SOD1启动子上调多肽筛选体系的建立
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摘要
超氧化物岐化酶(super oxide dismutase,SOD)是一种广泛存在于生物体内的金属酶,根据分布的不同主要分为SOD1(Cu/Zn SOD,主要分布于真核细胞胞浆),SOD2(Mn/Fe SOD,主要分布于真核细胞线粒体和原核细胞)和SOD3(EC-SOD,主要分布于细胞外基质),不同的SOD均有催化超氧阴离子自由基O_2~-发生歧化反应的功能,是体内重要的氧自由基清除剂。其中,SOD1是发现最早,研究最广泛的一类。
氧自由基是直接或间接由分子氧转化而成,外层轨道具有未配对电子的一类化学分子,包括超氧离子自由基、羟自由基、过氧化氢等。它们具有活泼的化学活性,极易发生得到或失去电子的反应,从而引发链式自由基反应。体内的氧自由基包括由于高温、辐射等原因导致某些含氧分子共价键断裂而产生的外源性自由基和体内各种代谢过程中产生的内源性氧自由基。氧自由基是机体代谢正常的中间产物,低浓度的氧自由基是机体执行正常生理功能所必需,但在氧自由基异常增多或机体抗氧化能力下降的情况下,氧自由基将通过链式自由基反应损伤细胞膜、DNA、蛋白质等多种细胞组分。目前已知氧自由基与衰老、炎症、缺血再灌注损伤、神经退行性病变等多种疾病相关,清除多余的氧自由基对延长动物寿命,增强其对氧应激的承受能力和改善炎症状况等多个方面具有显著的作用。
机体主要通过两阶段的反应来清除体内的氧自由基:第一阶段是超氧阴离子自由基在SOD的作用下转变为氧气和过氧化氢;第二阶段是过氧化氢在过氧化氢酶(catalase,CAT)的作用下转变为水和氧气。SOD在氧自由基的清除过程中起着至关重要的作用。SOD缺陷的大肠杆菌将无法在有氧条件下生存,而适量增加的SOD则能起到减少细胞内氧自由基含量,减轻脂质体过氧化乃至延缓衰老、控制炎症等多种作用。可见,通过控制SOD在体内的含量,可以调节氧自由基的浓度并从而改变多种相关的病理、生理状态,具有极其重要的临床意义。
皮肤皱纹的产生机制包括外源性因素和内源性因素两个方面。外源性因素主要有日光照射所致的光老化和其他环境因素如外伤、感染、空气污染等对皮肤的损伤;内源性因素主要有年龄增长导致的基因调控变化、内源性自由基对细胞的损伤和机体代谢的紊乱。目前国际上主要针对光老化和内源性氧自由基对皮肤的损伤这两个方面进行抗皱研究。光老化指日光长期照射所致的皮肤老化,日光中的紫外线可通过产生高度反应的外源性氧自由基损伤皮肤。此外,紫外线最早损伤的部位为调节SOD和过氧化氢酶的基因,使二者合成减少,致使氧自由基聚集而损伤细胞。由上述可见,在皮肤皱纹产生的内源性和外源性途径中,过高浓度的氧自由基都扮演着重要角色。因此,清除体内过高的氧自由基对于皮肤抗皱具有重要的意义。如果能适度提高皮肤细胞内SOD含量,则可以通过减少氧自由基的含量来减少对皮肤的损伤,从而保护皮肤,减少皱纹。
虽然针对SOD的研究已有多年,但目前还没有能够实际进入临床应用的SOD类药物,这是因为SOD作为一种蛋白质药物,具有制备价格昂贵,不能直接进入细胞发挥作用、半衰期短、易诱发免疫反应等缺陷,且具体使用剂量也不易控制。目前针对SOD药用方面的研究主要包括研究药物载体、发现SOD的小分子替代物和寻找能上调内源性SOD表达药物等途径,据此,我们设计了一个利用报告基因技术筛选具有上调内源性SOD1表达的随机多肽筛选系统,试图同时克服上述多种问题。
报告基因是指其表达产物易被检测且易与内源性背景蛋白相区别的基因。通过将特定基因的转录控制元件克隆到报告基因载体,可以通过报告基因的表达来直观地检测目标基因的表达情况。红色荧光蛋白(red fluorenscent protein,RFP)是一种分子量为28 kD的荧光蛋白,可自动发射荧光、无需任何辅助因子和底物的参与,且无需裂解细胞即可检测活体组织和细胞的基因表达,作为报告基因具有其特有的优越性。
本研究利用基因重组技术构建了含人SOD1启动子区的红色荧光蛋白报告基因质粒,并通过转染和抗生素筛选技术得到了能稳定表达该质粒的细胞系。此细胞系在适宜浓度的SOD1诱导剂卟啉醇肉豆蔻酸乙酸酯(phorbol myristateacetate,PMA)刺激下,红色荧光蛋白表达明显增加,红色荧光强度在一定范围内呈线性增长。这为筛选具有内源性SOD1表达上调功能的药物提供了一个方便、灵敏的工具。
为了克服蛋白质大分子常具有的免疫原性,我们使用连续的十二个随机氨基酸组成的多肽作为筛选的对象。由于细胞膜具有选择性通透性,大部分蛋白质分子无法直接进入细胞内部发挥生理学效应。我们选用Ⅰ型人类免疫缺陷病毒(human immunodeficiency virusⅠ,HIV-Ⅰ)Tat蛋白的蛋白转导域(proteintransduction domain,PTD)作为携带多肽入胞的载体。HIV-Tat是一种以非传统内吞途径、非能量依赖,非受体介导方式直接穿透细胞膜进入细胞的多肽,它能携带多种不同大小和性质的生物活性物质如核苷酸、多肽、蛋白质等进入细胞,是一种理想的多肽转运载体。此外,融合蛋白中的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)可作为蛋白入胞的指示剂,提示随机多肽在细胞内的分布情况。针对肽库的容量性和多样性问题,我们利用点突变技术引入36个随机碱基,形成随机表达文库,其理论容量达到20~(12),从而保证了随机多肽库的容量性和多样性。
为了能够进行高通量的多肽筛选,我们总结出一套切实可行的蛋白纯化及药物筛选流程。利用融合蛋白上的His标签纯化融合蛋白,将纯化后的融合蛋白与稳定表达报告基因质粒的细胞孵育一定时间后,融合蛋白进入细胞,通过测量细胞荧光强度的变化来判断多肽对SOD1启动子的影响,从而为后续大规模的筛选奠定了基础。
综上所述,本研究利用报告基因技术和细胞系筛选技术得到了能作为人SOD1启动子表达情况检测标准的细胞系,并利用基因重组和点突变技术得到了包含随机多肽序列、跨膜转运载体和示踪用绿色荧光蛋白的随机多肽库,在两者的基础上开发出一套能够用以筛选上调内源性SOD1表达的随机多肽药物的筛选流程,从而为后续的药物筛选工作提供了有力的支持。
The SODs are a family of enzymes that very efficiently catalyze the dismutation of the superoxide radical anion(O2~(·-)). There are mainly three categories of SOD according to their distributions in cells: SOD1 (Cu/Zn SOD, mainly located in eukaryotic cytoplasm), SOD2 (Mn/Fe SOD, mainly located in eukaryotic mitochondria) and SOD3 (EC-SOD, mainly located in the extracellular matrix). All the SODs have the ability to catalyze superoxide anion to have dismutation reaction and thus become an important scavenger for oxygen free radicals. Among them, SOD1 was the first discovered and studied widely.
