| Objective: Like most tumors, parts of cells of laryngocarcinoma without enough oxygen supplying, which induced resistance to radiation exposure of 60Coγ, but the mechanisms are still uncertain. To observe the relation between radioresistance of Hep-2 cells and hypoxia, our study simulated the microenvironment of cancer cell in vivo, establish models of Hep-2 laryngeal cancer cell line of different oxygen supplying, trying to investigate the impact of normoxia, hypoxia, reoxygenation after hypoxia on apoptosis and the expression of protein HIF-1αand p53 in Hep-2 human laryngeal cancer cell line induced by 60Coγ-ray. This study try to lay a experimental foundation on finding a new way with hypoxia as a target for laryngocarcinoma radiotherapy.Methods: Human laryngeal cancer cell Hep-2 was cultivated in the medium of RPMI-1640 contain 10% calf serum,penicilin(l00U/ml),Stmycin(l00μg/ml)in nomoxia condition of 37℃, 5%CO2,20%O2 and/or in hypoxia condition of 37℃,5%CO2,2%O2,93%N2. At the time of the cells got into exponential phase of growth, They were diviced into 6 groups : group A (normoxia), group B (hypoxia), group C (reoxygenation after hypoxia), group D (normoxia plus radiotherapy), group E (hypoxia plus radiotherapy), group F (reoxygenation after hypoxia plus radiotherapy). cancer cell Hep-2 of group B,C,E and F were cultivated in the condition of hypoxia for 24 hours, then group C and F were moved into normoxia, go on cultivating 4 hours. All of the cells of group D,E,F were exposured in the 5Gy dosage ofγ-ray. Flow cytometry (FCM) was used to measure the protein level of HIF-1αand p53 and to detect cell apoptosis. The protein level of HIF-1αand p53 was also determined by immunohistochemistry. HIF-1αmRNA level was determined by RT- PCR.Results: Flow cytometry result show that: the protein levels of HIF-1αof Hep-2 cell in group C(1.36±0.08)were significantly lower than that in group B(1.53±0.06)(p<0.05), but have no difference compared with group A(1.35±0.05)(P>0.05)The protein level of HIF-1αin Hep-2 cell in group C,F have no difference. Also in group B,E , in group A,D have no difference(P>0.05), and which is in accord with the result of immunohistochemistry. This result suggested: the levels of HIF-1αhave few expression in normoxia; hypoxia evidently increased the protein level of HIF-1α, compared to hypoxia, reoxygenation after hypoxia, the protein level of HIF-1αbecome lower too; 60Coγ-ray have no influence to the expression of HIF-1α.Same as the expression of protein HIF-1α, Flow cytometry result show that: the protein level of p53 in Hep-2 cell in group C(1.08±0.12)were significantly lower than that in group B(1.24±0.07)(p<0.05), but have no difference compared to group A(1.10±0.04)(P>0.05).the protein level of p53 in Hep-2 cell in group C,F have no difference. Also in group B,E , in group A,D have no difference(P>0.05).This result is in accord with with immunohistochemistry. These rusults suggest that p53 have some expression in normoxia, Hypoxia evidently increased the protein level of p53, compared to hypoxia, reoxygenation after hypoxia, the protein level of p53 become lower too, 60Coγ-ray have no influence to the expression of p53. Correlation analysis showed that the expression of protein p53 and HIF-1αhave conspicuous positive correlation, correlation coefficient is 0.9341, correlation coefficient probability value P=0.0064<0.01, have statistics significance. The expression of protein p53 and HIF-1αhave dependability, accompany to the high expression of protein HIF-1α, the expression of protein p53 become high too. Cell apoptosis was detected by flow cytometry (FCM), result show that hypoxia and reoxygenation after hypoxia have no influence to the apoptosis of Hep-2 cell. The apoptosis in group A,B,C have no difference(P>0.05), hypoxia lower the effect of 60Coγray induced apoptosis. The apoptosis in group D(31.68±1.27),E(4.31±0.35)have significant statistical significance(P<0.05), after reoxygenation the effect of 60Coγray induced apoptosis become higher. The apoptosis in group E(4.31±0.35),F(8.06±0.73)have significant statistical difference(P<0.05).The result of RT- PCR show that the expression of HIF-1αmRNA in group A(1.33±0.10),C(1.26±0.09)are lower than that in group B(1.74±0.13)(P<0.05). Hypoxia evidently increased the mRNA level of HIF-1α, after reoxygenation, the mRNA level of HIF-1αbecome lower too, The mRNA level of HIF-1αin Hep-2 cell in group C,F have no difference. Also in group B,E , in group A,D have no difference(P>0.05).Suggest that 60Coγ-ray have no influence to the mRNA expression of HIF-1α.Conclutions: 1 Hypoxia evidently increased the protein level of HIF-1αin Hep-2 cell, and it happened in the mRNA level. 2 The high express of protein HIF-1αincreased the protein level of p53, the expression of protein HIF-1αand p53 had visible positive correlation. 3 The 60Coγray have no effect on the expression of protein p53, HIF-1αand HIF-1αmRNA. 4 hypoxia decreased the effect of 60Coγ-ray induced apoptosis in Hep-2 human laryngeal cancer cell line. Compared to hypoxia, after reoxygenation the effect was obviously elevated. But at this time, the protein level of p53 did not show high expression. Guess the mechanism of high apoptosis induced by 60Coγ-ray in the condition of reoxygenation after hypoxiaon were related to some other apoptosis pathway except p53.
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