基于FoxMlc抗癌先導(dǎo)藥物設(shè)計與研究
發(fā)布時間：2018-10-11 08:27
【摘要】：FoxM1c作為轉(zhuǎn)錄因子Fox家族成員之一,對正常細胞的增殖分裂具有重要的調(diào)控作用,但同時FoxM1的異常表達也能導(dǎo)致腫瘤細胞的發(fā)生與發(fā)展。許多研究已經(jīng)證實,FoxM1在多種腫瘤細胞中持續(xù)高表達,而在正常靜止?fàn)顟B(tài)或高度分化的細胞中基本不表達。因此為通過靶向FoxM1蛋白來治療腫瘤提供了理論上的可行性,但是由于FoxM1轉(zhuǎn)錄因子的特性及其結(jié)構(gòu)上缺乏疏水性結(jié)構(gòu),傳統(tǒng)小分子化學(xué)藥物很難靶向。因此傳統(tǒng)藥物學(xué)研究通常將轉(zhuǎn)錄因子排除在腫瘤治療靶蛋白之外,但是隨著生物技術(shù)的發(fā)展,多肽藥物本身的優(yōu)點克服了傳統(tǒng)藥物固有的缺陷,從而使得靶向FoxM1成為可能性。另外天然植物藥中的許多成分具有抗腫瘤活性,因此使得其具有多靶點特性,這種特點與腫瘤多靶點治療思想和方法相吻合。 本研究首先在本實驗室前期利用噬菌體隨機十二肽庫篩選獲得的高親和多肽序列的基礎(chǔ)上,通過MEME模體分析軟件獲得一條高親和多肽序列共有的模體序列：WHLD。之后再將模體序列與穿膜肽結(jié)合并最終確定兩條多肽序列CT-788:rrrrrrrrGSGSWHLD(r為D型精氨酸)和CT-789:WHLDGSGSWHLD。在合成之前,通過計算機分子對接軟件Molegro.Virtual.Docker (MVD)模擬兩條多肽與受體蛋白FoxM1c DNA結(jié)合區(qū)的相互作用。結(jié)果顯示兩條多肽分子均能與FoxMlc DNA結(jié)合區(qū)穩(wěn)定結(jié)合。 在分子對接的基礎(chǔ)上,我們合成了CT-788和CT-789兩種多肽,并進行腫瘤細胞體外實驗,用MTT法檢測多肽分子對腫瘤細胞活力的影響,其中CT-788在濃度100ug/mL下對HEPG-2和MCF-7腫瘤細胞的抑制率分別為41.26%和34.26%,而相同濃度下CT-789對兩種細胞的抑制率分別為15.96%和17.8%。 通過顯微鏡直接觀察天然植物液處理過的腫瘤細胞形態(tài)學(xué),可以清楚觀察到天然植物液YN-01能顯著抑制HEPG-2細胞的生長,并且在4%的低濃度(含50%的乙醇)下就能殺死腫瘤細胞。MTT實驗數(shù)據(jù)也很好地說明了YN-01能夠很好抑制HEPG-2細胞的活力,其4%的低濃度抑制率就高達68%。之后通過AO-EB細胞雙染、電泳檢測DNA ladder、Annexin V-FITC/PI標(biāo)記流式細胞儀實驗進一步證明了YN-01能夠引起HEPG-2細胞的凋亡。最后,熒光定量PCR實驗數(shù)據(jù)顯示YN-01處理HEPG-224小時之后,FoxM1和p53的mRNA水平大幅上調(diào),但48小時后其mRNA水平均大幅下調(diào)。抗凋亡基因Survivin和Bcl-2在48小時之后其mRNA水平也同樣出現(xiàn)大幅下調(diào)的現(xiàn)象,因此根據(jù)文獻研究以及上述數(shù)據(jù)可以進行推論,YN-01可能通過抑制FoxM1和p53基因的表達,從而抑制其下游的靶基因Svrvivin和Bcl-2的轉(zhuǎn)錄,來誘導(dǎo)腫瘤細胞進入凋亡途徑。
[Abstract]:As a member of the transcription factor Fox family, FoxM1c plays an important role in regulating the proliferation and division of normal cells, but the abnormal expression of FoxM1 can also lead to the occurrence and development of tumor cells. Many studies have confirmed that FoxM1 remains highly expressed in various tumor cells, but not in normal or well-differentiated cells. Therefore, it is theoretically feasible to treat tumor by targeting FoxM1 protein. However, because of the characteristics of FoxM1 transcription factors and their lack of hydrophobic structure, traditional small molecular chemicals are difficult to target. Therefore, traditional pharmacological studies usually exclude transcription factors from tumor therapy target proteins, but with the development of biotechnology, the advantages of polypeptide drugs overcome the inherent defects of traditional drugs, which makes targeted FoxM1 possible. In addition, many components of natural plant drugs have anti-tumor activity, which makes them have multi-target characteristics, which is consistent with the idea and method of tumor multi-target therapy. In this study, on the basis of screening high affinity polypeptide sequences by phage random dodecapeptide library in the early stage of our laboratory, a common motif sequence of high affinity peptide sequence, WHLD., was obtained by MEME motif analysis software. Then the motif sequence was bound to the transmembrane peptide and the two polypeptide sequences CT-788:rrrrrrrrGSGSWHLD (r is D arginine) and CT-789:WHLDGSGSWHLD. were determined. Before synthesis, the interaction between two peptides and FoxM1c DNA binding region of receptor protein was simulated by computer molecular docking software Molegro.Virtual.Docker (MVD). The results showed that the two polypeptides could bind stably to the FoxMlc DNA binding region. On the basis of molecular docking, we synthesized two kinds of polypeptides, CT-788 and CT-789, and carried out in vitro experiments on tumor cells. The effect of polypeptide molecules on the viability of tumor cells was detected by MTT assay. The inhibitory rates of CT-788 on HEPG-2 and MCF-7 tumor cells were 41.26% and 34.26% at the same concentration of 100ug/mL, respectively, while the inhibition rates of CT-789 on the two kinds of cells were 15.96% and 17.8% at the same concentration, respectively. The morphology of tumor cells treated with natural plant liquid was observed directly by microscope. It was clearly observed that natural plant liquid YN-01 could significantly inhibit the growth of HEPG-2 cells. And the tumor cells could be killed at a low concentration of 4% (containing 50% ethanol). MTT data also showed that YN-01 could inhibit the viability of HEPG-2 cells very well, and the inhibition rate of 4% low concentration was as high as 68%. After that, the double staining of AO-EB cells and the detection of DNA ladder,Annexin V-FITC/PI labeling by flow cytometry further proved that YN-01 can induce apoptosis of HEPG-2 cells. Finally, fluorescence quantitative PCR data showed that mRNA levels of FoxM1 and p53 increased significantly after YN-01 treatment for HEPG-224 hours, but mRNA levels decreased significantly 48 hours later. The mRNA levels of anti-apoptotic genes Survivin and Bcl-2 were also significantly down-regulated after 48 hours. Therefore, based on literature studies and the above data, we can infer that YN-01 may inhibit the expression of FoxM1 and p53 genes. In order to induce tumor cells to enter the apoptotic pathway, it inhibited the transcription of Svrvivin and Bcl-2 genes downstream.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R91
