本文選題：基因傳遞 + 聚甲基丙烯酸-N　； 參考：《天津科技大學(xué)》2017年碩士論文

【摘要】：基因治療是指將基因片段(如DNA)導(dǎo)入靶細(xì)胞的一種治療疾病的方式,從而讓外源基因糾正或修復(fù)基因缺陷、關(guān)閉或抑制異常表達(dá)的基因,從而達(dá)到治愈疾病的目的�；蛑委熥鳛橐环N新型的治療癌癥的方法,在臨床方面已經(jīng)取得了重大進(jìn)展。聚甲基丙烯酸-N, N' -二甲氨基乙酯(PDMAEMA)作為一種研究細(xì)胞轉(zhuǎn)染效率與聚合物結(jié)構(gòu)之間關(guān)系的模型,在基因載體研究領(lǐng)域內(nèi)受到廣泛的關(guān)注,而且它具有優(yōu)良的轉(zhuǎn)基因性能,有望在未來(lái)成為基因傳遞領(lǐng)域的突破點(diǎn)。本文首先采用原子轉(zhuǎn)移自由基聚合法(ATRP)在水溶液中制備了一系列不同聚合度的PDMAEMA均聚物和PEG-b-PDMAEMA嵌段共聚物。通過(guò)紅外光譜、核磁共振譜等測(cè)試方法確定均聚物和共聚物在聚合物結(jié)構(gòu)、相對(duì)分子量等方面均符合預(yù)期。紫外可見(jiàn)分光光度計(jì)的光透射率-溫度測(cè)試表明PDMAEMA和PEG-b-PDMAEMA均具有溫度敏感性,并且研究了影響聚合物低臨界溶解溫度(LCST)的多種因素,從而驗(yàn)證聚合物的LCST是否會(huì)影響其作為基因載體的基因傳遞性能。本項(xiàng)目以人結(jié)腸癌細(xì)胞(HCT-116)作為研究對(duì)象,采用編碼有Flag蛋白標(biāo)簽的pCMV-3×Flag-LTA4H大腸桿菌進(jìn)行質(zhì)粒DNA的擴(kuò)培、提取,利用帶正電的聚合物能與帶負(fù)電的DNA靜電相互作用自組裝形成納米微球的原理,將合成的PDMAEMA均聚物和PEG-b-PDMAEMA嵌段共聚物應(yīng)用于基因傳遞方面,以商品化的Lipofectamine 2000 Reagent(Life)轉(zhuǎn)染劑為參照,研究所制備聚合物的基因傳遞性能,從細(xì)胞轉(zhuǎn)染效率和細(xì)胞毒性等多方面進(jìn)行了評(píng)價(jià)。結(jié)果表明,PDMAEMA均聚物有較高的體外轉(zhuǎn)染效率,同時(shí)也具有了一定的細(xì)胞毒性。PEG化可以改善基因載體的應(yīng)用,盡管PEG化后會(huì)降低轉(zhuǎn)染效率,但它能夠降低細(xì)胞毒性。更為重要的是,PEG可以作為一種連接體,為進(jìn)一步提高載體性能提供廣闊的發(fā)展空間。
[Abstract]:Gene therapy is a way to treat diseases by introducing gene fragments (such as DNA) into target cells, so that exogenous genes can correct or repair gene defects, turn off or inhibit abnormal expression of genes, and achieve the goal of curing diseases.Gene therapy, as a new method of cancer treatment, has made great progress in clinical practice.As a model to study the relationship between cell transfection efficiency and polymer structure, poly (methacrylate), N '-dimethylaminoethyl ester (PDMAEMA), has attracted extensive attention in the field of gene vector research, and it has excellent transgenic performance.It is expected to become a breakthrough point in the field of gene transmission in the future.In this paper, a series of PDMAEMA homopolymers and PEG-b-PDMAEMA block copolymers with different degrees of polymerization were prepared by atom transfer radical polymerization (ATRP) in aqueous solution.The structure and relative molecular weight of homopolymers and copolymers were determined by IR, NMR and other methods.The phototransmittance and temperature measurements of UV-Vis spectrophotometer show that both PDMAEMA and PEG-b-PDMAEMA are temperature-sensitive, and many factors affecting the low critical solution temperature of polymer are studied.To verify whether the polymer's LCST will affect its gene transfer performance as a gene vector.The human colon cancer cell line HCT-116) was used as the research object. The plasmid DNA was amplified and extracted by pCMV-3 脳 Flag-LTA4H E. coli with Flag protein tag.Based on the principle that polymer with positive charge can interact with negatively charged DNA to form nano-microspheres, the synthesized PDMAEMA homopolymers and PEG-b-PDMAEMA block copolymers were used in gene transfer, as compared with commercial Lipofectamine 2000 ReagentLife-based transcoloring agent.The gene transfer properties of the prepared polymers were evaluated in terms of cell transfection efficiency and cytotoxicity.The results showed that PDMAEMA homopolymer had a high transfection efficiency in vitro and a certain cytotoxicity. PEGylation could improve the application of the gene vector. Although the transfection efficiency was decreased after PEG, it could reduce the cytotoxicity.More importantly, PEG can be used as a kind of connectors, which provides a broad development space for further improving the performance of the carrier.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R450

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本文編號(hào)：1768105