本文選題：非病毒載體 + π-π堆積�。� 參考：《吉林大學(xué)》2014年碩士論文

【摘要】：基因治療作為一種新型的治療手段，對(duì)常規(guī)治療手段無法有效應(yīng)對(duì)的疑難疾�。ㄈ缒[瘤、免疫缺陷病及某些遺傳病）有良好的臨床應(yīng)用前景。為實(shí)現(xiàn)基因治療的目的，承載治療基因的核酸（DNA或RNA）必須能夠準(zhǔn)確、穩(wěn)定、高效地進(jìn)入目的細(xì)胞，并且在病理細(xì)胞及組織中正常表達(dá)，發(fā)揮治療作用。人體內(nèi)環(huán)境的生理生化條件較為復(fù)雜，治療用核酸如果暴露在其中，很可能出現(xiàn)不同程度的降解，進(jìn)而無法達(dá)成預(yù)期的治療效果。因此，尋求便利有效的基因傳遞手段成為了基因治療應(yīng)用于臨床的首要任務(wù)之一。 基因傳遞媒介主要分為物理手段、病毒載體和非病毒載體三大類。其中非病毒載體由于容量限制小、結(jié)構(gòu)多樣、安全可控、毒性低、生物相容性好、可降解等優(yōu)點(diǎn)受到科研領(lǐng)域的關(guān)注。非病毒載體需要具備以下特點(diǎn)：容易包裝治療基因形成穩(wěn)定的復(fù)合體，能有效進(jìn)入靶細(xì)胞，在細(xì)胞內(nèi)能迅速崩解釋放核酸，無明顯的生理或遺傳毒性。目前商品化的轉(zhuǎn)染試劑大多基于陽離子脂質(zhì)體或陽離子聚合物，其進(jìn)入細(xì)胞的途徑是胞吞作用或陽離子逃逸效應(yīng)，對(duì)細(xì)胞膜表現(xiàn)出了一定程度的損傷。而且其設(shè)計(jì)缺乏針對(duì)特定靶細(xì)胞的指向性，，這意味著在臨床應(yīng)用中這類試劑的治療效果很大程度上會(huì)依賴于劑量與給藥頻度，治療過程中很可能產(chǎn)生嚴(yán)重的副作用。因此，本文設(shè)計(jì)了一種利用短肽衍生物，利用其自組裝形成的納米結(jié)構(gòu)作為基因載體，該載體系統(tǒng)具有較高的核酸搭載效率，細(xì)胞毒性低于常規(guī)轉(zhuǎn)染試劑，同時(shí)有促進(jìn)基因靶向傳遞的可能性。 本文短肽衍生物FGPep的序列設(shè)計(jì)源于π-π堆積自組裝的典型基序之一Fmoc-Phe-Phe，C端選取了針對(duì)HER2進(jìn)行肽庫篩選獲得的配體序列之一(YSSPTQR)，以若干個(gè)丙氨酸殘基作為兩者的連接部位，使其親水端具有HER2靶向性。其序列為Fmoc-FFAAYSSPTQR-NH2。我們采用經(jīng)典的Fmoc固相肽合成法合成了該衍生肽，以HPLC對(duì)其進(jìn)行純化，目的產(chǎn)物收率達(dá)80%以上。FGPep能夠在超聲作用下在水相中均勻分散并自組裝，形成有序的納米線。通過瓊脂糖凝膠電泳評(píng)估了FGPep包覆核酸的能力，在一定的濃度與混合比下，F(xiàn)GPep凝膠能夠?qū)λ觅|(zhì)粒達(dá)到完全截留。選用SK-OV-3細(xì)胞作為考察衍生肽復(fù)合物細(xì)胞毒性的對(duì)象。當(dāng)FGPep在培養(yǎng)基中的分散濃度達(dá)到500μM時(shí)，仍未見細(xì)胞有明顯的凋亡，我們認(rèn)為FGPep呈現(xiàn)的低細(xì)胞毒性符合其作為轉(zhuǎn)染載體的要求。然而實(shí)際的轉(zhuǎn)染效果不夠理想。在同等pEGFP-N1加入量的條件下，實(shí)驗(yàn)組的目的蛋白GFP表達(dá)量遠(yuǎn)低于使用脂質(zhì)體2000的陽性對(duì)照組。我們推測可能是FGPep與核酸形成的復(fù)合物尺寸偏大，難以誘導(dǎo)胞吞作用，導(dǎo)致細(xì)胞的核酸攝入量低下。 綜上所述，本文基于新思路設(shè)計(jì)的衍生肽具有自組裝和包覆核酸的能力，而且其細(xì)胞毒性較低，安全性好。但距離作為轉(zhuǎn)染材料的實(shí)際應(yīng)用仍有一定差距，需要進(jìn)一步研究與改進(jìn)。
[Abstract]:As a new therapeutic method, gene therapy has a good prospect in clinical application for difficult diseases (such as tumor, immunodeficiency disease and some hereditary diseases) which can not be effectively dealt with by conventional therapy. In order to achieve the purpose of gene therapy, nucleic acid DNA or RNAs carrying therapeutic genes must be able to enter the target cells accurately, stably and efficiently, and express them normally in pathological cells and tissues to play a therapeutic role. The physiological and biochemical conditions of human body environment are more complex. If therapeutic nucleic acid is exposed to it, it may be degraded to different degrees, and the expected therapeutic effect can not be achieved. Therefore, it is one of the most important tasks for gene therapy to seek convenient and effective means of gene transmission. Gene transfer vectors are divided into three categories: physical means, viral vectors and non-viral vectors. Among them, non-viral vectors have attracted much attention due to their small capacity limitation, diverse structure, safe and controllable, low toxicity, good biocompatibility, biodegradability and so on. Non-viral vectors need to possess the following characteristics: it is easy to package therapeutic genes to form stable