Lycopene對皮膚腫瘤的化學(xué)預(yù)防效應(yīng)及相關(guān)機制的研究
發(fā)布時間：2019-03-03 17:34
【摘要】：[研究背景與目的]皮膚癌近年來受到研究者們非常廣泛的關(guān)注,原因有二:①皮膚癌是為數(shù)不多年發(fā)病率仍然呈逐年上升趨勢的腫瘤類型之一;②皮膚癌由于其發(fā)病部位的特殊性(直接可見),會對患者造成極大的心理創(chuàng)傷,由此引發(fā)一系列與腫瘤無關(guān)的重度心理性疾病(抑郁癥、自閉癥等)。因此,研究如何預(yù)防皮膚腫瘤的發(fā)生及發(fā)展,降低其逐年升高的發(fā)生率,是極為重要的。Lycopene是食物番茄中的重要活性物質(zhì)。課題組經(jīng)前期文獻(xiàn)調(diào)研發(fā)現(xiàn),Lycopene是被報道最多的在臨床上能逆轉(zhuǎn)皮膚腫瘤致癌劑(如UV和砷制劑)誘導(dǎo)的皮膚損傷甚至是癌前病變的效應(yīng)成分。因此,我們有理由相信Lycopene對皮膚腫瘤的發(fā)生發(fā)展理應(yīng)有很好的預(yù)防效應(yīng)。于是我們開展了一系列體內(nèi)外實驗并結(jié)合生物信息學(xué)分析以及分子生物學(xué)手段以證實Lycopene對皮膚腫瘤的預(yù)防效應(yīng)及其分子機制和生物學(xué)基礎(chǔ)。[研究方法與結(jié)果](1)本課題第一部分實驗研究運用DMBA/TPA誘導(dǎo)的小鼠背部乳突淋瘤模型和化學(xué)促癌劑(TPA)誘導(dǎo)的JB6 P+細(xì)胞軟瓊脂克隆形成實驗,通過不同時程下給予的Lycopene,分別從體內(nèi)整體動物水平和體外細(xì)胞水平確證了 Lycopene確有皮膚腫瘤預(yù)防相應(yīng),且具有階段選擇性(效應(yīng)的產(chǎn)生集中于皮膚腫瘤發(fā)展的促進(jìn)階段);(2)本課題第二部分實驗綜合利用多種生物信息學(xué)(綜合藥效團模型、分子對接模型、網(wǎng)絡(luò)拓?fù)鋵W(xué)分析、基因富集分析、基因芯片數(shù)據(jù)分析、蛋白質(zhì)間相互作用分析)的手段,分析出可能的直接或間接介導(dǎo)Lycopene預(yù)防皮膚癌發(fā)生發(fā)展促進(jìn)階段的關(guān)鍵靶標(biāo),并進(jìn)行基因富集分析,挖掘出藥效的可能分子機制及生物學(xué)基礎(chǔ):細(xì)胞內(nèi)氧化還原失衡、細(xì)胞自噬與衰老、Nrf2信號通路的轉(zhuǎn)錄激活以及泛素蛋白酶體降解途徑;(3)本課題第三部分實驗是基于之前的生物信息學(xué)分析的結(jié)果,檢測了 Lycopene在體內(nèi)、外對抗病理性氧化還原失衡(檢測8-oxo-dG、ROS、MDA及GSH/GSSG的水平)的效應(yīng)。并順藤摸瓜,挖掘出Lycopene對抗這一病理損傷以及發(fā)揮最終的皮膚腫瘤預(yù)防效應(yīng)的機制:可能與其維持病理狀態(tài)下耗竭的抗氧化酶系(SOD、CAT、GPx、GR)至正常生理水平的效應(yīng)有關(guān);(4)根據(jù)前期的生物信息學(xué)分析以及相關(guān)分子生物學(xué)實驗(對機體細(xì)胞氧化還原狀態(tài)和相關(guān)抗氧化酶系活性及表達(dá)水平的監(jiān)測)的探索,初步預(yù)測出了 Nrf2可能是Lycopene發(fā)揮皮膚腫瘤預(yù)防效應(yīng)的關(guān)鍵靶標(biāo)。