【摘要】：G3BP1(Ras GAP SH3-binding protein)是Ras活性的負(fù)反饋調(diào)節(jié)因子GTP酶激活蛋白(Ras GTPase activating protein,Ras GAP)SH3結(jié)構(gòu)域的特異結(jié)合蛋白。由466個(gè)氨基酸組成,位于胞質(zhì),是Ras信號(hào)通路中重要的調(diào)控分子及下游靶分子。G3BP1具有核酸內(nèi)切酶、DNA解旋酶活性,能誘導(dǎo)應(yīng)急顆粒形成,參與多種細(xì)胞生長(zhǎng)、分化、凋亡和RNA代謝的信號(hào)傳遞。因此G3BP1可能是腫瘤生長(zhǎng)的關(guān)鍵蛋白。本文研究目的是在前期Ras GAP與G3BP1的NTF2樣結(jié)構(gòu)域結(jié)合模型研究的基礎(chǔ)上,設(shè)計(jì)合成了全新多肽藥物GAP162,在分子和細(xì)胞水平上研究多肽藥物的抗腫瘤作用及可能的作用機(jī)制。對(duì)以G3BP1為作用靶點(diǎn)的的多肽藥物從體內(nèi)抗腫瘤藥效學(xué)和藥動(dòng)學(xué)方面進(jìn)行成藥性初步研究。第一部分:基于G3BP靶點(diǎn)的多肽藥物設(shè)計(jì)及體外抗腫瘤作用機(jī)理研究首先本課題與中科院大學(xué)合作在計(jì)算機(jī)模擬Ras GAP與G3BP1 NTF2樣結(jié)構(gòu)域的結(jié)合模型的基礎(chǔ)上,通過(guò)氨基酸殘基突變?cè)O(shè)計(jì)了理論上比天然Ras GAP多肽片段具有更強(qiáng)親和力的多肽片段P160,在此基礎(chǔ)上引入穿膜肽設(shè)計(jì)了多肽藥物GAP161和GAP162。首先利用ATP法研究GAP162對(duì)HCT116和A549細(xì)胞的體外抗腫瘤作用。結(jié)果表明GAP162對(duì)HCT116和A549的增殖均具有顯著的抑制作用,并且這種增殖抑制作用優(yōu)于GAP161,且不依賴于穿膜肽片段P167。為了探究GAP162的體外抑瘤作用機(jī)理,采用流式細(xì)胞術(shù)和Annxin V/PI雙染檢測(cè)給藥后HCT116的細(xì)胞凋亡情況;采用Western blot分析HCT116凋亡通路上的關(guān)鍵蛋白Caspase-3;用SPR技術(shù)測(cè)定GAP162與G3BP1親和力;免疫共沉淀法檢測(cè)GAP162對(duì)Ras GAP與G3BP1的結(jié)合的影響;免疫熒光法檢測(cè)GAP162對(duì)SG顆粒形成的影響;Western blot檢測(cè)GAP162對(duì)G3BP1蛋白總量,G3BP1蛋白Ser-149位磷酸化水平和C-myc蛋白表達(dá)的影響。實(shí)驗(yàn)結(jié)果表明GAP162能夠顯著引起HCT116細(xì)胞凋亡,并且證明這種凋亡是通過(guò)提高caspase-3的剪切活性來(lái)實(shí)現(xiàn)的。Biacore測(cè)定結(jié)果表明GAP162能夠特異地與G3BP1蛋白結(jié)合,并且親和力大于GAP161。在HCT116細(xì)胞經(jīng)GAP162處理后,分別用G3BP1和Ras GAP抗體進(jìn)行互逆免疫共沉淀后發(fā)現(xiàn)GAP162能夠減少Ras GAP與G3BP1的結(jié)合。這與我們基于G3BP蛋白與Ras GAP結(jié)合設(shè)計(jì)多肽藥物一致。用Western blot檢測(cè)不同濃度GAP162對(duì)HCT116細(xì)胞G3BP1總蛋白及Ser-149位磷酸化水平的影響,結(jié)果發(fā)現(xiàn)GAP162對(duì)G3BP1的總蛋白沒(méi)有影響,但能夠抑制Ser-149位去磷酸化水平。免疫熒光實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)在A549細(xì)胞中,GAP162能夠顯著抑制SG顆粒的形成,并且呈現(xiàn)濃度依賴性。最后在HCT116細(xì)胞中,GAP162能夠顯著降低C-myc蛋白的表達(dá)量。因此推測(cè)GAP162的腫瘤抑制作用可能的機(jī)制是:GAP162能夠與G3BP1特異結(jié)合,抑制G3BP1的Ser-149去磷酸化,進(jìn)而抑制SG顆粒的形成和C-myc蛋白表達(dá),引起細(xì)胞凋亡,起到對(duì)腫瘤細(xì)胞的增殖的抑制作用。第二部分:基于G3BP靶點(diǎn)的多肽藥物體內(nèi)藥效及藥動(dòng)學(xué)初步研究GAP161是第一個(gè)合成和進(jìn)行體內(nèi)外藥效研究的以G3BP1為靶點(diǎn)的多肽藥物。因此本文首先對(duì)GAP161進(jìn)行藥動(dòng)學(xué)研究。建立了HPLC-MS/MS法測(cè)定大鼠血漿中GAP161濃度。