本文選題：鼻咽癌 + 放射治療��； 參考：《大連大學》2017年碩士論文

【摘要】：目的:探索IGF-1R抑制劑OSI-906增強鼻咽癌細胞(NPC)放射敏感性的作用機制。方法:應用蛋白免疫印跡法(Western blot)檢測X線照射(IR)后5種鼻咽癌細胞pIGF-1R基礎表達水平;應用四甲基偶氮唑藍(MTT)實驗觀察單獨應用胰島素樣生長因子-1(IGF-1)、IGF-1R抑制劑OSI-906(Linsitinib)以及IGF-1聯(lián)合OSI-906對NPC增殖的影響;應用Western blot檢測IR、IGF-1、OSI-906、OSI-906聯(lián)合IGF-1、OSI-906聯(lián)合IR后IGF-1/IGF-1R信號轉(zhuǎn)導通路和相關蛋白表達活化情況;應用流式細胞術(FACS)檢測單獨應用OSI-906、IR、OSI-906聯(lián)合IR后NPC周期和凋亡情況;應用細胞克隆形成實驗觀察不同IR后IGF-1、OSI-906單獨應用以及OSI-906聯(lián)合IGF-1后細胞克隆形成的差異;最后應用gH2AX焦點成型實驗,觀察單獨應用OSI-906和OSI-906聯(lián)合X線照射后細胞DNA損傷情況。以上實驗數(shù)據(jù)采用GraphPad Prism 5進行統(tǒng)計學分析。結(jié)果:在鼻咽癌細胞系中,pIGF-1R的表達水平高低不一,其中CNE-2、SUNE-1細胞pIGF-1R蛋白表達屬于中游水平,且對OSI-906敏感性較高,作為本研究對象。MTT結(jié)果提示,IGF-1促進NPC增殖,OSI-906明顯抑制細胞增殖,且兩者的作用呈劑量依賴性。同時,OSI-906能夠逆轉(zhuǎn)IGF-1的促增殖作用;Western blot結(jié)果顯示,IR、IGF-1和OSI-906分別激活和抑制IGF-1/IGF-1R增殖信號轉(zhuǎn)導通路,pIGF-1R、pAKT、pERK表達明顯增高和降低,總IGF-1R、AKT、ERK蛋白均無明顯變化;同時,OSI-906可以逆轉(zhuǎn)IGF-1、IR介導的IGF-1/IGF-1R信號通路的激活;細胞周期結(jié)果顯示,單獨應用OSI-906、IR后細胞G2/M期比例增加不明顯,且細胞S期比例升高,而OSI-906聯(lián)合X線照射后顯著增加細胞G2/M期比例,細胞S期比例降低;單獨應用OSI-906、IR可以促進細胞凋亡,OSI-906聯(lián)合IR后進一步增加細胞凋亡率;克隆形成實驗結(jié)果表明,IR后,IGF-1提高了NPC的生存數(shù),而OSI-906明顯地降低了NPC的生存數(shù)并且可以逆轉(zhuǎn)IGF-1的作用;gH2AX焦點成形實驗提示OSI-906可以顯著增加X線照射細胞的gH2AX焦點數(shù),細胞DNA斷裂蛋白標記物gH2AX表達升高。結(jié)論:OSI-906增加NPC放射敏感性,其機制可能與抑制細胞增殖信號傳導通路的激活、減少DNA損傷的修復、改變細胞周期、促進細胞凋亡、誘導細胞基因組不穩(wěn)定性、且逆轉(zhuǎn)IGF-1介導的鼻咽癌放射抵抗有關。
[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.
【學位授予單位】：大連大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R739.63

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本文編號：1811402