基于FoxMlc抗癌先导药物设计与研究
发布时间：2018-10-11 08:27
【摘要】：FoxM1c作为转录因子Fox家族成员之一,对正常细胞的增殖分裂具有重要的调控作用,但同时FoxM1的异常表达也能导致肿瘤细胞的发生与发展。许多研究已经证实,FoxM1在多种肿瘤细胞中持续高表达,而在正常静止状态或高度分化的细胞中基本不表达。因此为通过靶向FoxM1蛋白来治疗肿瘤提供了理论上的可行性,但是由于FoxM1转录因子的特性及其结构上缺乏疏水性结构,传统小分子化学药物很难靶向。因此传统药物学研究通常将转录因子排除在肿瘤治疗靶蛋白之外,但是随着生物技术的发展,多肽药物本身的优点克服了传统药物固有的缺陷,从而使得靶向FoxM1成为可能性。另外天然植物药中的许多成分具有抗肿瘤活性,因此使得其具有多靶点特性,这种特点与肿瘤多靶点治疗思想和方法相吻合。 本研究首先在本实验室前期利用噬菌体随机十二肽库筛选获得的高亲和多肽序列的基础上,通过MEME模体分析软件获得一条高亲和多肽序列共有的模体序列：WHLD。之后再将模体序列与穿膜肽结合并最终确定两条多肽序列CT-788:rrrrrrrrGSGSWHLD(r为D型精氨酸)和CT-789:WHLDGSGSWHLD。在合成之前,通过计算机分子对接软件Molegro.Virtual.Docker (MVD)模拟两条多肽与受体蛋白FoxM1c DNA结合区的相互作用。结果显示两条多肽分子均能与FoxMlc DNA结合区稳定结合。 在分子对接的基础上,我们合成了CT-788和CT-789两种多肽,并进行肿瘤细胞体外实验,用MTT法检测多肽分子对肿瘤细胞活力的影响,其中CT-788在浓度100ug/mL下对HEPG-2和MCF-7肿瘤细胞的抑制率分别为41.26%和34.26%,而相同浓度下CT-789对两种细胞的抑制率分别为15.96%和17.8%。 通过显微镜直接观察天然植物液处理过的肿瘤细胞形态学,可以清楚观察到天然植物液YN-01能显著抑制HEPG-2细胞的生长,并且在4%的低浓度(含50%的乙醇)下就能杀死肿瘤细胞。MTT实验数据也很好地说明了YN-01能够很好抑制HEPG-2细胞的活力,其4%的低浓度抑制率就高达68%。之后通过AO-EB细胞双染、电泳检测DNA ladder、Annexin V-FITC/PI标记流式细胞仪实验进一步证明了YN-01能够引起HEPG-2细胞的凋亡。最后,荧光定量PCR实验数据显示YN-01处理HEPG-224小时之后,FoxM1和p53的mRNA水平大幅上调,但48小时后其mRNA水平均大幅下调。抗凋亡基因Survivin和Bcl-2在48小时之后其mRNA水平也同样出现大幅下调的现象,因此根据文献研究以及上述数据可以进行推论,YN-01可能通过抑制FoxM1和p53基因的表达,从而抑制其下游的靶基因Svrvivin和Bcl-2的转录,来诱导肿瘤细胞进入凋亡途径。
[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.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R91
本文编号：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.
【学位授予单位】：西南交通大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R91
【参考文献】
相关期刊论文 前4条
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2 陈贯虹;迟建国;邱维忠;王加宁;孙元军;;多肽药物的研究进展[J];山东科学;2008年03期
3 朱志远;张燕;李征;李金恒;芮建中;袁靖;;受体蛋白与药物分子对接的研究进展[J];中国临床药理学与治疗学;2009年11期
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