The oxygen free radicals(OFR) converted from oxygen molecules directly or indirectly. There are uncoupled electrons in their outer orbit, including superoxide anion free radical, hydroxyl radical, hydrogen peroxide. With reactive chemical properties , they gain or loss electrons easily and thus initiate free radical chain reaction. The free radicals in vivo are produced by the stimulus such as high temperature, radiation or just a by-product of normal metabolism process. Produced in proper amount, superoxide is a normal and useful metabolite, serving important roles as a signaling molecule in processes such as cell division, and even serving to act as a terminator of lipid peroxidation. When flagrantly overproduced, however, the radical can initiate lipid peroxidation, protein oxidation, and DNA damage, leading to cell dysfunction and death by apoptosis or necrosis. The radical and/or the SODs have been implicated in a broad range of disease states including inflammatory diseases, diseases of ischemia and reperfusion injury, neurodegenerative diseases, diabetes, cancer, and many others. It's helpful to those diseases when redundant free radicals cleaned out.
Two main reactions involved in the elimination of free radicals. The first is to transform superoxide anion to oxygen molecule and hydrogen peroxide by SOD, the second is to transform hydrogen peroxide into oxygen molecule and water. SOD plays an essential role in such process. If the major SOD genes were disrupted in E. coli, the organism could no longer grow in oxygen on minimal medium. Considerably increased SOD could decrease the amount of free radicals and ameliorate liposome peroxidation, inflammatory activity and aging. So it would be possible to regulate the amount of free radicals and thus control physical and pathological conditions by manipulating the SOD content.
The mechanism of wrinkle generating involves exogenous factors such as light aging, injury and infection and endogenous factors such as genetic regulation according to aging and injury caused by endogenous free radicals. The free radicals produced by the ultraviolet ray ( UV ) of sun light will cause skin aging. SOD and CAT genes also are damaged by UV, as a result, the production of SOD and CAT decrease and free radicals accumulate and the skin be damaged. So, the free radicals play a very important role in the wrinkle generating. If the amount of SOD could increase moderately, the skin would be protected and wrinkles diminished.
Despite the long time of the SOD study, SOD-based antioxidant therapy has not made a greater and more rapid impact on clinical medicine: the problem of oxidant-antioxidant balance. As a protein with large molecular weight, SOD is expensive, short-life, besides, it can not overcome the barrier of cell membrane to work inside the cells. The immunogenicity and dose problems are hard to solve too. Methods to overcome such problems includ drug vectors transporting the drugs into cells, SOD mimics and up-regulating the endogenous SOD production. In this study, a system was designed to screen out the polypeptide drugs which could up-regulate the endogenous SOD production.
