Lycopene对皮肤肿瘤的化学预防效应及相关机制的研究
发布时间：2019-03-03 17:34
【摘要】：[研究背景与目的]皮肤癌近年来受到研究者们非常广泛的关注,原因有二:①皮肤癌是为数不多年发病率仍然呈逐年上升趋势的肿瘤类型之一;②皮肤癌由于其发病部位的特殊性(直接可见),会对患者造成极大的心理创伤,由此引发一系列与肿瘤无关的重度心理性疾病(抑郁症、自闭症等)。因此,研究如何预防皮肤肿瘤的发生及发展,降低其逐年升高的发生率,是极为重要的。Lycopene是食物番茄中的重要活性物质。课题组经前期文献调研发现,Lycopene是被报道最多的在临床上能逆转皮肤肿瘤致癌剂(如UV和砷制剂)诱导的皮肤损伤甚至是癌前病变的效应成分。因此,我们有理由相信Lycopene对皮肤肿瘤的发生发展理应有很好的预防效应。于是我们开展了一系列体内外实验并结合生物信息学分析以及分子生物学手段以证实Lycopene对皮肤肿瘤的预防效应及其分子机制和生物学基础。[研究方法与结果](1)本课题第一部分实验研究运用DMBA/TPA诱导的小鼠背部乳突淋瘤模型和化学促癌剂(TPA)诱导的JB6 P+细胞软琼脂克隆形成实验,通过不同时程下给予的Lycopene,分别从体内整体动物水平和体外细胞水平确证了 Lycopene确有皮肤肿瘤预防相应,且具有阶段选择性(效应的产生集中于皮肤肿瘤发展的促进阶段);(2)本课题第二部分实验综合利用多种生物信息学(综合药效团模型、分子对接模型、网络拓扑学分析、基因富集分析、基因芯片数据分析、蛋白质间相互作用分析)的手段,分析出可能的直接或间接介导Lycopene预防皮肤癌发生发展促进阶段的关键靶标,并进行基因富集分析,挖掘出药效的可能分子机制及生物学基础:细胞内氧化还原失衡、细胞自噬与衰老、Nrf2信号通路的转录激活以及泛素蛋白酶体降解途径;(3)本课题第三部分实验是基于之前的生物信息学分析的结果,检测了 Lycopene在体内、外对抗病理性氧化还原失衡(检测8-oxo-dG、ROS、MDA及GSH/GSSG的水平)的效应。并顺藤摸瓜,挖掘出Lycopene对抗这一病理损伤以及发挥最终的皮肤肿瘤预防效应的机制:可能与其维持病理状态下耗竭的抗氧化酶系(SOD、CAT、GPx、GR)至正常生理水平的效应有关;(4)根据前期的生物信息学分析以及相关分子生物学实验(对机体细胞氧化还原状态和相关抗氧化酶系活性及表达水平的监测)的探索,初步预测出了 Nrf2可能是Lycopene发挥皮肤肿瘤预防效应的关键靶标。在本课题第四部分研究中,我们首先运用多种分子生物学手段(Western-blot,IHC,IF)等证实了 Lycopene在体内外对Nrf2蛋白核内聚集和转录活化的诱导效应。在此基础上,运用了 Nrf2-/-小鼠以及Nrf2KD细胞分别构建体内乳突淋瘤模型以及体外细胞恶性转化模型,均发现Lycopene的皮肤肿瘤预防效应的缺失。这一系列的结果都指向一个结论:Lycopene是通过诱导Nrf2的核内分布,从而刺激转录活化,继而促进下游抗氧化酶系活性及水平的增加,最终发挥体内外皮肤肿瘤预防效应的;(5)课题最后一部分的结果主要是通过多种分子生物学手段(Western-blot、realtime-PCR)探索Lycopene调控Nrf2入核和转录激活的分子机制:Lycopene是通过诱导p62介导的Keap1蛋白(胞浆内Nrf2的抑制因子)自噬性降解,从而解放Nrf2并促其入核发挥下游效应的;并通过检测Lycopene对p62敲降JB6P+细胞恶性转化的预防效应,明确了 p62/Keap1信号通路对Lycopene发挥皮肤肿瘤预防效应至关重要的作用。(6)除了课题主线外,还发现了一个现象:Lycopene仅对存在化学促癌剂TPA刺激的受损细胞存在系列生物学效应(对皮肤肿瘤的预防,对抗氧化酶系活性及水平的诱导,对Nrf2入核及转录活性的激活,对Keap1的自噬性降解)。这也许是Lycopene作为皮肤肿瘤预防药物的最大优势也应该是作为一个预防类药物最重要的特质。[结论与意义]预先给予Lycopene可以诱导病理状态下细胞(有促癌、致癌剂的刺激)内p62的蛋白水平的上调,从而促进p62与Keap1结合,诱导Keap1的降解,进而将Nrf2从与Keap1间形成的异源二聚体中解放出来,导致了 Nrf2在胞质中的蓄积以及进一步的核表达增多,继而启动下游靶蛋白的转录,上调机体细胞抗氧化酶系的活力及水平,逆转病理条件下细胞内的氧化还原失衡损伤状态,最终发挥皮肤肿瘤的预防作用。基于以上一系列实验结果及相关讨论,我们有理由相信在采取合适的防晒措施的同时联合使用一些含Lycopene的系列护肤产品有助于降低皮肤肿瘤高发人群的癌症发生率。
[Abstract]:[Study Background and Objective] Skin cancer has been widely concerned by the researchers in recent years, for two reasons: skin cancer is one of the few tumor types that the incidence of skin cancer is still increasing year by year; skin cancer is due to its particularity (directly visible). It can cause a great psychological trauma to the patient, thereby initiating a series of severe psychological diseases unrelated to the tumor (depression, autism, etc.). Therefore, it is of great importance to study how to prevent the occurrence and development of skin tumors and to reduce the incidence of increasing year by year. Lycopene is an important active ingredient in food tomatoes. The research group has found that Lycopene is the most reported effect component that can be used to reverse the skin damage induced by the skin tumor carcinogen (such as UV and arsenic preparation) and even the pre-cancerous lesion. Therefore, we have reason to believe that Lycopene should have a good preventive effect on the development of skin tumors. So we carried out a series of in-vivo experiments and combined with bioinformatics analysis and molecular biology to confirm the preventive effect of Lycopene on skin tumors and its molecular mechanism and biological basis. [Methods and results] (1) The first part of this study was to study the formation of the soft agar of JB6 P + cells induced by DMBA/ TPA-induced back-mastoid and chemical-promoting agent (TPA). In vivo, the level of the whole animal and the in vitro cell level have confirmed that Lycogene has the corresponding prevention of skin tumor, and has the stage