【摘要】：運用基因轉(zhuǎn)染技術(shù)可以實現(xiàn)對細胞蛋白表達的調(diào)控,其中構(gòu)建高效低毒的基因載體是關(guān)鍵。神經(jīng)營養(yǎng)因子-3(NT-3)能促進神經(jīng)干細胞(NSC)向神經(jīng)元的分化和繁殖,維持神經(jīng)元存活,但是其在體內(nèi)不穩(wěn)定,易被降解,而被轉(zhuǎn)染了NT-3基因的受體細胞可不斷地表達NT-3。本課題利用功能短肽修飾的陽離子聚合物構(gòu)建了一類基因載體,探討了將NT-3基因轉(zhuǎn)導(dǎo)入受體細胞的表達及對NSC分化的影響作用。具體工作如下:(一)將殼聚糖氧化開環(huán)后與PEI接枝(CS-g-PEI),具有雙官能團的PEG通過加成反應(yīng)與短肽接枝(PEG-PEP),最后CS-g-PEI和PEG-PEP經(jīng)酰胺反應(yīng)連接形成CS-g-PEI-PEG-PEP。實驗結(jié)果表明四元聚合物能成功合成,電位和粒徑分布適宜,呈現(xiàn)為結(jié)構(gòu)緊密的圓球形,能有效包裹DNA且對細胞的毒性較小,復(fù)合物進入NIH-3T3和Hela、293T細胞后報告基因能從中釋放出來并被轉(zhuǎn)錄表達。(二)利用CS-g-PEI-PEG-PEP將NT-3基因運載至受體細胞后,運用酶聯(lián)免疫吸附試驗(ELISA)檢測得受體細胞能有效表達NT-3,用含有轉(zhuǎn)染后受體細胞培養(yǎng)液的分化培養(yǎng)基培養(yǎng)NSC,NSC分化成神經(jīng)元的比例提高,且神經(jīng)元軸突長度增加,存活率更高。
[Abstract]:Gene transfer technology can be used to regulate the expression of cell protein, in which the construction of high efficiency and low toxicity gene vector is the key. Neurotrophic factor-3 (NT-3) can promote the differentiation and reproduction of neural stem cell (NSC) into neurons and maintain the survival of neurons, but it is unstable and easy to degrade in vivo. The recipient cells infected with NT-3 gene can constantly express NT-3.. In this study, a class of gene vectors were constructed by using cationic polymers modified by functional peptides to investigate the expression of NT-3 gene into receptor cells and its effect on the differentiation of NSC. The specific work is as follows: (1) chitosan was oxidized and opened up and grafting with PEI (CS-g-PEI). PEG with bifunctional groups was Graft with short peptides (PEG-PEP) by addition reaction. Finally, CS-g-PEI and PEG-PEP were connected by amide reaction to form CS-g-PEI-PEG-PEP.. The results showed that the quaternion polymer could be synthesized successfully, the potential and particle size distribution were suitable, and it showed a compact spherical structure, which could effectively encapsulate DNA and had little toxicity to cells. The reporter gene could be released and expressed in NIH-3T3 and Hela,293T cells after the complex entered NIH-3T3 and Hela,293T cells. (2) after NT-3 gene was carried to recipient cells by CS-g-PEI-PEG-PEP, (ELISA) assay was used to detect the effective expression of NT-3, into neurons. The proportion of NSC,NSC differentiated into neurons in differentiation medium containing transferred receptor cell culture medium was increased, and the axonal length of neurons was increased and the survival rate was higher.
【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R450

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