抑制IGF-1R增强鼻咽癌细胞放射敏感性的机制研究

发布时间：2018-04-27 16:08

本文选题：鼻咽癌 + 放射治疗　；参考：《大连大学》2017年硕士论文

【摘要】：目的:探索IGF-1R抑制剂OSI-906增强鼻咽癌细胞(NPC)放射敏感性的作用机制。方法:应用蛋白免疫印迹法(Western blot)检测X线照射(IR)后5种鼻咽癌细胞pIGF-1R基础表达水平;应用四甲基偶氮唑蓝(MTT)实验观察单独应用胰岛素样生长因子-1(IGF-1)、IGF-1R抑制剂OSI-906(Linsitinib)以及IGF-1联合OSI-906对NPC增殖的影响;应用Western blot检测IR、IGF-1、OSI-906、OSI-906联合IGF-1、OSI-906联合IR后IGF-1/IGF-1R信号转导通路和相关蛋白表达活化情况;应用流式细胞术(FACS)检测单独应用OSI-906、IR、OSI-906联合IR后NPC周期和凋亡情况;应用细胞克隆形成实验观察不同IR后IGF-1、OSI-906单独应用以及OSI-906联合IGF-1后细胞克隆形成的差异;最后应用gH2AX焦点成型实验,观察单独应用OSI-906和OSI-906联合X线照射后细胞DNA损伤情况。以上实验数据采用GraphPad Prism 5进行统计学分析。结果:在鼻咽癌细胞系中,pIGF-1R的表达水平高低不一,其中CNE-2、SUNE-1细胞pIGF-1R蛋白表达属于中游水平,且对OSI-906敏感性较高,作为本研究对象。MTT结果提示,IGF-1促进NPC增殖,OSI-906明显抑制细胞增殖,且两者的作用呈剂量依赖性。同时,OSI-906能够逆转IGF-1的促增殖作用;Western blot结果显示,IR、IGF-1和OSI-906分别激活和抑制IGF-1/IGF-1R增殖信号转导通路,pIGF-1R、pAKT、pERK表达明显增高和降低,总IGF-1R、AKT、ERK蛋白均无明显变化;同时,OSI-906可以逆转IGF-1、IR介导的IGF-1/IGF-1R信号通路的激活;细胞周期结果显示,单独应用OSI-906、IR后细胞G2/M期比例增加不明显,且细胞S期比例升高,而OSI-906联合X线照射后显著增加细胞G2/M期比例,细胞S期比例降低;单独应用OSI-906、IR可以促进细胞凋亡,OSI-906联合IR后进一步增加细胞凋亡率;克隆形成实验结果表明,IR后,IGF-1提高了NPC的生存数,而OSI-906明显地降低了NPC的生存数并且可以逆转IGF-1的作用;gH2AX焦点成形实验提示OSI-906可以显著增加X线照射细胞的gH2AX焦点数,细胞DNA断裂蛋白标记物gH2AX表达升高。结论:OSI-906增加NPC放射敏感性,其机制可能与抑制细胞增殖信号传导通路的激活、减少DNA损伤的修复、改变细胞周期、促进细胞凋亡、诱导细胞基因组不稳定性、且逆转IGF-1介导的鼻咽癌放射抵抗有关。
[Abstract]:Objective: to explore the mechanism of IGF-1R inhibitor OSI-906 in enhancing radiosensitivity of nasopharyngeal carcinoma (NPC) cells. Methods: Western blotting was used to detect the basic expression of pIGF-1R in 5 kinds of nasopharyngeal carcinoma cells after X-ray irradiation. The effects of insulin-like growth factor-1 IGF-1R inhibitor OSI-906 Linsitinib and IGF-1 combined with OSI-906 on the proliferation of NPC were observed by MTT. Western blot was used to detect the activation of IGF-1/IGF-1R signal transduction pathway and related protein expression after IRI-906 combined with IGF-1OSI-906 combined with IR, and flow cytometry was used to detect NPC cycle and apoptosis after using OSI-906 combined with IR alone. The difference of cell clone formation between IGF-1OSI-906 and OSI-906 combined with IGF-1 was observed by cell clone formation assay, and the cell DNA damage after OSI-906 and OSI-906 combined with X-ray irradiation was observed by gH2AX focus molding experiment. The above experimental data were statistically analyzed by GraphPad Prism 5. Results: the expression level of pIGF-1R in nasopharyngeal carcinoma cell line was different, and the expression of pIGF-1R protein in CNE-2SUNE-1 cell line was middle level, and the sensitivity to OSI-906 was high. The results showed that IGF-1 promoted NPC proliferation and OSI-906 significantly inhibited the proliferation of NPC cells. The effect of both was dose dependent. At the same time, OSI-906 could reverse the proliferative effect of IGF-1. The results of Western blot showed that IRI IGF-1 and OSI-906 activated and inhibited the expression of pIGF-1RnpAK-pERK respectively, but the total IGF-1RnAK-AKTERK protein did not change. At the same time, OSI-906 could reverse the activation of IGF-1IR mediated IGF-1/IGF-1R signaling pathway, and the cell cycle results showed that the proportion of G _ 2 / M phase was not significantly increased and the S phase ratio of cells was increased after the treatment of OSI-906 IR alone, while the ratio of G _ 2 / M phase was significantly increased after OSI-906 combined with X-ray irradiation. The ratio of S phase was decreased, the apoptosis rate was further increased by OSI-906 combined with IR alone, the results of clone formation showed that IGF-1 increased the survival rate of NPC after IR. However, OSI-906 significantly reduced the survival of NPC and reversed the role of IGF-1. The results showed that OSI-906 could significantly increase the number of gH2AX focal points of the cells irradiated by X-ray, and the gH2AX expression of DNA break protein marker was increased. Conclusion the mechanism of NPC radiosensitivity may be related to inhibiting the activation of cell proliferation signal transduction pathway, reducing the repair of DNA damage, changing cell cycle, promoting cell apoptosis and inducing cell genomic instability. And reversal of IGF-1-mediated radiation resistance in nasopharyngeal carcinoma.
【学位授予单位】：大连大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R739.63

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