【摘要】： 基因治療有三個(gè)重要環(huán)節(jié),即目的基因、轉(zhuǎn)基因載體和靶細(xì)胞�；�?qū)胂到y(tǒng)是基因治療的核心技術(shù)。目前,應(yīng)用于基因治療的載體主要有病毒載體系統(tǒng)和非病毒載體系統(tǒng)。病毒載體轉(zhuǎn)染效率高,是體內(nèi)基因治療的主要工具,但安全性存在隱患及有免疫原性,體內(nèi)不能反復(fù)應(yīng)用。而非病毒載體具有安全性高、免疫原性低、易于對DNA進(jìn)行操作等優(yōu)點(diǎn),故人們愈來愈重視人工合成的非病毒載體的研究,而解決轉(zhuǎn)染效率問題是非病毒中載體中最為關(guān)注的問題,理想的載體系統(tǒng)是能將治療基因輸送到并進(jìn)入特定的靶細(xì)胞,從而能在該細(xì)胞中得到有效表達(dá),這對于惡性腫瘤基因治療尤為重要。以受體靶向的非病毒載體系統(tǒng)是最常用和有效的策略。利用細(xì)胞表面表達(dá)特異性的受體或蛋白,將特定的配體分子或片段與載體連接形成分子偶聯(lián)體,使DNA能靶向性地轉(zhuǎn)到表達(dá)受體的細(xì)胞。同時(shí),針對非病毒載體的缺陷及DNA轉(zhuǎn)移過程中的內(nèi)吞小泡釋放問題、轉(zhuǎn)運(yùn)入核問題,根據(jù)具體情況選擇合適的載體,并對其作進(jìn)一步的優(yōu)化、改善以獲得滿意的治療或應(yīng)用效果。目前常用的非病毒載體包括裸DNA脂質(zhì)體載體及陽離子多聚物型載體等。 納米粒材料的選擇是成功進(jìn)行納米基因轉(zhuǎn)運(yùn)和治療的關(guān)鍵。所選擇的材料必須是生物可降解型或者易于從體內(nèi)排泄,而不產(chǎn)生有害的降解產(chǎn)物,且無免疫原性,不引起機(jī)體的免疫排斥反應(yīng)。高分子生物降解材料制備的納米顆粒具有穩(wěn)定、無毒、無抗原性、生物相容性好、對所轉(zhuǎn)運(yùn)基因的表達(dá)有緩釋作用及對基因有保護(hù)作用等優(yōu)點(diǎn),是良好的納米基因轉(zhuǎn)運(yùn)載體材料。聚乙烯亞胺(polyethylenimine,PEI)是最常用的陽離子多聚物非病毒載體,PEI可把質(zhì)粒DNA縮合(condense)成數(shù)百納米大小的顆粒,通過靜電作用吸附到細(xì)胞表面上,被動(dòng)內(nèi)吞。PEI在吞噬泡內(nèi)不能降解,同時(shí)保護(hù)DNA免受溶酶體降解;另外,PEI有滲透性腫脹效應(yīng),導(dǎo)致吞噬泡破裂,使DNA進(jìn)入胞漿,并促進(jìn)DNA進(jìn)入細(xì)胞核。 在本研究中,我們選擇聚乙烯亞胺(polyethylenimine,PEI)通過自由基聚合法來制備PEI納米粒(nanoparticles)作為基因轉(zhuǎn)運(yùn)載體。PEI納米粒由于表面帶正電荷而獲得結(jié)合帶負(fù)電DNA的能力及某些膠體特性。同時(shí),實(shí)驗(yàn)也證實(shí),PEI納米顆粒/DNA復(fù)合物能保護(hù)所攜帶的DNA免受核酸酶的降解。當(dāng)PEI納米顆粒濃度在＜10mg/ml時(shí)PEI納米顆粒對正常的肝細(xì)胞(L-02)以及HepG2細(xì)胞無細(xì)胞毒性,當(dāng)PEI納米顆粒濃度＞25 mg/ml時(shí)細(xì)胞毒性顯著增加。在體外基因轉(zhuǎn)運(yùn)中,該納米顆�？捎行мD(zhuǎn)運(yùn)EGFP(綠色熒光蛋白)報(bào)導(dǎo)基因表達(dá)質(zhì)粒進(jìn)入NIH3T3細(xì)胞,通過流式細(xì)胞儀的檢測,PEI納米粒轉(zhuǎn)運(yùn)效率達(dá)33%,強(qiáng)于相同條件下SuperFect~(TM)的轉(zhuǎn)染效率。綜上,PEI納米載體是一種具備良好的生物安全性,高轉(zhuǎn)染及靶向能力的基因載體系統(tǒng)。
[Abstract]:Gene therapy has three important links, namely, target gene, transgenic vector and target cell. Gene introduction system is the core technology of gene therapy. At present, the main vectors used in gene therapy are viral vector system and non-viral vector system. Virus vector transfer efficiency is high, is the main tool of gene therapy in vivo, but the safety has hidden dangers and immunogenicity, and can not be used repeatedly in vivo. However, non-viral vectors have the advantages of high safety, low immunogenicity and easy to operate DNA, so people pay more and more attention to the research of synthetic non-viral vectors. To solve the problem of transfer efficiency is the most concerned problem in the non-viral vector. The ideal vector system is that the therapeutic gene can be transported to and into a specific target cell, so that it can be effectively expressed in the cell. This is particularly important for gene therapy of malignant tumors. Receptor targeting non-viral vector system is the most commonly used and effective strategy. A specific receptor or protein was expressed on the cell surface, and a specific ligand molecule or fragment was connected with the vector to form a molecular coupling, so that DNA could be targeted to the cell expressing the receptor. At the same time, aiming at the defects of non-viral vectors and the release of endocytosis vesicles in the process of DNA transfer, the appropriate vectors were selected according to the specific conditions, and the appropriate vectors were further optimized. Improve to achieve satisfactory treatment or application results. At present, the commonly used non-viral vectors include naked DNA liposomes vector and cationic polymer vector. The selection of nanoparticles is the key to successful gene transfer and treatment. The selected material must be biodegradable or easily excreted from the body without harmful degradation products, no immunogenicity, and no immune rejection. The nanoparticles prepared by polymer biodegradable materials have the advantages of stability, non-toxicity, no antigenicity, good biocompatibility, sustained release effect on the expression of transporter genes and protective effect on genes. It is a good nano-gene transport vector material. Polyethylene imine (polyethylenimine,PEI) is the most commonly used cationic polymer non-viral vector. PEI can condensation the plasmid DNA into hundreds of nanometers of particles, which can be adsorbed on the cell surface by electrostatic action. Passive endocytosis. PEI could not be degraded in phagocytic vesicles and protected DNA from lysosome degradation. In addition, PEI has osmotic swelling effect, which leads to the rupture of phagocytic vesicles, the entry of DNA into the cytoplasm, and the promotion of DNA into the nucleus. In this study, we selected polyethylene imine (polyethylenimine,). PEI nanoparticles (nanoparticles) were prepared by free radical polymerization as gene transfer vector. PEI nanoparticles obtained the ability of binding negative DNA and some colloidal properties because of the positive charge on the surface. At the same time, it was also confirmed that PEI nanoparticles / DNA complex could protect the DNA from the degradation of nuclease. When the concentration of PEI nanoparticles was < 10mg/ml, PEI nanoparticles had no cytotoxicity to normal hepatocytes (L 鈮，

本文編號：2495181