complexes, which can effectively enter target cells, rapidly disintegrate and release nucleic acids in cells without obvious physiological or genetic toxicity. At present, most of the commercial transfection reagents are based on cationic liposomes or cationic polymers, which enter cells through cytosolic action or cationic escape effect, showing a certain degree of damage to cell membrane. Moreover, its design is not directed to specific target cells, which means that the therapeutic effect of these reagents in clinical application depends largely on the dosage and frequency of administration, and the treatment process is likely to have serious side effects. In this paper, we designed a novel nucleic acid carrier using short peptide derivatives and its self-assembled nanostructures as gene carriers. The carrier system has higher nucleic acid carrying efficiency and lower cytotoxicity than conventional transfection reagents. At the same time, it is possible to promote gene targeting transmission. In this paper, the sequence design of short peptide derivative FGPep originated from Fmoc-Phe-Phe-Phe-Phe-Phe-C terminal, which is one of the typical motifs of 蟺-蟺 stacking self-assembly. One of the ligand sequences obtained by peptide library screening for HER2, YSSPTQRN, was selected, and several alanine residues were used as the connecting sites. So that the hydrophilic end of HER2 target. Its sequence is Fmoc-FFAAYSSPTQR-NH2. The derivative peptide was synthesized by the classical Fmoc solid phase peptide synthesis method and purified by HPLC. The yield of the derivative peptide was over 80%. FGPep could be uniformly dispersed and self-assembled in aqueous phase under ultrasonic irradiation to form ordered nanowires. The ability of FGPep to coat nucleic acid was evaluated by agarose gel electrophoresis. SK-OV-3 cells were selected to study the cytotoxicity of derived peptide complexes. When the concentration of FGPep in the medium reached 500 渭 M, there was no obvious apoptosis. We think that the low cytotoxicity of FGPep meets the requirement of FGPep as a transfection vector. However, the actual transfection effect is not ideal. Under the same dosage of pEGFP-N1, the expression of target protein GFP in the experimental group was much lower than that in the positive control group using liposome 2000. We speculate that the complex formed by FGPep and nucleic acid is too large to induce cytosptosis, resulting in a low intake of nucleic acid. To sum up, the derived peptides designed based on the new idea have the ability of self-assembly and nucleic acid coating, and their cytotoxicity is low and their safety is good. However, there is still a gap in the application of distance as a transfection material, which needs further study and improvement.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R914

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Wei Qu;Si Chen;Shan Ren;Xue-jun Jiang;Ren-xi Zhuo;張先正;;A BIOREDUCIBLE POLYPEPTIDE FOR EFFICIENT GENE TRANSFECTION BOTH IN VITRO AND IN VIVO[J];Chinese Journal of Polymer Science;2013年05期

本文編號(hào)：2033497