本文選題：FoxM1 + 肝癌�。� 參考：《西南交通大學》2016年碩士論文

【摘要】：肝癌是一種常見的嚴重危害公眾健康的惡性腫瘤,我國每年約來13萬人死于肝癌,成為受肝癌影響最大的國家之一。FoxM1作為Fox家族的特殊成員,在多種人類癌癥細胞中過量表達,與癌癥晚期、癌細胞轉移、高增殖、化療耐藥性以及預后不良等密切相關,還參與多種癌癥相關基因的表達及信號傳導途徑。因此,FoxM1被認為是抗癌藥物治療與干預的重要藥靶與標記。以FOXM1cDNA結合域為靶標,通過噬菌體展示肽庫篩選出的高親和力多肽P201可能成為未來靶向抗腫瘤治療藥物。本研究通過一系列細胞、分子生物學實驗和計算機輔助藥物設計方法,初步探討P201多肽對肝癌細胞的殺傷作用和死亡途徑,再對多肽進行氨基酸水平的優(yōu)化設計,并檢驗優(yōu)化后多肽對肝癌細胞的殺傷作用,發(fā)現(xiàn)多肽的關鍵氨基酸。MTT細胞活力測定和形態(tài)觀察顯示,P201多肽能夠顯著抑制人肝癌HepG2細胞的活力,60μg/mL濃度作用48h抑制率達到96%,計算IC50為25.16μg/mL。P201多肽對MCF-7和293T細胞增殖也具有一定殺傷作用,60μg/mL濃度作用48小時抑制率分別為64.1%和61.9%,形態(tài)上變化明顯,并且在一定濃度范圍內具有時間劑量依賴性。在此基礎上,進一步定性實驗AO-EB細胞雙染以及定量實驗流式細胞術,發(fā)現(xiàn)P201多肽對HepG2細胞的殺傷作用與誘導細胞凋亡有關,但可能存在多種死亡途徑,需進一步研究證明。在實驗探討P201多肽的抗癌活性和可能機制基礎上,基于虛擬篩選對P201多肽進行優(yōu)化,模擬丙氨酸突變,運用LibDock、CDOCKER和MVD等分子對接方法,發(fā)現(xiàn)第10位氨基酸殘基為多肽優(yōu)化位點,并確定第10位氨基酸優(yōu)化為天冬氨酸；結合模體序列搜索、反義氨基酸分析、虛擬丙氨酸突變及分子對接的結果,確定第1、2、4、7、9位5個P201關鍵氨基酸。最后通過CCK-8法檢測發(fā)現(xiàn)優(yōu)化后的多肽M10aa-D對HepG2細胞殺傷作用顯著低于P201多肽,100μg/mL濃度作用48h抑制率僅26.3%,在低濃度下甚至促進細胞增殖。多肽對FOXM1的親和力與其對細胞的殺傷作用呈相關性。綜上所述,P201多肽一方面具有成為靶向抗腫瘤治療藥物的潛力,同時尚待進一步分子作用機理研究和其他水平的優(yōu)化,以滿足抗癌多肽生物技術藥物研發(fā)的要求。
[Abstract]:Liver cancer is a common malignant tumor that seriously endangers public health. About 130000 people die of liver cancer every year in China. FoxM1, as a special member of the Fox family, has become one of the countries most affected by liver cancer, and it is overexpressed in many kinds of human cancer cells. It is closely related to advanced cancer metastasis, high proliferation, chemotherapeutic resistance and poor prognosis. It is also involved in the expression and signal transduction pathway of many kinds of cancer-related genes. Therefore, FoxM1 is regarded as an important drug target and marker for anticancer drug therapy and intervention. Using FOXM1 cDNA binding domain as the target, P201, a high affinity peptide screened from phage display peptide library, may be used as a target antitumor drug in the future. In this study, a series of cell, molecular biological experiments and computer-aided drug design methods were used to study the killing effect and death pathway of P201 polypeptide on hepatoma cells, and then to optimize the amino acid level of the peptide. The killing effect of the optimized polypeptide on hepatoma cells was tested. It was found that the activity of the key amino acid, MTT cell, and morphological observation showed that P201 polypeptide could significantly inhibit the activity of HepG2 cells. The inhibitory rate of P201 peptide on MCF-7 and 293T cell proliferation was calculated to be 25.16 渭 g / mL.P201 polypeptide for 48 h after exposure to 60 渭 g / mL concentration. The inhibitory rates of 60 渭 g / mL for 48 hours were 64.1% and 61.9%, respectively. And in a certain concentration range of time and dose dependent. On this basis, further qualitative experiments of AO-EB cell double staining and quantitative flow cytometry showed that the killing effect of P201 polypeptide on HepG2 cells was related to the induction of apoptosis, but there may be many death pathways, which need to be further studied and proved. On the basis of the experimental study on the anticancer activity and possible mechanism of P201 polypeptide, the P201 polypeptide was optimized based on virtual screening, and the alanine mutation was simulated. The 10th amino acid residue was found to be the optimal peptide site by using the molecular docking methods such as LibDocktl CDOCKER and MVD. The 10th amino acid was optimized as aspartic acid, combined with motif sequence search, antisense amino acid analysis, virtual alanine mutation and molecular docking results, 5 P201 key amino acids were determined at the 9th position. Finally, CCK-8 assay showed that the cytotoxicity of the optimized polypeptide M10aa-D to HepG2 cells was significantly lower than that of P201 polypeptide 100 渭 g / mL for 48 h. The inhibition rate of M10aa-D on HepG2 cells was only 26.3g / mL at low concentration, and even promoted the proliferation of HepG2 cells at low concentration. The affinity of polypeptide to FOXM1 was correlated with its cytotoxicity. In conclusion, P201 polypeptide has the potential to be a target antitumor drug, and needs further molecular mechanism research and other optimization to meet the requirements of anti-cancer polypeptide biotechnology drug development.
【學位授予單位】：西南交通大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R735.7

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