由于多肽藥物的特殊性,方法的選擇優(yōu)化包括:1)選擇EP管而非玻璃管作為樣品處理的容器,工作液配制的過(guò)程中加入了0.5%大鼠空白血漿來(lái)解決非特異性吸附的問(wèn)題;2)非酶切進(jìn)行整體多肽分析;3)加入0.5%的DMSO作為電荷聚集試劑;4)使用離子交換固相萃取96孔微板Oasis MAX進(jìn)行前處理;5)選擇孔徑較大的C4色譜柱進(jìn)行分離。本文建立的生物分析方法,線性范圍為5~2000 ng·m L-1,線性良好;最低定量下限為5ng·m L-1,選擇性良好;GAP161和內(nèi)標(biāo)GAP120的保留時(shí)間分別為1.51 and 1.50 min,樣品的進(jìn)樣時(shí)間3 min以內(nèi);樣品處理用96孔板進(jìn)行,提高了樣品前處理的速度,樣品回收率大于57%;日間和日內(nèi)精密度,準(zhǔn)確度滿足研究需求;在大鼠血漿中-80℃凍融2次、冰上放置12 h、樣品處理后在自動(dòng)進(jìn)樣器上放置24 h均不影響其穩(wěn)定性,但是在常溫下放置4 h,穩(wěn)定性會(huì)下降。因此整個(gè)實(shí)驗(yàn)處理過(guò)程在冰上進(jìn)行。方法學(xué)驗(yàn)證表明大鼠血漿中GAP161測(cè)定的LC-MS/MS方法滿足藥物代謝動(dòng)力學(xué)研究的要求。Sprague-Dawley大鼠單次靜脈注射5 mg·kg-1 GAP161后測(cè)定不同時(shí)間的血漿藥物濃度,并用非房室模型進(jìn)行參數(shù)計(jì)算。末端相半衰期T1/2為1.84±0.14 h,而MRT為0.258±0.053 h,表觀分布容積為35894±1621 m L·kg-1。結(jié)果表明GAP161半衰期較短、在體內(nèi)較易降解、穩(wěn)定性差。部分地解釋GAP161體內(nèi)腫瘤抑制作用不強(qiáng)。在GAP161生物分析方法的基礎(chǔ)上,建立了HPLC-MS/MS測(cè)定大鼠血漿中GAP162濃度的生物分析方法,并對(duì)GAP162在大鼠體內(nèi)的藥動(dòng)學(xué)進(jìn)行研究。Sprague-Dawley大鼠單次靜脈注射5 mg·kg-1 GAP162后測(cè)定不同時(shí)間的血漿藥物濃度,用非房室模型進(jìn)行參數(shù)計(jì)算。末端相半衰期T1/2為1.43±0.34 h,而MRT為0.649±0.053 h,表觀分布容積為7872±1061 m L·kg-1。結(jié)果表明GAP162與GAP161有相似的半衰期。本文采用Iodogen標(biāo)記法結(jié)合三氯醋酸沉淀法和分子篩排阻HPLC來(lái)研究GAP162在HCT116腫瘤裸鼠體內(nèi)重要器官及腫瘤組織的分布及其隨時(shí)間的變化。采用總放射性進(jìn)行定量。HCT116腫瘤裸鼠靜脈注射125I-GAP162后總放射性分布的AUC排序顯示GAP162在肺中的暴露水平最高,腦濃度最低,不經(jīng)血腦屏障。由數(shù)據(jù)可看出心臟,血清和腫瘤均在給藥后2 min達(dá)到組織的Cmax,并且在腫瘤組織當(dāng)中消除慢。為了評(píng)價(jià)GAP162的體內(nèi)抗腫瘤藥效,本課題用小鼠結(jié)腸癌C26細(xì)胞構(gòu)建小鼠移植腫瘤模型。接種后24 h,采用兩種給藥方式進(jìn)行劑量范圍的腹腔和皮下連續(xù)給藥GAP162(每天一次),或者劑量范圍的GAP162與順鉑劑量1 mg·kg-1聯(lián)合用藥后,比較動(dòng)物體重、荷瘤變化,計(jì)算瘤重抑制率指標(biāo)。結(jié)果顯示單獨(dú)腹腔和皮下給藥后,GAP162對(duì)C26腫瘤腫瘤模型具有顯著的抑制作用。腹腔給藥的有效劑量為40 mg·kg-1,皮下給藥的有效劑量為160 mg·kg-1。此外,GAP162與其他抗腫瘤藥物聯(lián)合給藥治療將會(huì)提高抗腫瘤效果。與GAP161相比,相同劑量下,抑瘤率提高了最少2倍。
[Abstract]:G3BP1 (Ras GAP SH3-binding protein) is a specific binding protein of the Ras GTPase activating protein (Ras GAP) SH3 domain. It is composed of 466 amino acids and is located in the cytoplasm. It is an important regulatory molecule and downstream target molecule in the Ras signal pathway. G3BP1 has endonuclease and DNase activity, can induce the formation of emergency particles, and participates in the signal transmission of various cell