Reporter genes are a kind of genes whose products could be detected easily and could be distinguished from background proteins. The transcription elements of certain gene cloned into reporter gene vector, the expression level of the gene can be detected directly by the expression of reporter gene. Red fluorescent protein gene encodes a red fluorescent protein that has been optimized for high expression in mammalian cells (excitation maximum = 558 nm; emission maxmum = 583 nnm) without any co-factor or substrate, thus it could detect the gene expression of living tissue and cells without cell disruption.
In this study, a human SOD1 promoter was cloned into the pDsRED1-1 vector containing red fluorescence protein reporter gene and transfected into NIH 3T3 cell line, subsequently a permanent cell line was screened out. The red fluorescence of this cell line remarkably increased after stimulated by PMA, and the value of fluorescence increased in a linear manner. The cell line provided a convenient and sensitive tool to screen the drugs which could up-regulate the expression of endogenous SOD1.
In order to avoid the immunogenicity problem by protein, a peptide library was constructed for drug screening, which contains 12 random amino with a theoretical capacity of 2012 to guarantee the capability and diversity of the library. Since most of the large molecules could not entry the cell membrane, HIV-Tat PTD was chosen as the drug vector to transport the fusion protein into the cells. HIV-Tat can penetrate the cell membrane in a energy independent, receptor independent and classical internalization independent way. It can carry various cargoes including nucleotide, polypeptide and protein into the cells and is an ideal transporter for polypeptide. Besides, EGFP in the fusion protein can be a taq to tell the location of the protein in the cells.
An effective work-flow has been established to purify the polypeptide drug candidates and to screen out the potential drugs. The histidine taq was selected to purify the proteins and then the purified proteins were incubated with the certain premanent cells for a certain time, and then the value of fluorescence inside the cells was detected to judge the effect of the peptide on human SOD1 promoter.
To sum up, this study used the gene technology and cell line screening technology to obtain a permanent cell line that can be a tool to detect the expression level of human SOD1 promoter, and used genetic recombination and mutagenesis technology to construct a random polypeptide library containing 12 continuous random polypeptides, transmembrane transporting carrier and EGFP. Based on the cell line and random polypeptide library, a work-flow was established to screen out the polypeptide drug which could up-regulate the endogenous SOD1 expression, thereby provided a system for the following drug screening work.
氧自由基是直接或间接由分子氧转化而成,外层轨道具有未配对电子的一类化学分子,包括超氧离子自由基、羟自由基、过氧化氢等。它们具有活泼的化学活性,极易发生得到或失去电子的反应,从而引发链式自由基反应。体内的氧自由基包括由于高温、辐射等原因导致某些含氧分子共价键断裂而产生的外源性自由基和体内各种代谢过程中产生的内源性氧自由基。氧自由基是机体代谢正常的中间产物,低浓度的氧自由基是机体执行正常生理功能所必需,但在氧自由基异常增多或机体抗氧化能力下降的情况下,氧自由基将通过链式自由基反应损伤细胞膜、DNA、蛋白质等多种细胞组分。目前已知氧自由基与衰老、炎症、缺血再灌注损伤、神经退行性病变等多种疾病相关,清除多余的氧自由基对延长动物寿命,增强其对氧应激的承受能力和改善炎症状况等多个方面具有显著的作用。
机体主要通过两阶段的反应来清除体内的氧自由基:第一阶段是超氧阴离子自由基在SOD的作用下转变为氧气和过氧化氢;第二阶段是过氧化氢在过氧化氢酶(catalase,CAT)的作用下转变为水和氧气。SOD在氧自由基的清除过程中起着至关重要的作用。SOD缺陷的大肠杆菌将无法在有氧条件下生存,而适量增加的SOD则能起到减少细胞内氧自由基含量,减轻脂质体过氧化乃至延缓衰老、控制炎症等多种作用。可见,通过控制SOD在体内的含量,可以调节氧自由基的浓度并从而改变多种相关的病理、生理状态,具有极其重要的临床意义。
