| Part one Cancer Stem Cells Promotes Resistance of Laryngeal Squamous Cancer to Irradiation Mediated by HypoxiaObject:To study whether cancer stem cells promotes resistance of laryngeal squamous cancer to irradiation mediated by hypoxia. Methods:Hep-2cells were respectively cultured in hypoxia and normoxia enviroment, and the expression of HIF-la was detected by western blot. Then they were radiated with different doses of γ-rays. After that we detected growth inhibition ratio with MTT assay, cell circle and ratio of CD133+cells with Flow cytometry (FCM) at defferent times. Results:MTT assay showed that inhibition ratio of the hypoxia group was lower than that of the normoxia group after different doses of γ-rays at each time point, and the difference was significant24h after lOGy irradiation (P<0.05). The results of flow cytometry (FCM) demonstated that cells of the two groups were arrested at G1phase, and cells ratio in G1phase of the hypoxia group was higher than that of normoxia group after lOGy irradiation. The ratio of CD133+cells was higher in the hypoxia group than in the normoxia group after radiation. In each group, the ratio of CD133+cells raised after radiation.(P<0.05). Conclusion:we can conclude that cancer stem cells play an important role in radioresistance mediated by hypoxia.Part Two The Mechanism of the Role Cancer Stem Cells Played in Resistance of Laryngeal Squamous Cancer to Irradiation Mediated by HypoxiaObject:To study whether laryngeal cancer stem cells have the characteristic of resistance to irradiation in hypoxia enviroment. Methods:CD133+cells were separated from Hep-2cells and HIF-la-silenced-Hep-2cells by cell sorting with a flow cytometer and the purity was over92%. CD133+cells were respectively cultured in serum-free medium in hypoxia and normoxia enviroment, Then CD133+cells were divided into4groups accordingly:group A, B, C and D. They were radiated with different doses of X-rays. After that we detected growth inhibition ratio of each group with MTT assay at defferent time point; Soft agar colony formation assay were performed to dectect colony formation ratio of each group. Expression of DNA-PKcs, ATM, Survivin, P53were detected by FCM. Results:MTT assay showed that inhibition ratio of group A was lower than that of the other3groups at24h after lOGy irradiation (P<0.05). Soft agar colony formation assay showed that colony formation ratio of CD133+cells was higher in group A than in the other3groups before and after irradiation (P<0.05). The colony formation ratio of group A beared no difference before and after irradiation. However, this ratio of group B, C and D after irradiation was lower than that before irradiation (P<0.05). The results of FCM demonstrated that after radiation, the expression of DNA-PKcs and Survivin of group A was higher than that of the other3groups (P<0.05). But there were no defference in the expression of ATM and P53among the4groups. Conclusion:Cancer stem cells play an important role in radioresistance mediated by hypoxia.Part Three Study of the Role of Cancer Stem Cells in Radioresistance of Laryngeal Squamous Cancer Mediated by Hypoxia in vivoObjective:To elucidate the mechanism of radioresistance by cancer stem cell, and whether hypoxia promote it. Methods:The animal models of xenotransplanted tumor of CD133+cells from human laryngeal carcinoma cell line Hep-2were set up in16nude mice, and tumor formed in12of them which were distributed into4groups at random:nude mice implanted with CD133+cells and received lOGy radiation were in group A, those received OGy radiation were in group Ac, which was control group of group A in fact; nude mice transplanted with HIF-la-silenced CD133+cells and received lOGy radiation were in group B, received OGy radiation were in group Bc which was control group of group B in fact. Tumor volume was measured regularly. Two weeks after radiation, the mice were sacrificed and the tumors were weighted. Then CD133+cells rate in tumor was measured by FCS. Moreover the expression of DNA-PKcs, ATM, p53, Survivin were investigated by immunohistochemistry. Results:There was a significant difference in tumor volume between group A and B, and compared to their control groups (P<0.01). The difference of tumor inhibition rate between group A and group B was significent. The expression of DNA-PKcs, ATM, P53, Survivin increased significantly after lOGy radiation. There was a significent difference of expression of DNA-PKcs and Survivin between group A and B (P<0.05), while the expression of ATM and P53had no difference. Conclusion:Cancer stem cells play an important role in tumor radioresistance. The machanism may be that injured DNA was repaired throuth NHEJ way in which DNA-PK play the main role and HR way in which ATM play the main part. The promotion of radioresistance induced by hypoxia depends on enhanced activiation of DNA-PK. |
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