selectivity (the effect is focused on the promotion stage of the development of the skin tumor); (2) The second part of the study comprehensively uses a variety of bioinformatics (integrated pharmacophore model, molecular docking model, network topology analysis, gene enrichment analysis, gene chip data analysis and protein-to-protein interaction analysis). The potential molecular mechanism and biological basis of gene enrichment analysis, cell autophagy and aging are analyzed, and the possible molecular mechanism and biological basis of the drug effect are also carried out, (3) The third part of the study was based on the results of the previous bioinformatics analysis, and the effect of lycoene in the in vivo and out-of-vivo anti-pathological oxidative reduction (detection of the levels of 8-oxo-dG, ROS, MDA and GSH/ GSSG) was detected. The mechanism of Lyoprene to counter this pathological injury and to play a final role in the skin-tumor prevention is shown in this paper. It may be related to the effect of the antioxidant enzyme system (SOD, CAT, Gx, GR) which can be exhausted in the pathological state to the normal physiological level. (4) According to the bioinformatics analysis of the early stage and the related molecular biology experiment (the monitoring of the redox state of the organism and the activity of the related anti-oxidation enzyme system and the expression level), it is preliminarily predicted that Nrf2 may be the key target of the skin tumor prevention effect of lycoene. In the fourth part of this study, we first used a variety of molecular biological methods (Western-blot, IHC, IF) to confirm the induction effect of lycoene on the aggregation and transcription of Nrf2 protein in vivo. On this basis, Nrf2-/-mice and Nrf2KD cells were used to construct the model of the in-vivo mastoid and the malignant transformation of the cells in vitro. The results of this series point to a conclusion: Lycopene is the core distribution of Nrf2, which can stimulate the transcription and activation, then promote the activity and level of the downstream anti-oxidation enzyme system, and finally play a role in the prevention of external skin tumor. (5) The final part of the project is to explore the molecular mechanism of the regulation of Nrf2 in nuclear and transcriptional activation by means of a variety of molecular biological means (Western-blot, time-PCR): Lycopene is a self-phagocytic degradation by inducing p62-mediated Kappa 1 protein (inhibitory factor of Nrf2 in the cytoplasm), So as to release Nrf2 and promote its entry into the nucleus to play a downstream effect, and the effect of the p62/ Keap1 signaling pathway on the skin tumor prevention effect of Lycopene is determined by detecting the preventive effect of the Lyoprene on the malignant transformation of the p62 knock-down JB6P + cell. (6) In addition to the main line of the subject, a phenomenon was found: Lycopene only has a series of biological effects on the damaged cells that are stimulated by the chemical-promoting agent (TPA) (the prevention of skin tumors, the induction of the activity and the level of the anti-oxidase system, the activation of Nrf2 in the nuclear and transcriptional activity, Self-phagocytosis to Keap1). This may be the greatest advantage of lycoene as a drug for skin tumor prevention, and should be the most important feature of a preventive drug. [Conclusion and significance] The pre-administration of Lycoprene to the up-regulation of the protein level of p62 in the cells (in the stimulation of cancer and cancer) in the pathological state, thus promoting the