皮肤皱纹的产生机制包括外源性因素和内源性因素两个方面。外源性因素主要有日光照射所致的光老化和其他环境因素如外伤、感染、空气污染等对皮肤的损伤;内源性因素主要有年龄增长导致的基因调控变化、内源性自由基对细胞的损伤和机体代谢的紊乱。目前国际上主要针对光老化和内源性氧自由基对皮肤的损伤这两个方面进行抗皱研究。光老化指日光长期照射所致的皮肤老化,日光中的紫外线可通过产生高度反应的外源性氧自由基损伤皮肤。此外,紫外线最早损伤的部位为调节SOD和过氧化氢酶的基因,使二者合成减少,致使氧自由基聚集而损伤细胞。由上述可见,在皮肤皱纹产生的内源性和外源性途径中,过高浓度的氧自由基都扮演着重要角色。因此,清除体内过高的氧自由基对于皮肤抗皱具有重要的意义。如果能适度提高皮肤细胞内SOD含量,则可以通过减少氧自由基的含量来减少对皮肤的损伤,从而保护皮肤,减少皱纹。
虽然针对SOD的研究已有多年,但目前还没有能够实际进入临床应用的SOD类药物,这是因为SOD作为一种蛋白质药物,具有制备价格昂贵,不能直接进入细胞发挥作用、半衰期短、易诱发免疫反应等缺陷,且具体使用剂量也不易控制。目前针对SOD药用方面的研究主要包括研究药物载体、发现SOD的小分子替代物和寻找能上调内源性SOD表达药物等途径,据此,我们设计了一个利用报告基因技术筛选具有上调内源性SOD1表达的随机多肽筛选系统,试图同时克服上述多种问题。
报告基因是指其表达产物易被检测且易与内源性背景蛋白相区别的基因。通过将特定基因的转录控制元件克隆到报告基因载体,可以通过报告基因的表达来直观地检测目标基因的表达情况。红色荧光蛋白(red fluorenscent protein,RFP)是一种分子量为28 kD的荧光蛋白,可自动发射荧光、无需任何辅助因子和底物的参与,且无需裂解细胞即可检测活体组织和细胞的基因表达,作为报告基因具有其特有的优越性。
本研究利用基因重组技术构建了含人SOD1启动子区的红色荧光蛋白报告基因质粒,并通过转染和抗生素筛选技术得到了能稳定表达该质粒的细胞系。此细胞系在适宜浓度的SOD1诱导剂卟啉醇肉豆蔻酸乙酸酯(phorbol myristateacetate,PMA)刺激下,红色荧光蛋白表达明显增加,红色荧光强度在一定范围内呈线性增长。这为筛选具有内源性SOD1表达上调功能的药物提供了一个方便、灵敏的工具。
为了克服蛋白质大分子常具有的免疫原性,我们使用连续的十二个随机氨基酸组成的多肽作为筛选的对象。由于细胞膜具有选择性通透性,大部分蛋白质分子无法直接进入细胞内部发挥生理学效应。我们选用Ⅰ型人类免疫缺陷病毒(human immunodeficiency virusⅠ,HIV-Ⅰ)Tat蛋白的蛋白转导域(proteintransduction domain,PTD)作为携带多肽入胞的载体。HIV-Tat是一种以非传统内吞途径、非能量依赖,非受体介导方式直接穿透细胞膜进入细胞的多肽,它能携带多种不同大小和性质的生物活性物质如核苷酸、多肽、蛋白质等进入细胞,是一种理想的多肽转运载体。此外,融合蛋白中的增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)可作为蛋白入胞的指示剂,提示随机多肽在细胞内的分布情况。针对肽库的容量性和多样性问题,我们利用点突变技术引入36个随机碱基,形成随机表达文库,其理论容量达到20~(12),从而保证了随机多肽库的容量性和多样性。
为了能够进行高通量的多肽筛选,我们总结出一套切实可行的蛋白纯化及药物筛选流程。利用融合蛋白上的His标签纯化融合蛋白,将纯化后的融合蛋白与稳定表达报告基因质粒的细胞孵育一定时间后,融合蛋白进入细胞,通过测量细胞荧光强度的变化来判断多肽对SOD1启动子的影响,从而为后续大规模的筛选奠定了基础。
综上所述,本研究利用报告基因技术和细胞系筛选技术得到了能作为人SOD1启动子表达情况检测标准的细胞系,并利用基因重组和点突变技术得到了包含随机多肽序列、跨膜转运载体和示踪用绿色荧光蛋白的随机多肽库,在两者的基础上开发出一套能够用以筛选上调内源性SOD1表达的随机多肽药物的筛选流程,从而为后续的药物筛选工作提供了有力的支持。
The SODs are a family of enzymes that very efficiently catalyze the dismutation of the superoxide radical anion(O2~(·-)). There are mainly three categories of SOD according to their distributions in cells: SOD1 (Cu/Zn SOD, mainly located in eukaryotic cytoplasm), SOD2 (Mn/Fe SOD, mainly located in eukaryotic mitochondria) and SOD3 (EC-SOD, mainly located in the extracellular matrix). All the SODs have the ability to catalyze superoxide anion to have dismutation reaction and thus become an important scavenger for oxygen free radicals. Among them, SOD1 was the first discovered and studied widely.
The oxygen free radicals(OFR) converted from oxygen molecules directly or indirectly. There are uncoupled electrons in their outer orbit, including superoxide anion free radical, hydroxyl radical, hydrogen peroxide. With reactive chemical properties , they gain or loss electrons easily and thus initiate free radical chain reaction. The free radicals in vivo are produced by the stimulus such as high temperature, radiation or just a by-product of normal metabolism process. Produced in proper amount, superoxide is a normal and useful metabolite, serving important roles as a signaling molecule in processes such as cell division, and even serving to act as a terminator of lipid peroxidation. When flagrantly overproduced, however, the radical can initiate lipid peroxidation, protein oxidation, and DNA damage, leading to cell dysfunction and death by apoptosis or necrosis. The radical and/or the SODs have been implicated in a broad range of disease states including inflammatory diseases, diseases of ischemia and reperfusion injury, neurodegenerative diseases, diabetes, cancer, and many others. It's helpful to those diseases when redundant free radicals cleaned out.
