| Background:Nasopharyngeal carcinoma(NPC) is one of the common tumors in China,especially in Southern China regions, such as guangdong, guangxi.The incidence of it account for about 80% of the world. In clinical pathology classification Nasopharyngeal carcinoma mainly is low differentiated squamous carcinoma. It is sensitive to X radiation. Surgical treatment is difficult to cure,radiotherapy presently ranks first in the integral treatment of NPC. NPC can be easily occur local recurrence and distant metastasis, it is the main cause of treatment failure.One of the reasons why treatment failure because there is a certain proportion of radiation-resist cells in NPC. tissue. Exploring the mechanisms of this resistance and determining the various radio-sensitization approaches may help improve the recovery rate of patients.Due to tumor cell heterogeneity exists, even though of the same histologically differentiated squamous carcinoma cell will also show different radiosensitivity.Obviously, if a pair of cells with the same source but with different radiosensitivity for radiation sensitivity study, will have more advantages.Objective:Establish a radio-resistance cell line CNE1/70R by exposing to a large number of X-ray, to study the radiation sensitivity, drug sensitivity, cell apoptosis, cell cycle distribution of the two cell lines.To study the the different growth speed between the two cell CNE1 and CNE1/70R by nude mice experiment. Discuss the metastasis between the expression of cell cycle related protein CUEDC2 and the radio sensitivity. The application of Affymetrix Geneship microarray technique for analysis of gene expression profiles is a pwoerful strategy to identify novel radioresistance-associated genes of the two cell lines with different radioresistance.Methods:To recovery nasopharyngeal carcinoma cell CNE1, cultivates and batches, using the cells growth in logarithmic phase to do the experiment. Using a radical cure dose (6Mv X ray, 70Gy/35 days, 2Gy/day, 1 time/day, 5 times peer week) of clinical routine segmentation in vitro irradiation to establish nasopharyngeal carcinoma cell line model CNE1 and CNE1/70R. The double time of the two cells was measured by cell growth curve. MTT was used to test the effect of different concentration of Docetaxel (100, 10, 1, 0.1, 0.01μmol/L) and Cisplatin(1, 0.1, 0.01, 0.001, 0.0001μg/ml ) on CNE1 and CNE1/70R. The radiobiological characteristics of the two cells lines were analyzed by the colony forming assay. Apoptosis index after 0, 4, 8, 12Gy irradiation was analyzed by AnnexinV/PI dye flow cytometry. And test the cell cycle distribution after 8Gy irradiation by flow cytometry. Nude mice experiment is used to identify the relationship between tumor rate and the radiation resistance of the two tumor cells.Microarray analyses wasa used to detect the different expression of the two cell lines. Western blot and qRT-PCR was applied to detect the expression of CUEDC2.Results:(1)The doubling time of CNE1/70R cells was longer than that of there parental generation CNE1 were obtained, the doubling time were (1.483±0.046)d vs ( 1.586±0.069) d. (2)The result of colony formation assay showed that cells CNE1/70R formed colony ability stronger than CNE1, the radioresistance of CNE1/70R cells was stronger than that of CNE1/70R, obvious difference was observed (p<0.05). (3)Docetaxel and Cisplat in significantly inhibited the growth of human NPC CNE1 and CNE1/70R cells and its IC50 was 0.049μmol/L vs 0.001μmol/L, 1.008μg/ mL vs 0.337μg/ mL, CNE1/70R was more sensitive to Docetaxel and Cisplat. (4)Cell cycle distribution showed that After 8 Gy irradiating in 24 hours. Cell number of S and G0/G1 phase decreased, while G2/M phase increased, both two cells was significantly arrested in phase G2/M,CNE1 cells arrested more obvious than CNE1/70R, the result were different statistically (p<0.05). (5)After irradiation of OGy, 4Gy, 8Gy, 12 Gy, the rate of apoptosis was positively correlated to the dose of X radiation, the cell apoptosis rate of CNE1 cells was more than CNE1/70R. The apoptosis rate of the two cells were different statistically (p<0.05). ( 6 ) radiation resistant strains of nasopharyngeal carcinoma (NPC) cell transplantation tumor growth slower than human nasopharyngeal carcinoma radiosensitive cells transplantation tumor. (7)In comprehensive,differenttly gene expression profile was obtained for the two different radioresitance cells.Totally,among 1191 genes, 1172 genes was obtained up-regulate ,19 genes down-regulated. These genes function involving in different molecular function, and many biological processes. (8)Compared to the CNE1 tumors, the CNE1/70R tumors grew more slowly. The expression level of CUEDC2 was decreased in CNE1/70R detected by Western Blot and qRT-PCR.Conclusion:(1)Using clinical normal segmentation means (2Gy/day, 35days) can build a stable radioresistant strains NPC CNE1/70R.(2)Growth speed: growth curve experiment shows that the growth speed of radio-resistent NPC CNE1/70R is slow than radio-sensitivy NPC CNE1.(3)Drug sensitivity: radio-resistent NPC CNE1/70R is more sensitive to tumor chemotherapy medicine Docetaxel and Cispltain. Provide new theoretical basis for recurrent nasopharyngeal carcinoma patients in Clinical treatment.(4)CNE1/70R cell lines have stronger ability in the phase of G2/M phase retardation, radiation resistance and apoptosis resistance than their parents cell lines.(5)The up-regulate of protein CUEDC2 may be one of the main reason associates with radio-resistant.Which provides the new molecular target andtheoretical foundation for further research on radiation resistance mechanism.(6) Our results suggest that the radioresistnaee process of NPC is a complieated and multifactorial process and may involves some of all of the following changes activation of stress response, inhibition of apotosis pathway disturbance of cell cycle, signal transduction and,cell apotosis. |
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