growth, differentiation, apoptosis and RNA metabolism. Therefore, G3BP1 may be the key protein of tumor growth. The purpose of this paper is to study the anti-tumor effect and possible mechanism of the polypeptide drug on the molecular and cellular level. In order to inhibit the formation of SG particles and the expression of C-myc protein, the effect of the expression of C-myc protein on the proliferation of the tumor cells was studied. The second part: The drug effect and the pharmacokinetics of the polypeptide drug based on the G3BP target were the first to synthesize and carry out the in-vivo drug-effect study. The GAP161 was the target of G3BP1. In this paper, the pharmacokinetic study of GAP161 was carried out in this paper. In this paper, the pharmacokinetics of GAP161 was studied in this paper. In this paper, the concentration of GAP161 in the rat plasma was determined by HPLC-MS/ MS. The selection of the method includes:1) selecting EP tube instead of glass tube as the container for sample treatment, adding 0.5% of blank plasma to solve the problem of non-specific adsorption in the process of preparation of working fluid;2) performing whole polypeptide analysis by non-enzyme digestion;3) adding 0.5% DMSO as the charge aggregation reagent;4) extracting 96-hole microplate Oasis MAX with ion exchange solid-phase extraction for pretreatment; and 5) selecting a C4 chromatographic column with a larger pore diameter to separate. In this paper, a bioanalytical method for determining the concentration of GAP162 in plasma of rat plasma by HPLC-MS/ MS was established based on the bioanalytical method of GAP161, and the pharmacokinetics of GAP162 in rats were studied. In Sprague-Dawley rats, a single intravenous injection of 5 mg 路 kg-1 GAP162 was used to determine the plasma drug concentration in different time, and the parameters were calculated with non-compartmental model. The half-life of the end phase was 1.43-0.34 h, and the MRT was 0.649-0.053 h. The apparent distribution volume was 7872-1061 m...
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R96

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相關(guān)期刊論文 前2條

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本文編號(hào)：2505702