Two main reactions involved in the elimination of free radicals. The first is to transform superoxide anion to oxygen molecule and hydrogen peroxide by SOD, the second is to transform hydrogen peroxide into oxygen molecule and water. SOD plays an essential role in such process. If the major SOD genes were disrupted in E. coli, the organism could no longer grow in oxygen on minimal medium. Considerably increased SOD could decrease the amount of free radicals and ameliorate liposome peroxidation, inflammatory activity and aging. So it would be possible to regulate the amount of free radicals and thus control physical and pathological conditions by manipulating the SOD content.
The mechanism of wrinkle generating involves exogenous factors such as light aging, injury and infection and endogenous factors such as genetic regulation according to aging and injury caused by endogenous free radicals. The free radicals produced by the ultraviolet ray ( UV ) of sun light will cause skin aging. SOD and CAT genes also are damaged by UV, as a result, the production of SOD and CAT decrease and free radicals accumulate and the skin be damaged. So, the free radicals play a very important role in the wrinkle generating. If the amount of SOD could increase moderately, the skin would be protected and wrinkles diminished.
Despite the long time of the SOD study, SOD-based antioxidant therapy has not made a greater and more rapid impact on clinical medicine: the problem of oxidant-antioxidant balance. As a protein with large molecular weight, SOD is expensive, short-life, besides, it can not overcome the barrier of cell membrane to work inside the cells. The immunogenicity and dose problems are hard to solve too. Methods to overcome such problems includ drug vectors transporting the drugs into cells, SOD mimics and up-regulating the endogenous SOD production. In this study, a system was designed to screen out the polypeptide drugs which could up-regulate the endogenous SOD production.
Reporter genes are a kind of genes whose products could be detected easily and could be distinguished from background proteins. The transcription elements of certain gene cloned into reporter gene vector, the expression level of the gene can be detected directly by the expression of reporter gene. Red fluorescent protein gene encodes a red fluorescent protein that has been optimized for high expression in mammalian cells (excitation maximum = 558 nm; emission maxmum = 583 nnm) without any co-factor or substrate, thus it could detect the gene expression of living tissue and cells without cell disruption.
In this study, a human SOD1 promoter was cloned into the pDsRED1-1 vector containing red fluorescence protein reporter gene and transfected into NIH 3T3 cell line, subsequently a permanent cell line was screened out. The red fluorescence of this cell line remarkably increased after stimulated by PMA, and the value of fluorescence increased in a linear manner. The cell line provided a convenient and sensitive tool to screen the drugs which could up-regulate the expression of endogenous SOD1.
In order to avoid the immunogenicity problem by protein, a peptide library was constructed for drug screening, which contains 12 random amino with a theoretical capacity of 2012 to guarantee the capability and diversity of the library. Since most of the large molecules could not entry the cell membrane, HIV-Tat PTD was chosen as the drug vector to transport the fusion protein into the cells. HIV-Tat can penetrate the cell membrane in a energy independent, receptor independent and classical internalization independent way. It can carry various cargoes including nucleotide, polypeptide and protein into the cells and is an ideal transporter for polypeptide. Besides, EGFP in the fusion protein can be a taq to tell the location of the protein in the cells.
An effective work-flow has been established to purify the polypeptide drug candidates and to screen out the potential drugs. The histidine taq was selected to purify the proteins and then the purified proteins were incubated with the certain premanent cells for a certain time, and then the value of fluorescence inside the cells was detected to judge the effect of the peptide on human SOD1 promoter.
To sum up, this study used the gene technology and cell line screening technology to obtain a permanent cell line that can be a tool to detect the expression level of human SOD1 promoter, and used genetic recombination and mutagenesis technology to construct a random polypeptide library containing 12 continuous random polypeptides, transmembrane transporting carrier and EGFP. Based on the cell line and random polypeptide library, a work-flow was established to screen out the polypeptide drug which could up-regulate the endogenous SOD1 expression, thereby provided a system for the following drug screening work.
引文
1 Kindo S. The roles of cytokines in photoaging[J]. J Dermatol Sci, 2000, 23: 30 -36.
2 Giacomoni PU, Rein G. Factors of skin ageing share common mechanisms[J].Biogerontology,2001,2:219-229.
3 Marian V, Dieter L, Jan M, et al. Free radicals and antioxidants in normal physiological functions and human disease[J]. Int J Biochem Cell Biol, 2007, 39:44 - 84.
4 Yasui K, Baba A. Therapeutic potential of superoxide dismutase (SOD) for resolution of inflammation[J]. Inflamm Res, 2006, 55: 359 - 363.
5 Kagan VE, Kisin ER, Kawai K, et al. Toward mechanism based antioxidant interventions: lessons from natural antioxidants[J]. Ann N Y Acad Sci, 2002,959: 188-198.
6 Fridovich I. Superoxide radical and superoxide dismutases[J]. Annu Rev Biochem, 1995,64:97-112.
7 Carlioz A, Touati D. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life[J]. EMBO, 1986, 5: 623-630.
8 Gao B, Flores SC, Bose SK, et al. A novel Escherichia coli vector for oxygen-inducible high level expression of foreign genes[J]. Gene 1996, 176: 269-72.
