电荷修饰流感疫苗脂质体的免疫效果  

Immune effect of electric charge modified influenza vaccine liposomes

作　　者：李漂 刘艳红 刘晓琳 杨艾 张茜 鲁卫东 LI Piao;LIU Yanhong;LIU Xiaolin;YANG Ai;ZHANG Qian;LU Weidong(School of Pharmaceutical Science and Yunnan Key Laboratory of Pharmacology for Natural Products,Kunming Medical University,Kunming 650500,Yunnan Province,China)

机构地区：[1]昆明医科大学药学院暨云南省天然药物药理重点实验室,云南昆明650500

出　　处：《中国生物制品学杂志》2023年第5期518-523,共6页Chinese Journal of Biologicals

基　　金：云南省生物医药重大科研专项基金资助项目(2018ZF006)。

摘　　要：目的检测电荷修饰脂质体的理化性质,并评价其作为流感疫苗佐剂的免疫增强效果。方法以大豆卵磷脂、胆固醇、2,3-二油氧基丙基-三甲基氯化铵[N-1-(2,3-Dioleyloxy)propyl-N,N,N-trimethylam-moniumchloride,DOTAP]与1,2-二油酰基磷脂酰胆碱(1,2-dioleoyl-sn-glycero-3-phosphocholine,DOPC)(DOTAP与DOPC质量比为1∶4、2∶3、3∶2、4∶1)为膜材,制备4种电荷修饰的脂质体。将四价流感疫苗分别与制备的4种脂质体按1.4∶1.0的体积分数混合,通过冻融-冻干法将混合液制备为阳离子修饰的流感疫苗脂质体冻干粉。用PBS复溶脂质体冻干粉,采用激光粒度仪测定粒径及Zeta电位,Lowry法测定脂质体包封率。将231只雌性KM小鼠随机分为阴性对照组(PBS)、阳性对照组(四价疫苗原液)、阳离子脂质体组(4种电荷修饰的流感疫苗脂质体)和中性脂质体组(未经电荷修饰的流感疫苗脂质体),均经腹腔注射,0.2 m L/只,免疫后7、14、28 d,采用MTT法和T淋巴表面标记法评价其细胞免疫效果;免疫后3、7、14、28、42、56 d,采用血凝抑制(hemagglutinin inhibition,HI)试验评价其体液免疫效果。结果DOTAP与DOPC质量比为4∶1时,阳离子修饰脂质体的粒径和Zeta电位达最大,分别为529.65 nm和12.05 mV,此时包封率也较高,为81.82%。4个比例的阳离子脂质体组小鼠脾细胞的刺激指数(stimulus index,SI)及CD4^(+)/CD8^(+)值均明显高于阴性对照组(F=4.651~25.866,P均<0.05),其中4∶1比例组的两项指标均高于其他3个比例组;且该组小鼠于免疫后3 d即产生了早期体液免疫效果,免疫后42 d抗体滴度达最高,在整个免疫周期内均维持较高水平。结论不同电荷修饰的阳离子脂质体均能提高流感疫苗的免疫效果,其中DOTAP与DOPC质量比为4∶1时的免疫增强效果最好,免疫效应持续时间最长。Objective To explore the physical and chemical properties of electric charge modified liposomes and the immune enhancement effect as influenza vaccine adjuvants.Methods Four kinds of electric charge-modified liposomes were prepared with soybean lecithin,cholesterol,N-1-(2,3-dioleyloxy)propyl-N,N,N-trimethyllam-moniumchloride(DOTAP)and 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC)(DOTAP and DOPC mass ratio 1:4,2:3,3:2 and 4:1).The tetravalent influenza vaccine was mixed with the four liposomes according to the volume fraction of 1.4:1.0,which was prepared into cationic modified influenza vaccine liposome freeze-dried powder by freeze-thaw freeze-drying method.The liposome freeze-dried powder was redissolved with PBS,the particle size and Zeta potential were measured by laserparticle size analyzer,and the encapsulation efficiency ofliposome was measuredby Lowry method.231 female KM mice were randomly divided into negative control group(PBS),positive control group(tetravalent vaccine bulk),cationic liposome group(four electric charge modified influenza vaccine liposomes)and neutral liposome group(non-electric charge modified influenza vaccine liposomes),which were injected intraperitoneally at 7,14 and 28 d,0.2 mL per mouse.The cellular immune effect was evaluated by MTT assay and T lymphatic surface labeling,and the humoral immune effect was evaluated by hemagglutinin inhibition(HI)at 3,7,14,28,42 and 56 d of immunization.Results When the mass ratio of DOTAP to DOPC was 4:1,the particle size and Zeta potential of cationic modified liposomes reached the maximum,which were 529.65 nm and 12.05 mV respectively,and the encapsulation efficiency was also high,which was 81.82%.Stimulus index(SI)and CD4^(+)/CD8^(+)value of spleen cells of mice in the four cationic liposome groups were significantly higher than those in the negative control group(F=4.651~25.866,each P<0.05),among which,the two indexes in 4:1 cationic liposome group were higher than those in the other three groups;mice in this group produced early umoral imm

关 键 词：脂质体 电荷修饰 2 3-二油氧基丙基-三甲基氯化铵 1 2-二油酰基磷脂酰胆碱 流感疫苗 免疫原性 

分 类 号：R392-3[医药卫生—免疫学]