本文選題：抗菌肽 + 穿膜肽�。� 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：惡性腫瘤已經(jīng)成為繼心血管疾病之后的第二大致死性疾病,嚴(yán)重危害人類健康、制約社會(huì)經(jīng)濟(jì)發(fā)展。針對(duì)惡性腫瘤的治療藥物以細(xì)胞毒性藥物為主,但是選擇性差、伴隨較大的毒副作用以及容易出現(xiàn)耐藥性等限制了它們的應(yīng)用。近年來(lái)基因治療也已經(jīng)成為某些腫瘤的有效治療方法之一。然而由于核酸分子不能透過(guò)細(xì)胞膜,所以發(fā)展高效安全的核酸藥物載體是推進(jìn)腫瘤基因治療的關(guān)鍵�？咕�(AMPs)因其獨(dú)特的作用方式而不易出現(xiàn)耐藥性,研究發(fā)現(xiàn)有些AMPs也具有抗腫瘤活性。AMPs一般是通過(guò)破壞腫瘤細(xì)胞的細(xì)胞膜來(lái)殺死腫瘤細(xì)胞,也有些AMPs是穿過(guò)細(xì)胞膜進(jìn)入細(xì)胞后通過(guò)作用于細(xì)胞器來(lái)殺死腫瘤細(xì)胞,因此有些AMPs可以有效地穿過(guò)細(xì)胞膜而作為穿膜肽(CPPs)來(lái)應(yīng)用。十八烷酸修飾可以顯著提高AMPs的轉(zhuǎn)染效率,基于此我們實(shí)驗(yàn)小組發(fā)展了18-melittin和18-K6L9等CPPs,它們具有接近于Lipofectamin 2000的轉(zhuǎn)染活性。Camel是由melittin和cecropin A組成的嵌合AMPs,具有比melittin更強(qiáng)的膜擾動(dòng)性。同時(shí)研究發(fā)現(xiàn)Camel也具有穿過(guò)細(xì)胞膜破壞線粒體導(dǎo)致細(xì)胞死亡的抗腫瘤活性。因此,我們研究了Camel的穿膜活性,并且在Camel氨基端接上不同長(zhǎng)度(C4、C8、C12、C16、C18)的脂肪酸鏈來(lái)探究脂肪酸鏈長(zhǎng)度對(duì)Camel抗腫瘤活性和細(xì)胞穿膜活性的影響。我們的研究結(jié)果表明Camel以及脂�；疌amel具有很強(qiáng)的抗腫瘤活性。其中接入短脂肪酸鏈的4-Camel、8-Camel具有快速破壞腫瘤細(xì)胞膜的活性,所以具有快速抗腫瘤活性;而接入較長(zhǎng)脂肪酸鏈的16-Camel和18-Camel則失去了快速破壞細(xì)胞膜的活性,但是在作用長(zhǎng)時(shí)間72h后具有更強(qiáng)的抗腫瘤活性,這是可能因?yàn)樗鼈冏越M裝進(jìn)入細(xì)胞后通過(guò)作用于細(xì)胞器而發(fā)揮的抗腫瘤作用。對(duì)脂�；疌amel穿膜活性的研究結(jié)果表明接十二個(gè)脂肪酸鏈后穿膜活性開(kāi)始增強(qiáng),16-Camel和18-Camel的穿膜活性最強(qiáng),其中18-Camel具有比18-melittin更強(qiáng)的轉(zhuǎn)染活性。我們的結(jié)果也表明18-Camel是以小窩蛋白介導(dǎo)的內(nèi)吞為主、以網(wǎng)格蛋白介導(dǎo)的內(nèi)吞為輔的內(nèi)吞方式進(jìn)入細(xì)胞的,并且18-Camel可以有效地促進(jìn)內(nèi)吞體逃逸達(dá)到更強(qiáng)的轉(zhuǎn)染活性。此外,血清對(duì)18-Camel的穿膜活性基本沒(méi)有影響,說(shuō)明18-Camel具有體內(nèi)應(yīng)用的潛力。進(jìn)一步的研究表明18-Camel可以攜帶p53質(zhì)粒進(jìn)入腫瘤細(xì)胞中通過(guò)增加p53蛋白的表達(dá)來(lái)提高抗腫瘤活性,同時(shí)18-Camel和p53質(zhì)粒以及MDM2抑制劑Nultin-3a的協(xié)同抗腫瘤效果更強(qiáng);此外,18-Camel也可以攜帶survivin si RNA進(jìn)入MCF-7和MDA-MB-231細(xì)胞提高抗腫瘤活性�？傊�,我們發(fā)展了一種高效抗腫瘤多肽(ACPs),它不僅可以通過(guò)直接殺死作用抑制腫瘤生長(zhǎng),也可以通過(guò)攜帶基因起到腫瘤基因治療的作用,為抗腫瘤藥物的研發(fā)提供了新的策略。
[Abstract]:Malignant tumor has become the second most fatal disease after cardiovascular disease, which seriously endangers human health and restricts social and economic development. Cytotoxic drugs are the main therapeutic drugs for malignant tumors, but their application is limited by poor selectivity, large side effects and drug resistance. In recent years, gene therapy has become one of the effective treatments for some tumors. However, because nucleic acid molecules can not penetrate the cell membrane, the development of efficient and safe nucleic acid drug carriers is the key to promote tumor gene therapy. Antimicrobial peptides (AMPs) are not susceptible to drug resistance due to their unique way of action. Studies have found that some AMPs also have anti-tumor activity. AMPs generally kill tumor cells by destroying the cell membrane of tumor cells. Some AMPs kill