binding of p62 to Keap1, inducing the degradation of Keap1, and further freeing Nrf2 from the heterodimer formed between Keap1, Leading to the accumulation of Nrf2 in the cytoplasm and the increase of the further nuclear expression, and then the transcription of the downstream target protein is started, the activity and the level of the anti-oxidation enzyme system of the body cell are up-regulated, the damage state of the oxidative reduction in the cells under the pathological conditions is reversed, and the prevention effect of the skin tumor is finally played. Based on the above series of experimental results and related discussions, we have reason to believe that simultaneous use of some of the Lycopene-containing family of skin care products while taking appropriate sun protection measures will help to reduce the incidence of cancer in the high-risk population of skin tumors.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285
，
本文编号：2433943
[Abstract]:[Study Background and Objective] Skin cancer has been widely concerned by the researchers in recent years, for two reasons: skin cancer is one of the few tumor types that the incidence of skin cancer is still increasing year by year; skin cancer is due to its particularity (directly visible). It can cause a great psychological trauma to the patient, thereby initiating a series of severe psychological diseases unrelated to the tumor (depression, autism, etc.). Therefore, it is of great importance to study how to prevent the occurrence and development of skin tumors and to reduce the incidence of increasing year by year. Lycopene is an important active ingredient in food tomatoes. The research group has found that Lycopene is the most reported effect component that can be used to reverse the skin damage induced by the skin tumor carcinogen (such as UV and arsenic preparation) and even the pre-cancerous lesion. Therefore, we have reason to believe that Lycopene should have a good preventive effect on the development of skin tumors. So we carried out a series of in-vivo experiments and combined with bioinformatics analysis and molecular biology to confirm the preventive effect of Lycopene on skin tumors and its molecular mechanism and biological basis. [Methods and results] (1) The first part of this study was to study the formation of the soft agar of JB6 P + cells induced by DMBA/ TPA-induced back-mastoid and chemical-promoting agent (TPA). In vivo, the level of the whole animal and the in vitro cell level have confirmed that Lycogene has the corresponding prevention of skin tumor, and has the stage selectivity (the effect is focused on the promotion stage of the development of the skin tumor); (2) The second part of the study comprehensively uses a variety of bioinformatics (integrated pharmacophore model, molecular docking model, network topology analysis, gene enrichment analysis, gene chip data analysis and protein-to-protein interaction analysis). The potential molecular mechanism and biological basis of gene enrichment analysis, cell autophagy and aging are analyzed, and the possible molecular mechanism and biological basis of the drug effect are also carried out, (3) The third part of the study was based on the results of the previous bioinformatics analysis, and the effect of lycoene in the in vivo and out-of-vivo anti-pathological oxidative reduction (detection of the levels of 8-oxo-dG, ROS, MDA and GSH/ GSSG) was detected. The mechanism of Lyoprene to counter this pathological injury and to play a final role in the skin-tumor prevention is shown in this paper. It may be related to the effect of