9 Elroy-Stein O, Bernstein Y, Groner Y. Overproduction of human Cu/Zn superoxide dismutase in transfected cells: extenuation of paraquat-mediated cytotoxicity and enhancement of lipid peroxidation[J]. EMBO J, 1986, 5: 615 —622.
10 Melov S, Ravenscroft J, Malik S, et al. Extension of life-span with superoxide dismutase/catalase mimetics[J]. Sci, 2000, 289: 1567 - 1569.
11 Schriner S, Linford N, Martin G, et al. Extension of Murine Life Span by Overexpression of Catalase Targeted to Mitochondria[J]. Sci, 2005, 308: 1909 -1911.
12 Igor NZ, Thomas JM, Rodney JF. Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression[J]. Free Radical Bio Med. 2002, 33:337-349.
13 Marberger H, Huber W, Bartsch G, et al. Orgotein: a new antiinflammatory metalloprotein drug evaluation of clinical efficacy and safety in inflammatory conditions of the urinary tract[J]. Int Urol Nephrol, 1974, 6: 61 - 74.
14 Delanian S, Martin M, Bravard A, et al. Cu/Zn superoxide dismutase modulates phenotypic changes in cultured fibroblasts from human skin with chronic radiotherapy damage[J]. Radiother Oncol 2001, 58: 325 - 331.
15 Vozenin-Brotons MC, Sivan V, Gault N, et al. Antifibrotic action of Cu/Zn SOD is mediated by TGF-betal repression and phenotypic reversion of myofibroblasts[J]. Free Radic Biol Med, 2001, 30: 30 - 42.
16 田春美,钟秋平.超氧化物歧化酶的现状研究进展[J].中国热带医学,2005,5:1730-1732.
17 Ana IR,Marta S,Daniel M,et al.Regulation of Cu/Zn-superoxide dismutase expression via the phosphatidylinositol 3 kinase/akt pathway and nuclear factor-κB [J].J Neurosci,2004,24(33):7324 - 7334.
18 Mine E,Coppet P,Masson P,et al.The human copper-zinc superoxide dismutase gene(SOD1) proximal promoter is regulated by spl,egr-1,and wt1via non-canonical binding sites[J].J Biol Chem,1999,274:503 - 509.
19 McCord J.Therapeutic control of free radicals[J].Drug Discov Today,2004,9:781-782.
20 黄劭,刘亚伟,姜勇.细胞穿透肽在肿瘤治疗中的应用[J].生理科学进展,2007,38:301-306.
21 Baird GS,Zacharias DA,Tsien RY.Biochemistry,mutanenesis,and oligomerization of DsRed,a red fluorescent protein from coral[J].Proc Natl Acad Sci USA,2000,7:1984- 1989.
22 郭爱华,刘志峰,孙学刚,等.一种基于HIV-TAT蛋白质转导结构域细胞内转导系统的成功改建[J].南方医科大学学报,2006,26:545-548.
1 Pontus Lundberg, Ulo Langel. A brief introduction to cell-penetrating peptides[J]. J Mol Recognit, 2003, 16: 227-233.
2 Selivanova G, Iotsova V, Okan I, et al. Restoration of the growth suppression function of mutant p53 by a synthetic peptide derived from the p53 C-terminal domain[J]. Nat Med, 1997, 3: 632-638.
3 harbour JW, Worley L, Ma D, et al. Transducible peptide therapy for uveal melanoma and retinoblastoma[J]. Arch Ophthalmol, 2002, 120: 1341-1346.
4 Snyder EL, Meade BR, Saenz CC, et al. Treatment of terminal peritoneal carcinomatosis by a transducible p53-activating peptide[J]. PLoS Biol, 2004, 2:186-193.
5 Snyder E, Saenz C, Denicourt C, et al. Enhanced targeting and Killing of Tumor cells Expressing the CXC chemokine Receptor 4 by Transducible Anticancer Peptides[J]. Cancer Res, 2005, 65: 10646-10650.
6 Bashour AA, Meng JJ, Ip W, et al. The neurofibromatosis type 2 gene product,merlin, reverses the F-Actin Cytoskeletal defects in primary human[J]. Mol Cell Biol, 2002, 22: 1150-1157.
7 Datta K, Nambudripad R, Pal S, et al. Inhibition of insulin-like growth factor I mediated cell signalling by the von Hippel-Lindau gene product in renal cancer[J]. J Biol Chem, 2000, 275: 20700-20706.
8 Arnt C, Chiorean M, Heldebrant M, et al. Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAPl in Situ[J]. J Biol Chem,2002, 277: 44236-44243.
9 Yan H, Thomas J, Liu T, et al. Induction of melanoma cell apoptosis and inhibition of tumor growth using a cell-permeable Survivin antagonist[J].Oncogene, 2006, 25: 6968-6974.
10 Fulda S, Wick W, Weller M, et al. Smac agonists sensitize for Apo2L/TRAIL-or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo[J]. Nat Med,2002, 8: 808-815.
11 Holinger E, Chittenden T, Lutz R, et al. Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases[J]. J Biol Chem,1999, 274: 13298-13304.
12 Ezhevsky SA, Nagahara H, Vocero-Akbani AM, et al. Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb[J]. Proc Natl Acad Sci USA, 1997, 94: 10699-10704.
