本文選題：藥物篩選　切入點(diǎn)：細(xì)胞穿透肽　出處：《南方醫(yī)科大學(xué)》2008年碩士論文　論文類型：學(xué)位論文

【摘要】：超氧化物岐化酶(super oxide dismutase,SOD)是一種廣泛存在于生物體內(nèi)的金屬酶,根據(jù)分布的不同主要分為SOD1(Cu/Zn SOD,主要分布于真核細(xì)胞胞漿),SOD2(Mn/Fe SOD,主要分布于真核細(xì)胞線粒體和原核細(xì)胞)和SOD3(EC-SOD,主要分布于細(xì)胞外基質(zhì)),不同的SOD均有催化超氧陰離子自由基O_2~-發(fā)生歧化反應(yīng)的功能,是體內(nèi)重要的氧自由基清除劑。其中,SOD1是發(fā)現(xiàn)最早,研究最廣泛的一類。 氧自由基是直接或間接由分子氧轉(zhuǎn)化而成,外層軌道具有未配對(duì)電子的一類化學(xué)分子,包括超氧離子自由基、羥自由基、過氧化氫等。它們具有活潑的化學(xué)活性,極易發(fā)生得到或失去電子的反應(yīng),從而引發(fā)鏈?zhǔn)阶杂苫磻?yīng)。體內(nèi)的氧自由基包括由于高溫、輻射等原因?qū)е履承┖醴肿庸矁r(jià)鍵斷裂而產(chǎn)生的外源性自由基和體內(nèi)各種代謝過程中產(chǎn)生的內(nèi)源性氧自由基。氧自由基是機(jī)體代謝正常的中間產(chǎn)物,低濃度的氧自由基是機(jī)體執(zhí)行正常生理功能所必需,但在氧自由基異常增多或機(jī)體抗氧化能力下降的情況下,氧自由基將通過鏈?zhǔn)阶杂苫磻?yīng)損傷細(xì)胞膜、DNA、蛋白質(zhì)等多種細(xì)胞組分。目前已知氧自由基與衰老、炎癥、缺血再灌注損傷、神經(jīng)退行性病變等多種疾病相關(guān),清除多余的氧自由基對(duì)延長動(dòng)物壽命,增強(qiáng)其對(duì)氧應(yīng)激的承受能力和改善炎癥狀況等多個(gè)方面具有顯著的作用。 機(jī)體主要通過兩階段的反應(yīng)來清除體內(nèi)的氧自由基:第一階段是超氧陰離子自由基在SOD的作用下轉(zhuǎn)變?yōu)檠鯕夂瓦^氧化氫;第二階段是過氧化氫在過氧化氫酶(catalase,CAT)的作用下轉(zhuǎn)變?yōu)樗脱鯕狻OD在氧自由基的清除過程中起著至關(guān)重要的作用。SOD缺陷的大腸桿菌將無法在有氧條件下生存,而適量增加的SOD則能起到減少細(xì)胞內(nèi)氧自由基含量,減輕脂質(zhì)體過氧化乃至延緩衰老、控制炎癥等多種作用�？梢�,通過控制SOD在體內(nèi)的含量,可以調(diào)節(jié)氧自由基的濃度并從而改變多種相關(guān)的病理、生理狀態(tài),具有極其重要的臨床意義。 皮膚皺紋的產(chǎn)生機(jī)制包括外源性因素和內(nèi)源性因素兩個(gè)方面。外源性因素主要有日光照射所致的光老化和其他環(huán)境因素如外傷、感染、空氣污染等對(duì)皮膚的損傷;內(nèi)源性因素主要有年齡增長導(dǎo)致的基因調(diào)控變化、內(nèi)源性自由基對(duì)細(xì)胞的損傷和機(jī)體代謝的紊亂。目前國際上主要針對(duì)光老化和內(nèi)源性氧自由基對(duì)皮膚的損傷這兩個(gè)方面進(jìn)行抗皺研究。光老化指日光長期照射所致的皮膚老化,日光中的紫外線可通過產(chǎn)生高度反應(yīng)的外源性氧自由基損傷皮膚。此外,紫外線最早損傷的部位為調(diào)節(jié)SOD和過氧化氫酶的基因,使二者合成減少,致使氧自由基聚集而損傷細(xì)胞。由上述可見,在皮膚皺紋產(chǎn)生的內(nèi)源性和外源性途徑中,過高濃度的氧自由基都扮演著重要角色。因此,清除體內(nèi)過高的氧自由基對(duì)于皮膚抗皺具有重要的意義。如果能適度提高皮膚細(xì)胞內(nèi)SOD含量,則可以通過減少氧自由基的含量來減少對(duì)皮膚的損傷,從而保護(hù)皮膚,減少皺紋。 雖然針對(duì)SOD的研究已有多年,但目前還沒有能夠?qū)嶋H進(jìn)入臨床應(yīng)用的SOD類藥物,這是因?yàn)镾OD作為一種蛋白質(zhì)藥物,具有制備價(jià)格昂貴,不能直接進(jìn)入細(xì)胞發(fā)揮作用、半衰期短、易誘發(fā)免疫反應(yīng)等缺陷,且具體使用劑量也不易控制。