the antioxidant enzyme system (SOD, CAT, Gx, GR) which can be exhausted in the pathological state to the normal physiological level. (4) According to the bioinformatics analysis of the early stage and the related molecular biology experiment (the monitoring of the redox state of the organism and the activity of the related anti-oxidation enzyme system and the expression level), it is preliminarily predicted that Nrf2 may be the key target of the skin tumor prevention effect of lycoene. In the fourth part of this study, we first used a variety of molecular biological methods (Western-blot, IHC, IF) to confirm the induction effect of lycoene on the aggregation and transcription of Nrf2 protein in vivo. On this basis, Nrf2-/-mice and Nrf2KD cells were used to construct the model of the in-vivo mastoid and the malignant transformation of the cells in vitro. The results of this series point to a conclusion: Lycopene is the core distribution of Nrf2, which can stimulate the transcription and activation, then promote the activity and level of the downstream anti-oxidation enzyme system, and finally play a role in the prevention of external skin tumor. (5) The final part of the project is to explore the molecular mechanism of the regulation of Nrf2 in nuclear and transcriptional activation by means of a variety of molecular biological means (Western-blot, time-PCR): Lycopene is a self-phagocytic degradation by inducing p62-mediated Kappa 1 protein (inhibitory factor of Nrf2 in the cytoplasm), So as to release Nrf2 and promote its entry into the nucleus to play a downstream effect, and the effect of the p62/ Keap1 signaling pathway on the skin tumor prevention effect of Lycopene is determined by detecting the preventive effect of the Lyoprene on the malignant transformation of the p62 knock-down JB6P + cell. (6) In addition to the main line of the subject, a phenomenon was found: Lycopene only has a series of biological effects on the damaged cells that are stimulated by the chemical-promoting agent (TPA) (the prevention of skin tumors, the induction of the activity and the level of the anti-oxidase system, the activation of Nrf2 in the nuclear and transcriptional activity, Self-phagocytosis to Keap1). This may be the greatest advantage of lycoene as a drug for skin tumor prevention, and should be the most important feature of a preventive drug. [Conclusion and significance] The pre-administration of Lycoprene to the up-regulation of the protein level of p62 in the cells (in the stimulation of cancer and cancer) in the pathological state, thus promoting the binding of p62 to Keap1, inducing the degradation of Keap1, and further freeing Nrf2 from the heterodimer formed between Keap1, Leading to the accumulation of Nrf2 in the cytoplasm and the increase of the further nuclear expression, and then the transcription of the downstream target protein is started, the activity and the level of the anti-oxidation enzyme system of the body cell are up-regulated, the damage state of the oxidative reduction in the cells under the pathological conditions is reversed, and the prevention effect of the skin tumor is finally played. Based on the above series of experimental results and related discussions, we have reason to believe that simultaneous use of some of the Lycopene-containing family of skin care products while taking appropriate sun protection measures will help to reduce the incidence of cancer in the high-risk population of skin tumors.
【学位授予单位】：南京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R285
，
本文编号：2433943
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