃ 100 min, the number of intracellular autophagosome than the control group and heating 42℃ was significantly reduced, and43℃ when heated to 100 min, the number of intracellular autophagosome is 42℃ 100 min heating has decreased, and mitochondrial swelling in the present condition, some cells appeared apoptosis morphological changes. (3) With Annexin V-FITC labeling and analysis by flow cytometry, the apoptosis rate in the control group was (1.29±0.05)%. The apoptosis rate in rapamycin treated group was (2.18±0.04)%;The apoptosis rate in 3-MA treated group was (3.71±0.10)%,compared with the control group,no statistically difference(P>0.05);The hyperthermia-induced(43℃ 100min) group was (29.63±0.03)%,There was statistically significant differences compared with that of the group treated with control group(P<0.05). The rapamycin+hyperthermia-induced (43℃ 100min) group was(18.98±0.06)% at 24h after treatment,compared with hyperthermia-induced(43℃ 100min) group,apotosis rate significantly decreased, and the 3-MA+hyperthermia-induced(43℃ 100min) group was(38.92±0.28)% at 24h after treatment, the apoptosis rate was higher than hyperthermia-induced(43℃ 100min) alone.There was statistically significant differences compared with that of the group treated with hyperthermia-induced(43℃ 100min) alone (P<0.05). (4) Western blot experiments found that:①rpamycin alone or 3-MA alone, increase or decrease cell autophagy, LC3II/LC3I ratio and the expression of Beclin-1,Atg5, Bax compared with the control group, no significant change, there was no statistically difference (P< 0.05);②hyperthermia-induced(43℃ 100min) group alone LC3Ⅱ/LC3Ⅰ ratio, the expression of Atg5,Beclin-l in the control group, compared to significantly lower, the difference was statistically significant (P<0.05),the expression of Bax separate heating group, the difference was statistically significant (P< 0.05);(3) rapamycin+ hyperthermia-induced(43℃ 100min) group, LC3Ⅱ/LC3I ratio, the expression of Atg5 Beclin-1 separate heating group increased significantly, the difference was statistically significant (P< 0.05),the expression of Bax heating alone group was obviously lower than the control group,the difference was statistically significant (P< 0.05);(4)3-MA+ hyperthermia-induced(43℃ 100min) group, LC3II/LC3I ratio, the expression of Atg5,Beclin-1 hyperthermia-induced group decreased obviously, the difference was statistically significant (P< 0.05), the expression of Bax separate heating group, the difference was statistically significant (P< 0.05).[Conclusion] Autophagy occurs in tongue squamous cell carcinoma Cal-27 cells are in a thermal stimulus conditions. Improve the level of cell autophagy, inhibits apoptosis induced by hyperthermia-induced, reduce heat killing tumor cells;On the other hand, the lower levels of cell autophagy, promotes apoptosis induced byhyperthermia-induced, heat killing tumor cells. |
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