目前針對(duì)SOD藥用方面的研究主要包括研究藥物載體、發(fā)現(xiàn)SOD的小分子替代物和尋找能上調(diào)內(nèi)源性SOD表達(dá)藥物等途徑,據(jù)此,我們?cè)O(shè)計(jì)了一個(gè)利用報(bào)告基因技術(shù)篩選具有上調(diào)內(nèi)源性SOD1表達(dá)的隨機(jī)多肽篩選系統(tǒng),試圖同時(shí)克服上述多種問題。 報(bào)告基因是指其表達(dá)產(chǎn)物易被檢測(cè)且易與內(nèi)源性背景蛋白相區(qū)別的基因。通過將特定基因的轉(zhuǎn)錄控制元件克隆到報(bào)告基因載體,可以通過報(bào)告基因的表達(dá)來直觀地檢測(cè)目標(biāo)基因的表達(dá)情況。紅色熒光蛋白(red fluorenscent protein,RFP)是一種分子量為28 kD的熒光蛋白,可自動(dòng)發(fā)射熒光、無需任何輔助因子和底物的參與,且無需裂解細(xì)胞即可檢測(cè)活體組織和細(xì)胞的基因表達(dá),作為報(bào)告基因具有其特有的優(yōu)越性。 本研究利用基因重組技術(shù)構(gòu)建了含人SOD1啟動(dòng)子區(qū)的紅色熒光蛋白報(bào)告基因質(zhì)粒,并通過轉(zhuǎn)染和抗生素篩選技術(shù)得到了能穩(wěn)定表達(dá)該質(zhì)粒的細(xì)胞系。此細(xì)胞系在適宜濃度的SOD1誘導(dǎo)劑卟啉醇肉豆蔻酸乙酸酯(phorbol myristateacetate,PMA)刺激下,紅色熒光蛋白表達(dá)明顯增加,紅色熒光強(qiáng)度在一定范圍內(nèi)呈線性增長。這為篩選具有內(nèi)源性SOD1表達(dá)上調(diào)功能的藥物提供了一個(gè)方便、靈敏的工具。 為了克服蛋白質(zhì)大分子常具有的免疫原性,我們使用連續(xù)的十二個(gè)隨機(jī)氨基酸組成的多肽作為篩選的對(duì)象。由于細(xì)胞膜具有選擇性通透性,大部分蛋白質(zhì)分子無法直接進(jìn)入細(xì)胞內(nèi)部發(fā)揮生理學(xué)效應(yīng)。我們選用Ⅰ型人類免疫缺陷病毒(human immunodeficiency virusⅠ,HIV-Ⅰ)Tat蛋白的蛋白轉(zhuǎn)導(dǎo)域(proteintransduction domain,PTD)作為攜帶多肽入胞的載體。HIV-Tat是一種以非傳統(tǒng)內(nèi)吞途徑、非能量依賴,非受體介導(dǎo)方式直接穿透細(xì)胞膜進(jìn)入細(xì)胞的多肽,它能攜帶多種不同大小和性質(zhì)的生物活性物質(zhì)如核苷酸、多肽、蛋白質(zhì)等進(jìn)入細(xì)胞,是一種理想的多肽轉(zhuǎn)運(yùn)載體。此外,融合蛋白中的增強(qiáng)型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)可作為蛋白入胞的指示劑,提示隨機(jī)多肽在細(xì)胞內(nèi)的分布情況。針對(duì)肽庫的容量性和多樣性問題,我們利用點(diǎn)突變技術(shù)引入36個(gè)隨機(jī)堿基,形成隨機(jī)表達(dá)文庫,其理論容量達(dá)到20~(12),從而保證了隨機(jī)多肽庫的容量性和多樣性。 為了能夠進(jìn)行高通量的多肽篩選,我們總結(jié)出一套切實(shí)可行的蛋白純化及藥物篩選流程。利用融合蛋白上的His標(biāo)簽純化融合蛋白,將純化后的融合蛋白與穩(wěn)定表達(dá)報(bào)告基因質(zhì)粒的細(xì)胞孵育一定時(shí)間后,融合蛋白進(jìn)入細(xì)胞,通過測(cè)量細(xì)胞熒光強(qiáng)度的變化來判斷多肽對(duì)SOD1啟動(dòng)子的影響,從而為后續(xù)大規(guī)模的篩選奠定了基礎(chǔ)。 綜上所述,本研究利用報(bào)告基因技術(shù)和細(xì)胞系篩選技術(shù)得到了能作為人SOD1啟動(dòng)子表達(dá)情況檢測(cè)標(biāo)準(zhǔn)的細(xì)胞系,并利用基因重組和點(diǎn)突變技術(shù)得到了包含隨機(jī)多肽序列、跨膜轉(zhuǎn)運(yùn)載體和示蹤用綠色熒光蛋白的隨機(jī)多肽庫,在兩者的基礎(chǔ)上開發(fā)出一套能夠用以篩選上調(diào)內(nèi)源性SOD1表達(dá)的隨機(jī)多肽藥物的篩選流程,從而為后續(xù)的藥物篩選工作提供了有力的支持。
