Experimental Study Of Apoptosis And Thermotolerance Induced By Hyperthermia Between 39℃-45℃ Of CNE28 Cells In Vivo And In Vitro

Purpose:To study apoptosis and thermotolerance produced and affected by hyperthermia between 39℃-45℃ and the production and reduction of HSP70 during this process of CNE28 cells.To investigate xenograft tumor growth inhibition and apoptosis and the expression of HSP70 treated by different temperature in vitro. Methods:The CNE28 cells cultured by 37℃ were heated by 39℃, 41℃,43℃, 45℃ for 50 minutes separately.Part of the cells were heated again separately with 45℃/50 minutes after 2,8,24,48,72 hours, then the thermotolerance ratio (TTR) was calculated .We also measured the apoptosis and HSP70 in different temperature and interval. Apoptosis was detected by FCM and TUNEL and the exposure of Phasphatidylserine measured by FCM. The HSP70 labeled by two antibodies was measured by FCM in different temperature and interval. Building CNE-28 xenograft tumors model, the tumors were treated by different temperature .Apoptosis was measured by TUNEL.The expression of HSP70 was detected by IHC. Results: 1 in vitroAs the temperature = 43℃ ,the apoptosis increased with the temperature higher.The peak appeared in 43℃/50 minutes after 8 hours.Apoptosis reduced sharply when the temperature was 45℃ .The expression of HSP70 increases with the temperature arising, but there is no significant difference in different group below 43℃ and the peak appeared in 45℃/50minutes after 8 hours. As its effect to thermotolerance,the TTR is always < 5 in all the interval when the temperature = 43 ℃ .But the TTR > 5when the interval was 8 hours and 24 hours in 45℃ group .There is positive correlation between the expression of HSP70 andthermotolerance. 2 in vivo(1) By the time of the nude mice were killed ,the weight of tumor and the curve of tumor growth in different groups has no significant difference.(2) The rate of apoptosis in different groups measured by TUNEL has significant difference .The rate of apoptosis in the 41℃ and 43 ℃ group is higher than the group of 39℃ . But compared the 41 ℃ group with the 43 ℃ group and the 39℃ group with the control group, the rate has no significant difference.(3) Compared with the control group ,HSP70 is higher in 39℃, 41℃ ,43℃ groupthan the control group from 2 hours to 48 hours after hyperthermia. But to these three groups, the difference is not significant. All the groups have not significant difference in 72 hours .HSP was performed by IHC. Conclution:Mild hyperthermia can induce apoptosis in CNE-28 cell line. 43 ℃ was a temperature with high clinical value. The hyperthermia-induced apoptosis reached the maximal on this condition but it affect the thermotolerance slightly.We should pay more attention to its clinical value. There is positive correlation between the expression of HSP70 and thermotolerance . Measuring the expression of HSP70 can direct the temperature and interval of hyperthermia. In vivo,apoptosis was higher in 41℃,43℃ group than the 39℃ group and the control group,but there is no significant difference in the weight of tumor and tumor growth inhibition. So mild hyperthermia should be combined with other treatment in the clinical treatment.