在本課題第四部分研究中,我們首先運用多種分子生物學(xué)手段(Western-blot,IHC,IF)等證實了 Lycopene在體內(nèi)外對Nrf2蛋白核內(nèi)聚集和轉(zhuǎn)錄活化的誘導(dǎo)效應(yīng)。在此基礎(chǔ)上,運用了 Nrf2-/-小鼠以及Nrf2KD細(xì)胞分別構(gòu)建體內(nèi)乳突淋瘤模型以及體外細(xì)胞惡性轉(zhuǎn)化模型,均發(fā)現(xiàn)Lycopene的皮膚腫瘤預(yù)防效應(yīng)的缺失。這一系列的結(jié)果都指向一個結(jié)論:Lycopene是通過誘導(dǎo)Nrf2的核內(nèi)分布,從而刺激轉(zhuǎn)錄活化,繼而促進(jìn)下游抗氧化酶系活性及水平的增加,最終發(fā)揮體內(nèi)外皮膚腫瘤預(yù)防效應(yīng)的;(5)課題最后一部分的結(jié)果主要是通過多種分子生物學(xué)手段(Western-blot、realtime-PCR)探索Lycopene調(diào)控Nrf2入核和轉(zhuǎn)錄激活的分子機制:Lycopene是通過誘導(dǎo)p62介導(dǎo)的Keap1蛋白(胞漿內(nèi)Nrf2的抑制因子)自噬性降解,從而解放Nrf2并促其入核發(fā)揮下游效應(yīng)的;并通過檢測Lycopene對p62敲降JB6P+細(xì)胞惡性轉(zhuǎn)化的預(yù)防效應(yīng),明確了 p62/Keap1信號通路對Lycopene發(fā)揮皮膚腫瘤預(yù)防效應(yīng)至關(guān)重要的作用。(6)除了課題主線外,還發(fā)現(xiàn)了一個現(xiàn)象:Lycopene僅對存在化學(xué)促癌劑TPA刺激的受損細(xì)胞存在系列生物學(xué)效應(yīng)(對皮膚腫瘤的預(yù)防,對抗氧化酶系活性及水平的誘導(dǎo),對Nrf2入核及轉(zhuǎn)錄活性的激活,對Keap1的自噬性降解)。這也許是Lycopene作為皮膚腫瘤預(yù)防藥物的最大優(yōu)勢也應(yīng)該是作為一個預(yù)防類藥物最重要的特質(zhì)。[結(jié)論與意義]預(yù)先給予Lycopene可以誘導(dǎo)病理狀態(tài)下細(xì)胞(有促癌、致癌劑的刺激)內(nèi)p62的蛋白水平的上調(diào),從而促進(jìn)p62與Keap1結(jié)合,誘導(dǎo)Keap1的降解,進(jìn)而將Nrf2從與Keap1間形成的異源二聚體中解放出來,導(dǎo)致了 Nrf2在胞質(zhì)中的蓄積以及進(jìn)一步的核表達(dá)增多,繼而啟動下游靶蛋白的轉(zhuǎn)錄,上調(diào)機體細(xì)胞抗氧化酶系的活力及水平,逆轉(zhuǎn)病理條件下細(xì)胞內(nèi)的氧化還原失衡損傷狀態(tài),最終發(fā)揮皮膚腫瘤的預(yù)防作用�；谝陨弦幌盗袑嶒灲Y(jié)果及相關(guān)討論,我們有理由相信在采取合適的防曬措施的同時聯(lián)合使用一些含Lycopene的系列護(hù)膚產(chǎn)品有助于降低皮膚腫瘤高發(fā)人群的癌癥發(fā)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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R285
，
本文編號：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.
【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R285
，
本文編號：2433943
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