[Abstract]:Superoxide dismutase (super oxide, dismutase, SOD) is a kind of metal enzyme widely existed in organisms, according to the different distribution of mainly divided into SOD1 (Cu/Zn SOD, mainly distributed in the cytoplasm of eukaryotic cells (Mn/Fe), SOD2 SOD, is mainly distributed in eukaryotic and prokaryotic cells and mitochondria) SOD3 (EC-SOD, mainly distributed in the extracellular matrix, SOD) can catalyze the superoxide anion free radical O_2~- dismutation reaction function is an important oxygen free radical scavenger in vivo. Among them, SOD1 was the first discovered and studied widely.
Oxygen free radicals are directly or indirectly by molecular oxygen into, the outer track has a class of chemical molecules with unpaired electrons, including superoxide anion free radical, hydroxyl radical, hydrogen peroxide. They are chemically active and lively, prone to gain or lose electrons reaction, causing a chain reaction of free radical oxygen. Including free radicals because of high temperature, endogenous oxygen free radicals of exogenous free radicals and various metabolic processes inside radiation causes some oxygen molecules resulting in the covalent bond. Oxygen free radicals are often intermediate body on behalf of Xie Zheng, a low concentration of oxygen free radical is the body's normal physiological function of the executive necessary, but the decline in the number of abnormal oxygen free radicals and antioxidant ability under the condition of oxygen free radicals by reacting damage to the cell membrane, free radical chain DNA, proteins and other cellular components. Previously known oxygen free radicals are related to many diseases, such as aging, inflammation, ischemia reperfusion injury, neurodegenerative diseases, etc., clearing excess oxygen free radicals plays a significant role in prolonging the life span of animals, enhancing their tolerance to oxygen stress and improving the inflammatory status.