tumor cells by acting on organelles after entering the cells through the cell membrane, so some AMPs can effectively cross the cell membrane and be used as transmembrane peptides (CPPs). Octadecanoic acid modification can significantly improve the transfection efficiency of 18-melittin and 18-K6L9. CPPs, such as 18-melittin and 18-K6L9, have the same transfection activity as Lipofectamin 2000. Camel is a chimeric AMPs composed of melittin and cecropin A, and has stronger membrane disturbance than melittin. It was also found that Camel also had the anti-tumor activity of destroying mitochondria through the cell membrane and resulting in cell death. Therefore, we studied the transmembrane activity of Camel, and attached the fatty acid chains of different lengths to Camel amino terminal to explore the effect of the length of fatty acid chain on Camel's antitumor activity and cell membrane penetration activity. Our results show that Camel and acylated Camel have strong anti-tumor activity. 4-Camelan8-Camel with short fatty acid chains had rapid anti-tumor activity due to its rapid destruction of tumor cell membrane, while 16-Camel and 18-Camel with longer fatty acid chains lost the activity of rapid destruction of cell membrane. However, the antitumor activity was stronger after 72 h of treatment, which may be due to their antitumor effect by acting on organelles after self-assembly into the cells. The results showed that the transmembrane activity of acylated Camel began to enhance the transmembrane activity of 16-Camel and 18-Camel, and 18-Camel had stronger transfection activity than 18-melittin. Our results also showed that 18-Camel was mainly endocytosis mediated by fossa protein and endocytosis supplemented by grid protein. 18-Camel could effectively promote endocytosis escape to achieve stronger transfection activity. In addition, serum had no effect on the transmembrane activity of 18-Camel, indicating that 18-Camel has the potential to be used in vivo. Further studies showed that 18-Camel could carry p53 plasmid into tumor cells to increase the anti-tumor activity by increasing the expression of p53 protein, and the synergistic effect of 18-Camel and p53 plasmids and MDM2 inhibitor Nultin-3a was stronger. In addition, C18-Camel could also carry survivin si RNA into MCF-7 and MDA-MB-231 cells to enhance antitumor activity. In a word, we have developed an effective antitumor polypeptide, ACPsN, which can not only inhibit tumor growth through direct killing, but also provide a new strategy for the development of antitumor drugs by carrying genes to play the role of tumor gene therapy.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R730.5

【參考文獻(xiàn)】

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1 趙瑞君;程t熛，

本文編號(hào)：1987927