13 Fahraeus R, Paramio JM, Ball KL, et al. Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide derived from p16CDKN2/INK4A[J]. Curr Biol, 1996, 6: 84-91.
14 Gius DR, Ezhevsky SA, Becker-Hapak M, et al. Transduced p16INK4a peptides inhibit hypophosphorylation of the retinoblastoma protein and cell cycle progression prior to acti- vation of Cdk2 complexes in late G1[J]. Cancer Res,1999,59:2577-2580.
15 el-Deiry WS,Harper JW,O'Connor JM,et al.WAF1/CIP1 is induced in p53-ymediated G1 arrest and apoptosis[J].Cancer res,1994,54:1169-1174.
16 Bonfanti M,Taverna S,Salmona M,et al.p21 WAFl-derived peptides linked to an internal- ization peptide inhibit human cancer cell growth[J].Cancer Res,1997,57:1442-1446.
17 Nagahara H,Vocero-Akbani AM,Snyder EL,et al.Transduction of full-length TAT fusionproteins into mammalian cells:TAT-p27Kipl induces cell migration[J].Nat Med,1998,4:1449-1452.
18 Parada Y,Banerji L,Glassford J,et al.BCR-ABL and intedeukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2and p27Kipl expression[J].JBiol Chem,2001,276:23572-23580.
19 Harada H,Hiraoka M,Kizaka-Kondoh S.Antitumor effect of TAT-oxygen-dependent degradation-caspase-3 fusion protein specifically stabilized and activated in hypoxic tumorcells[J].Cancer Res,2002,62:2013-2018.
20 Batchu R,Moreno A,Szmania SS,et al.Protein transduction of dendritic cells for NY-ESO-1-Based immunotherapy of myeloma[J].Cancer Res,2005,65:10041-10049.
2 Giacomoni PU, Rein G. Factors of skin ageing share common mechanisms[J].Biogerontology,2001,2:219-229.
3 Marian V, Dieter L, Jan M, et al. Free radicals and antioxidants in normal physiological functions and human disease[J]. Int J Biochem Cell Biol, 2007, 39:44 - 84.
4 Yasui K, Baba A. Therapeutic potential of superoxide dismutase (SOD) for resolution of inflammation[J]. Inflamm Res, 2006, 55: 359 - 363.
5 Kagan VE, Kisin ER, Kawai K, et al. Toward mechanism based antioxidant interventions: lessons from natural antioxidants[J]. Ann N Y Acad Sci, 2002,959: 188-198.
6 Fridovich I. Superoxide radical and superoxide dismutases[J]. Annu Rev Biochem, 1995,64:97-112.
7 Carlioz A, Touati D. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life[J]. EMBO, 1986, 5: 623-630.
8 Gao B, Flores SC, Bose SK, et al. A novel Escherichia coli vector for oxygen-inducible high level expression of foreign genes[J]. Gene 1996, 176: 269-72.
9 Elroy-Stein O, Bernstein Y, Groner Y. Overproduction of human Cu/Zn superoxide dismutase in transfected cells: extenuation of paraquat-mediated cytotoxicity and enhancement of lipid peroxidation[J]. EMBO J, 1986, 5: 615 —622.
10 Melov S, Ravenscroft J, Malik S, et al. Extension of life-span with superoxide dismutase/catalase mimetics[J]. Sci, 2000, 289: 1567 - 1569.
11 Schriner S, Linford N, Martin G, et al. Extension of Murine Life Span by Overexpression of Catalase Targeted to Mitochondria[J]. Sci, 2005, 308: 1909 -1911.
12 Igor NZ, Thomas JM, Rodney JF. Superoxide dismutase multigene family: a comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression[J]. Free Radical Bio Med. 2002, 33:337-349.
13 Marberger H, Huber W, Bartsch G, et al. Orgotein: a new antiinflammatory metalloprotein drug evaluation of clinical efficacy and safety in inflammatory conditions of the urinary tract[J]. Int Urol Nephrol, 1974, 6: 61 - 74.
14 Delanian S, Martin M, Bravard A, et al. Cu/Zn superoxide dismutase modulates phenotypic changes in cultured fibroblasts from human skin with chronic radiotherapy damage[J]. Radiother Oncol 2001, 58: 325 - 331.
15 Vozenin-Brotons MC, Sivan V, Gault N, et al. Antifibrotic action of Cu/Zn SOD is mediated by TGF-betal repression and phenotypic reversion of myofibroblasts[J]. Free Radic Biol Med, 2001, 30: 30 - 42.
16 田春美,钟秋平.超氧化物歧化酶的现状研究进展[J].中国热带医学,2005,5:1730-1732.
17 Ana IR,Marta S,Daniel M,et al.Regulation of Cu/Zn-superoxide dismutase expression via the phosphatidylinositol 3 kinase/akt pathway and nuclear factor-κB [J].J Neurosci,2004,24(33):7324 - 7334.
18 Mine E,Coppet P,Masson P,et al.The human copper-zinc superoxide dismutase gene(SOD1) proximal promoter is regulated by spl,egr-1,and wt1via non-canonical binding sites[J].J Biol Chem,1999,274:503 - 509.
