P53磷酸化与鼻咽癌放射敏感性的相关性研究

发布时间：2018-06-27 05:22

本文选题：P53磷酸化 + 鼻咽癌　；参考：《中南大学》2013年硕士论文

【摘要】：目的：建立鼻咽癌HNE2放射不敏感细胞系,即HNE2-NRC,研究HNE2-NRC与HNE2细胞系中P53磷酸化的差异性表达,并在鼻咽部组织芯片中检测P53Ser376位点磷酸化水平,探讨P53蛋白磷酸化与鼻咽癌治疗反应的关系。方法：通过亚致死射线剂量、递增射线剂量反复照射人鼻咽癌细胞株HNE2的方法建立放射不敏感细胞株HNE2-NRC以HNE2-NRC及HNE2为研究对象,观察不同的放射剂量照射前后细胞形态、细胞增殖、细胞凋亡及细胞周期的变化。倒置显微镜下观察细胞形态的变化,克隆形成实验观察细胞克隆形成率及生长曲线、MTS法测定放射线照射后细胞增殖抑制。流式细胞术检测HNE2-NRC及HNE2细胞周期的差异。Western Blotting测定HNE2-NRC与HNE2细胞系中P53磷酸化的差异性表达,免疫组化法检测鼻咽部组织芯片中P53Ser376位点磷酸化水平。结果：克隆形成实验表明HNE2-NRC较HNE2克隆形成力强,放疗增敏比SER为0.7961。MTS实验检测到HNE2与HNE2-NRC相比,生长受到抑制,在6h、2d、6d、8d时差别均有统计学意义。流式细胞检测两株细胞周期具有差异,HNE2G2/M期细胞比率为23.88%,而HNE2-NRC G2/M期细胞比率为10.84%,且HNE2-NRC G0/G1期细胞比率增加。Western Blot结果显示HNE2-NRC与HNE2相比,P53、P53Ser15、Thr81、Thr55、 Ser389磷酸化水平升高,而Ser6、Ser9、Ser315、Ser376磷酸化水平降低。免疫组化染色检测鼻咽部组织芯片中P53Ser376表达,放疗后生存期5年的患者与放疗后生存期5年的患者相比较,P53Ser376磷酸化水平有降低的趋势。结论： 1、本研究建立了放射不敏感细胞系HNE2-NRC,与亲本细胞HNE2相比,具有耐放射的生长特性。 2、在HNE2-NRC细胞中,P53Serl5、Thr55磷酸化水平升高；而P53Ser315、Ser376发生去磷酸化,提示P53相关位点的磷酸化特征谱与鼻咽癌放射抵抗相关。 3、在组织芯片水平,P53Ser376磷酸化水平与放射治疗的鼻咽癌病人预后呈一定的相关性,进一步提示P53磷酸化谱作为放疗敏感性预测的可能性。图10幅,表4个,参考文献124篇。
[Abstract]:Objective: to establish a radiosensitive HNE2 cell line, HNE2-NRC, to study the differential expression of p53 phosphorylation in HNE2-NRC and HNE2 cell lines, and to detect the phosphorylation level of P53Ser376 in nasopharyngeal tissue microarray. To investigate the relationship between p53 protein phosphorylation and the therapeutic response of nasopharyngeal carcinoma (NPC). Methods: human nasopharyngeal carcinoma cell line HNE2 was irradiated repeatedly by sublethal and incremental radiation doses. HNE2-NRC and HNE2-NRC were used to observe the morphology of HNE2-NRC and HNE2 cells before and after irradiation. Cell proliferation, apoptosis and cell cycle changes. The cell morphology was observed under inverted microscope, the colony formation rate was observed and the growth curve was used to determine the inhibition of cell proliferation after irradiation. The difference of HNE2-NRC and HNE2 cell cycle was detected by flow cytometry. Western blotting was used to detect the expression of p53 phosphorylation in HNE2-NRC and HNE2 cell lines. The phosphorylation level of P53Ser376 in nasopharyngeal tissue microarray was detected by immunohistochemistry. Results: the clone forming ability of HNE2-NRC was stronger than that of HNE2. The sensitivity ratio of HNE2-NRC was 0.7961.MTS assay showed that HNE2 was inhibited compared with HNE2-NRC, and the difference was significant at 6h / 2d ~ 6d ~ (-1). The cell ratio of HNE2-NRC G _ 2 / M phase was 23.88, while that of HNE2-NRC G _ 2 / M phase was 10.84 and the ratio of HNE2-NRC G _ 0 / G _ 1 phase increased. Western blot analysis showed that the phosphorylation level of P53P53SerSer15Thr81Thr55, Ser389 was increased, while Ser6Ser9Ser315Ser376 was decreased in HNE2-NRC G0 / G1 phase compared with HNE2. The expression of P53Ser376 in nasopharyngeal tissue microarray was detected by immunohistochemical staining. The phosphorylation level of P53Ser376 was decreased in patients with 5 years survival after radiotherapy and 5 years after radiotherapy. Conclusion: 1. The radiosensitive cell line HNE2-NRC2.Compared with parent HNE2, HNE2-NRC2.2The phosphorylation level of P53Serl5hr55 in HNE2-NRC cells was increased, while that of P53Ser315Ser376 was dephosphorylated. It was suggested that the phosphorylation characteristic spectrum of p53 related sites was related to the radiation resistance of nasopharyngeal carcinoma. 3. The phosphorylation level of p53 Ser376 was correlated with the prognosis of patients with nasopharyngeal carcinoma after radiotherapy at the tissue microarray level. It is further suggested that P 53 phosphorylation spectrum can be used as a predictor of radiotherapy sensitivity. Ten figures, four tables and 124 references.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R739.63

【共引文献】