The main body of oxygen free radicals through the two stage of the reaction to remove the body: the first stage is the superoxide anion radical into oxygen and hydrogen peroxide in the presence of SOD; the second stage is the hydrogen peroxide in catalase (catalase, CAT) under the action of water and oxygen gas into.SOD in the removal of oxygen free radicals in the process the role of.SOD critical defects of Escherichia coli cannot survive under aerobic conditions, and increase the amount of SOD can be used to reduce the content of oxygen free radical in cells, reduce lipid peroxidation and aging, control of inflammation and other role. Therefore, by controlling the content of SOD in the body, can adjust the oxygen concentration free radical and pathological changes, several related physiological condition, it has important clinical significance.
Mechanism of skin wrinkles includes two aspects of exogenous and endogenous factors. Exogenous factors are mainly caused by sunlight light aging and other environmental factors such as trauma, infection, injury of air pollution on the skin; the main factors of endogenous gene regulation changes age caused by endogenous free radical damage to cells and the body's metabolic disorders. The mainly injury to skin photoaging and endogenous oxygen free radicals in the two aspects of Anti Wrinkle research. Light aging refers to sunlight long-term exposure caused by skin aging, on the ultraviolet rays can be produced by highly reactive exogenous oxygen free radical damage to the skin. In addition, the earliest ultraviolet the site of injury to the regulation of SOD and catalase genes, two reduced the synthesis of oxygen free radicals, resulting in accumulation of damaged cells. It can be seen from the above, the skin wrinkles The endogenous and exogenous pathway, oxygen free radical concentration plays an important role. Therefore, to clear the body of excessive oxygen free radical has an important significance for the skin wrinkle. If can appropriately increase the content of SOD in skin cells, can reduce skin damage by reducing the content of oxygen free radicals. In order to protect the skin, reduce wrinkles.
Although the SOD has been studied for many years, SOD class of drugs but not yet able to enter the actual clinical application, this is because the SOD as a protein drug, which is prepared by the price is expensive, can not directly enter the cells, short half-life, easy to induce immune response and other defects, and the specific dosage is not easy to control. The present research on SOD mainly includes the study of pharmaceutical drug carrier, the discovery of small molecules and looking for alternative SOD could up regulate the expression of endogenous SOD drugs and other ways, accordingly, we design a report by gene technology screening of random peptide screening system expression of endogenous SOD1, while trying to overcome the various problems.
The report refers to the gene and its expression product is easy to detect and easy to distinguish background and endogenous protein gene. The gene specific transcriptional control element was cloned into the reporter gene vector, can directly detect the expression of target genes through the expression of a reporter gene. The red fluorescent protein (red fluorenscent, protein, RFP) is a fluorescent protein with a molecular weight of 28 kD, which can automatically emit fluorescence, without any cofactor and substrate involved, and the expression can be detected without cell lysis in vivo tissue and cell gene as a reporter gene has its unique advantages.
The establishment of red fluorescent protein reporter gene plasmid containing human SOD1 promoter by gene recombination technology, and through the transfection and antibiotic screening technology has been to the plasmid stable expression cell lines. This cell line induced phorbol myristate acetate in the appropriate concentration of SOD1 (phorbol myristateacetate PMA) stimulation. The expression of red fluorescent protein increased significantly, red fluorescence intensity increases linearly in a certain range. It provides a convenient for the screening of drugs with endogenous SOD1 expression function, sensitive tool.
In order to overcome the immunogenicity of protein molecules is, we use twelve random amino acid polypeptide composed of sequential as screening objects. Because the cell membrane with selective permeability, most protein molecules can not directly enter the cell play a physiological effect. We use the human immunodeficiency virus type 1 (human immunodeficiency, virus I, HIV- I) the protein transduction domain of Tat protein (proteintransduction domain, PTD.HIV-Tat) as a carrier carrying polypeptide into cells is a kind of non conventional endocytic pathways, non energy dependent, non receptor mediated directly through cell membrane and enter the cell polypeptide, it can carry a variety of different sizes and properties of bioactive substances such as nucleotide. Peptides, proteins into cells, is a kind of ideal peptide transporter. In addition, the fusion of enhanced green fluorescent protein (enha protein in nced green fluorescent protein,EGFP)鍙綔涓鴻泲鐧藉叆鑳?yōu)鐨勬寚绀哄墸?

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