载铜绿假单胞菌DNA疫苗温敏水凝胶系统的构建及体内评价

Construction and in vivo evaluation of a thermosensitive hydrogel system loading with Pseudomonas aeruginosa DNA vaccine

构建载铜绿假单胞菌(Pseudomonas aeruginosa,PA)DNA疫苗的PLGA-PEG-PLGA温敏水凝胶系统,评价该系统的体内免疫效力。通过简单物理混合法制备PA DNA疫苗的PLGA-PEG-PLGA温敏水凝胶,测量其胶凝温度;评价其体外毒性、体外累计释放率;通过凝胶体积变化评价水凝胶的体内降解;将小鼠分为对照组(PBS)、水凝胶组(Hydrogel)、in vivo-jetPEI/质粒DNA组(in vivo-jetPEI/p DNA)、水凝胶+in vivo-jetPEI/质粒DNA组(Gel+in vivo-jetPEI/pDNA),免疫3次,每次间隔10 d,末次免疫2周后处死小鼠,检测血清特异性IgG抗体、脾淋巴细胞增殖情况及细胞上清液中IFN-γ水平。结果显示,PLGA-PEG-PLGA水凝胶具有温敏性,相变温度为(32±0.5)℃;体外对DC 2.4细胞无明显毒性;体外释放7 d时约释放80%的质粒DNA;PLGA-PEG-PLGA在体内具有可降解性,大约15 d几乎完全降解;体内免疫实验表明in vivo-jetPEI/pDNA及Gel+in vivo-jetPEI/pDNA组小鼠脾淋巴细胞明显增殖、特异性IgG抗体及IFN-γ水平升高,且水凝胶可增强DNA疫苗诱导的免疫反应水平。结果表明,载PA DNA疫苗的温敏水凝胶系统是研发PA疫苗有前景的一种新策略。

A thermosensitive hydrogel system consisting of PLGA-PEG-PLGA hydrogel and Pseudomonas aeru⁃ginosa DNA vaccine was constructed and the immune efficacy of the system was evaluated.The PLGA-PEGPLGA thermosensitive hydrogel containing Pseudomonas aeruginosa DNA vaccine was prepared by a simple physical mixing method.The gelation temperature,cytotoxicity,and release curve in vitro were tested.The degradability of hydrogel in vivo was evaluated.The mice were divided into control group(PBS),hydrogel group(Hydrogel),in vivo-jetPEI/plasmid DNA group(in vivo-jetPEI/pDNA),and hydrogel+in vivo-jetPEI/plasmid group(Gel+in vivo-jetPEI/pDNA).Mice were immunized three times with a ten-day interval.Two weeks after the last immunization,the mice were sacrificed.The proliferation of splenic lymphocytes,serum specific IgG anti⁃bodies and IFN-γin supernatant of splenic lymphocytes were detected.The gelation temperature of PLGA-PEGPLGA hydrogel was(32±0.5)℃.There was no obvious toxicity to cells in vitro,and about 80%of plasmid DNA was released after 7 days in vitro.PLGA-PEG-PLGA hydrogel was biodegradable,and degraded almost completely after 15 days in vivo.The spleen lymphocytes proliferated;the levels of specific IgG antibodies and IFN-γof in vivo-jetPEI/pDNA and Gel+in vivo-jetPEI/pDNA groups increased.The hydrogel could enhance the immune response induced by DNA vaccine.Results suggest that the thermosensitive hydrogel system consisting of PLGA-PEG-PLGA hydrogel and Pseudomonas aeruginosa DNA vaccine is a promising new strategy for the development of PA vaccine.