經玻尿酸包覆之雙硫鍵低分子直鏈聚乙烯亞胺攜帶綠螢光蛋白序列核酸的口服成效及引發之腸道黏膜免疫反應

Oral efficacy and intestinal mucosal immune responses induced by the complex of DNA encoding Green Fluorescent Protein disulfide-crosslinked low molecular linear polyetheleneimine, and sodium hyaluronate

吳冠龍(Kuan-Long Wu)

指導教授 ： 陳怡寧

中原大學/理學院/生物科技研究所/碩士(2015年)

https://doi.org/10.6840/cycu201500871

摘要

基因疫苗是目前疫苗發展的主流，而一種新型的基因傳遞系統，以非病毒的方式攜帶抗原蛋白之基因片段進入宿主體內表達，並強烈刺激黏膜免疫及全身免疫反應，以對抗腸道致病菌。本實驗使用一個由質體DNA、雙硫鍵低分子直鏈聚乙烯亞胺(disulfide-crosslinked low molecular linear　polyetheleneimine, CLPEI)、以及玻尿酸(hyaluronic acid, HA)所組成的三元複合物（DNA-CLPEI-HA），是一種在細胞培養系統中表現出較高的基因轉染效率和低毒性的複合物。實驗中利用電子顯微鏡觀察DNA-CLPEI-HA複合物的物理特性、以DNA電泳以及限制酵素處理的方式測試口服基因傳遞系統包覆DNA的穩定性、在腸道上皮細胞的細胞培養系統中細胞轉染效率、細胞存活率、體外腸道循環以及體內接踵的小鼠實驗。要確認表達所攜帶綠色螢光蛋白(green fluorescence protein, GFP)的黏膜及全身性免疫反應，將取決於血清中的抗GFP的IgG抗體量以及GFP特異性的細胞免疫反應。實驗結果證實在DNA與CLPEI及HA結合後，會形成直徑300~400nm左右的顆粒大小，而且複合物在混和比例N/P比值為100時可以有效保護DNA不被限制酵素切除，在細胞實驗中有良好的轉染效率以及低細胞毒性，但在動物實驗中複合物仍然會對腸道造成些微的損傷。在這項研究中所開發的口服基因傳遞系統，對於用來對抗腸道致病菌及食入和排泄物途徑傳染的病原體的口服疫苗，將成為一個重要的發展平台。

關鍵字

玻尿酸,聚乙烯亞胺,口服疫苗,黏膜免疫

並列摘要

A gene delivery system carrying the gene segment of the antigen protein into the host body to express in non-viral way to stimulate mucosal and systemic immune responses has been developed. The current study used the ternary complex（DNA-CLPEI-HA）composed of plasmid DNA, disulfide-crosslinked low molecular linear polyetheleneimine(CLPEI) ,and hyaluronic acid (HA), which exhibited high gene transfection efficiency and low toxicity in cell culture systems. The examination by electron microscopes showed the physical properties of DNA-CLPEI-HA complex. The stability of DNA in the complex after oral delivery was determined by DNA electrophoresis and the digestions of restriction enzyme. The transfection efficiency of green fluorescence protein (GFP) gene delivered by DNA-CLPEI-HA complex in the intestinal epithelial cells in cell culture systems and the cell viability of the complex were examined in vitro. The expression of GFP and the immune responses induced in mice was evaluated by the levels of anti-GFP IgG antibody. The DNA-CLPEI-HA complex had a particle size of about 300 ~ 400nm. The DNA binding with CLPEI and HA at the mixing ratio N / P ratio 100 can be effectively protected from the digestion of restriction enzymes. Good transfection efficiency and low cytotoxicity were observed in cell culture system. The complex caused mild to no damage to the intestine in mice ingested orally the complex. Thus, the oral gene delivery system developed in this study had potentials to become a platform for oral vaccines used to fight intestinal pathogens.

並列關鍵字

sodium hyaluronate,polyetheleneimine,oral vaccine,mucosal immune

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