本文編號：2263517
[Abstract]:As a member of the transcription factor Fox family, FoxM1c plays an important role in regulating the proliferation and division of normal cells, but the abnormal expression of FoxM1 can also lead to the occurrence and development of tumor cells. Many studies have confirmed that FoxM1 remains highly expressed in various tumor cells, but not in normal or well-differentiated cells. Therefore, it is theoretically feasible to treat tumor by targeting FoxM1 protein. However, because of the characteristics of FoxM1 transcription factors and their lack of hydrophobic structure, traditional small molecular chemicals are difficult to target. Therefore, traditional pharmacological studies usually exclude transcription factors from tumor therapy target proteins, but with the development of biotechnology, the advantages of polypeptide drugs overcome the inherent defects of traditional drugs, which makes targeted FoxM1 possible. In addition, many components of natural plant drugs have anti-tumor activity, which makes them have multi-target characteristics, which is consistent with the idea and method of tumor multi-target therapy. In this study, on the basis of screening high affinity polypeptide sequences by phage random dodecapeptide library in the early stage of our laboratory, a common motif sequence of high affinity peptide sequence, WHLD., was obtained by MEME motif analysis software. Then the motif sequence was bound to the transmembrane peptide and the two polypeptide sequences CT-788:rrrrrrrrGSGSWHLD (r is D arginine) and CT-789:WHLDGSGSWHLD. were determined. Before synthesis, the interaction between two peptides and FoxM1c DNA binding region of receptor protein was simulated by computer molecular docking software Molegro.Virtual.Docker (MVD). The results showed that the two polypeptides could bind stably to the FoxMlc DNA binding region. On the basis of molecular docking, we synthesized two kinds of polypeptides, CT-788 and CT-789, and carried out in vitro experiments on tumor cells. The effect of polypeptide molecules on the viability of tumor cells was detected by MTT assay. The inhibitory rates of CT-788 on HEPG-2 and MCF-7 tumor cells were 41.26% and 34.26% at the same concentration of 100ug/mL, respectively, while the inhibition rates of CT-789 on the two kinds of cells were 15.96% and 17.8% at the same concentration, respectively. The morphology of tumor cells treated with natural plant liquid was observed directly by microscope. It was clearly observed that natural plant liquid YN-01 could significantly inhibit the growth of HEPG-2 cells. And the tumor cells could be killed at a low concentration of 4% (containing 50% ethanol). MTT data also showed that YN-01 could inhibit the viability of HEPG-2 cells very well, and the inhibition rate of 4% low concentration was as high as 68%. After that, the double staining of AO-EB cells and the detection of DNA ladder,Annexin V-FITC/PI labeling by flow cytometry further proved that YN-01 can induce apoptosis of HEPG-2 cells. Finally, fluorescence quantitative PCR data showed that mRNA levels of FoxM1 and p53 increased significantly after YN-01 treatment for HEPG-224 hours, but mRNA levels decreased significantly 48 hours later. The mRNA levels of anti-apoptotic genes Survivin and Bcl-2 were also significantly down-regulated after 48 hours. Therefore, based on literature studies and the above data, we can infer that YN-01 may inhibit the expression of FoxM1 and p53 genes. In order to induce tumor cells to enter the apoptotic pathway, it inhibited the transcription of Svrvivin and Bcl-2 genes downstream.
【學(xué)位授予單位】：西南交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R91
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相關(guān)期刊論文 前4條
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2 陳貫虹;遲建國;邱維忠;王加寧;孫元軍;;多肽藥物的研究進展[J];山東科學(xué);2008年03期
3 朱志遠;張燕;李征;李金恒;芮建中;袁靖;;受體蛋白與藥物分子對接的研究進展[J];中國臨床藥理學(xué)與治療學(xué);2009年11期
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，本文編號：2263517
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