19 McCord J.Therapeutic control of free radicals[J].Drug Discov Today,2004,9:781-782.
20 黄劭,刘亚伟,姜勇.细胞穿透肽在肿瘤治疗中的应用[J].生理科学进展,2007,38:301-306.
21 Baird GS,Zacharias DA,Tsien RY.Biochemistry,mutanenesis,and oligomerization of DsRed,a red fluorescent protein from coral[J].Proc Natl Acad Sci USA,2000,7:1984- 1989.
22 郭爱华,刘志峰,孙学刚,等.一种基于HIV-TAT蛋白质转导结构域细胞内转导系统的成功改建[J].南方医科大学学报,2006,26:545-548.
1 Pontus Lundberg, Ulo Langel. A brief introduction to cell-penetrating peptides[J]. J Mol Recognit, 2003, 16: 227-233.
2 Selivanova G, Iotsova V, Okan I, et al. Restoration of the growth suppression function of mutant p53 by a synthetic peptide derived from the p53 C-terminal domain[J]. Nat Med, 1997, 3: 632-638.
3 harbour JW, Worley L, Ma D, et al. Transducible peptide therapy for uveal melanoma and retinoblastoma[J]. Arch Ophthalmol, 2002, 120: 1341-1346.
4 Snyder EL, Meade BR, Saenz CC, et al. Treatment of terminal peritoneal carcinomatosis by a transducible p53-activating peptide[J]. PLoS Biol, 2004, 2:186-193.
5 Snyder E, Saenz C, Denicourt C, et al. Enhanced targeting and Killing of Tumor cells Expressing the CXC chemokine Receptor 4 by Transducible Anticancer Peptides[J]. Cancer Res, 2005, 65: 10646-10650.
6 Bashour AA, Meng JJ, Ip W, et al. The neurofibromatosis type 2 gene product,merlin, reverses the F-Actin Cytoskeletal defects in primary human[J]. Mol Cell Biol, 2002, 22: 1150-1157.
7 Datta K, Nambudripad R, Pal S, et al. Inhibition of insulin-like growth factor I mediated cell signalling by the von Hippel-Lindau gene product in renal cancer[J]. J Biol Chem, 2000, 275: 20700-20706.
8 Arnt C, Chiorean M, Heldebrant M, et al. Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAPl in Situ[J]. J Biol Chem,2002, 277: 44236-44243.
9 Yan H, Thomas J, Liu T, et al. Induction of melanoma cell apoptosis and inhibition of tumor growth using a cell-permeable Survivin antagonist[J].Oncogene, 2006, 25: 6968-6974.
10 Fulda S, Wick W, Weller M, et al. Smac agonists sensitize for Apo2L/TRAIL-or anticancer drug-induced apoptosis and induce regression of malignant glioma in vivo[J]. Nat Med,2002, 8: 808-815.
11 Holinger E, Chittenden T, Lutz R, et al. Bak BH3 peptides antagonize Bcl-xL function and induce apoptosis through cytochrome c-independent activation of caspases[J]. J Biol Chem,1999, 274: 13298-13304.
12 Ezhevsky SA, Nagahara H, Vocero-Akbani AM, et al. Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb[J]. Proc Natl Acad Sci USA, 1997, 94: 10699-10704.
13 Fahraeus R, Paramio JM, Ball KL, et al. Inhibition of pRb phosphorylation and cell-cycle progression by a 20-residue peptide derived from p16CDKN2/INK4A[J]. Curr Biol, 1996, 6: 84-91.
14 Gius DR, Ezhevsky SA, Becker-Hapak M, et al. Transduced p16INK4a peptides inhibit hypophosphorylation of the retinoblastoma protein and cell cycle progression prior to acti- vation of Cdk2 complexes in late G1[J]. Cancer Res,1999,59:2577-2580.
15 el-Deiry WS,Harper JW,O'Connor JM,et al.WAF1/CIP1 is induced in p53-ymediated G1 arrest and apoptosis[J].Cancer res,1994,54:1169-1174.
16 Bonfanti M,Taverna S,Salmona M,et al.p21 WAFl-derived peptides linked to an internal- ization peptide inhibit human cancer cell growth[J].Cancer Res,1997,57:1442-1446.
17 Nagahara H,Vocero-Akbani AM,Snyder EL,et al.Transduction of full-length TAT fusionproteins into mammalian cells:TAT-p27Kipl induces cell migration[J].Nat Med,1998,4:1449-1452.
18 Parada Y,Banerji L,Glassford J,et al.BCR-ABL and intedeukin 3 promote haematopoietic cell proliferation and survival through modulation of cyclin D2and p27Kipl expression[J].JBiol Chem,2001,276:23572-23580.
19 Harada H,Hiraoka M,Kizaka-Kondoh S.Antitumor effect of TAT-oxygen-dependent degradation-caspase-3 fusion protein specifically stabilized and activated in hypoxic tumorcells[J].Cancer Res,2002,62:2013-2018.
20 Batchu R,Moreno A,Szmania SS,et al.Protein transduction of dendritic cells for NY-ESO-1-Based immunotherapy of myeloma[J].Cancer Res,2005,65:10041-10049.