狂犬疫苗壳聚糖微球制备及细胞免疫特性研究
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摘要
通过喷雾干燥法制备狂犬疫苗壳聚糖微球冻干粉,以微球吸附疫苗原液的吸附率为主要评价指标,运用均匀设计试验确定制备微球的最佳处方并对最佳处方得到的微球进行粒度分布、吸附率及免疫原性考查;用狂犬疫苗壳聚糖微球、狂犬疫苗脂质体、空白壳聚糖微球和生理盐水分别免疫小鼠,通过脾淋巴细胞增殖试验检测脾淋巴细胞增殖能力。结果表明:采用优化工艺制备的狂犬疫苗壳聚糖微球冻干粉复溶后于显微镜下观察呈圆形或近似球形,分散性好,粒度分布均匀,平均粒径为4.71μm,吸附率为74.21%;在小鼠免疫第7天时狂犬疫苗壳聚糖微球组和狂犬疫苗脂质体组产生的脾淋巴细胞增殖水平(SI值)无显著差异,分别与空白微球组和生理盐水组比较均有极显著差异,说明壳聚糖微球作为疫苗佐剂也可增强机体的细胞免疫。
Rabies vaccine chitosan microspheres was prepared by the spray drying method.The uniform design experiment was adopted to optimize the formulation by using adsorption rate as the main index.The particle distribution,adsorption rate and immunogenicity of the resulted product were investigated.Mice were immunized with rabies vaccine chitosan microspheres,rabies vaccine lyophilized liposome,blank chitosan microspheres and normal saline respectively.Then the splenic lymphocyte proliferation ability was studied by spleen lymphocyte proliferation test.The results showed that:the shapes of prepared rabies vaccine chitosan microspheres were round or nearly spherical,and the particle sizes were evenly distributed with the mean size of 4.71μm and the adsorption rate of 74.21%.After seven days of immunization,the proliferation level of splenic lymphocyte(SI value)of chitosan microsphere group and liposome group had no significant difference.While compared with that of blank microsphere group and saline group,there were great differences(P<0.01).The data revealed that chitosan microsphere as rabies vaccine adjuvant can strengthen the cellular immunity of the body as liposome did based on previous study.
Rabies vaccine chitosan microspheres was prepared by the spray drying method.The uniform design experiment was adopted to optimize the formulation by using adsorption rate as the main index.The particle distribution,adsorption rate and immunogenicity of the resulted product were investigated.Mice were immunized with rabies vaccine chitosan microspheres,rabies vaccine lyophilized liposome,blank chitosan microspheres and normal saline respectively.Then the splenic lymphocyte proliferation ability was studied by spleen lymphocyte proliferation test.The results showed that:the shapes of prepared rabies vaccine chitosan microspheres were round or nearly spherical,and the particle sizes were evenly distributed with the mean size of 4.71μm and the adsorption rate of 74.21%.After seven days of immunization,the proliferation level of splenic lymphocyte(SI value)of chitosan microsphere group and liposome group had no significant difference.While compared with that of blank microsphere group and saline group,there were great differences(P<0.01).The data revealed that chitosan microsphere as rabies vaccine adjuvant can strengthen the cellular immunity of the body as liposome did based on previous study.
引文
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[20]屈娜,郭立玮,朱华旭,等.离子交联-喷雾干燥联用技术制备鼻用黄芩苷微球及其体外性能评价[J].中国实验方剂学杂志,2016,22(7):1-5.
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[22]徐菀佚,乔建斌,马波,等.冻融-冻干法制备的流感疫苗脂质体的细胞免疫研究[J].中国药科大学学报,2015,46(6):730-733.
[23]刘洁,马波,鲁卫东,等.单价流感疫苗脂质体干粉细胞免疫研究[J].南京工业大学学报(自然科学版),2011,33(6):102-106.
[24]叶红艳,徐菀佚,张益丽,等.狂犬疫苗脂质体冻干粉制备及免疫原性研究[J].扬州大学学报(农业与生命科学版),2016,37(3):11-14.
[2]DREESEN D W.A global review of rabies vaccines for human use[J].Vaccine,1997,15(Suppl):2-6.
[3]PIYASIRISILP S.Neurological adverse events associated with vaccination[J].Change Current Opinion in Neurology,2002,15(3):333-338.
[4]鲁卫东,林意菊,马波,等.流感疫苗脂质体干粉的稳定性[J].中国药科大学学报,2010,41(4):360-362.
[5]高立栋,王秀梅,尹铁娜.恶性胸水的局部药物治疗[J].临床医学,1997(3):31-34.
[6]邓苏宏,刘君杰,胡四海.细胞因子佐剂对核酸疫苗免疫效果的影响及作用机制[J].细胞与分子免疫学杂志,2013,29(9):1004-1007.
[7]邱少辉,方鑫,张然,等.氢氧化铝佐剂对重组乙型肝炎疫苗诱导的免疫相关细胞因子的影响[J].中国生物制品学杂志,2015,28(9):889-893.
[8]岳晓琳,殷冬冬,王瑞,等.小鹅瘟病毒“自杀性”DNA疫苗对小鼠免疫原性分析[J].扬州大学学报(农业与生命科学版),2016,37(1):6-10.
[9]KO J A,PARK H J,HWANG S J,et al.Preparation and characterization of chitosan microparticles intended for controlled drug delivery[J].International Journal of Pharmaceutics,2002,249(1/2):165-174.
[10]SINHA V R,SINGLA A K,WADHAWAN S,et al.Chitosan microspheres as a potential carrier for drugs[J].International Journal of Pharmaceutics,2004,274(1/2):1-33.
[11]DIAZ A G,QUINTEROS D A,LLABOT J M,et al.Spray dried microspheres based on chitosan:apromising new carrier for intranasal administration of polymeric antigen BLSOmp31for prevention of ovine brucellosis[J].Materials Science&Engineering,Materials for Biological Applications,2016,62:489-496.
[12]OCHIUZ L.Preparation and characterisation of alendronate-loaded chitosan microparticles obtained through the spray drying technique[J].Medicinal Chemistry(United Arab Emirates),2009,5(2):191-196.
[13]KANG M L,KANG S G,JIANG H L,et al.Chitosan microspheres containing Bordetella bronchiseptica antigens as novel vaccine against atrophic rhinitis in pigs[J].Journal of Microbiology and Biotechnology,2008,18(6):1179-1185.
[14]VAN DER LUBBEN I M,VERHOEF J C,BORCHARD G.Chitosan for mucosal vaccination[J].Advanced Drug Delivery Reviews,2001,52(2):139-144.
[15]ALPAR H O,SOMAVARAPU S,ATUAH K N.Biodegradable mucoadhesive particulates for nasal and pulmonary antigen and DNA delivery[J].Advanced Drug delivery Reviews,2005,57(3):411-430.
[16]MOHAMMADPOURDOUNIGHI N,BEHFAR A,EZABADI A,et al.Preparation of chitosan nanoparticles containing Naja naja oxiana snake venom[J].Nanomedicine:Nanotechn-ology,Biology,and Medicine,2010,6(1):137-143.
[17]LI Y,AI L,YOKOYAMA W,et al.Properties of chitosan-microencapsulated orange oil prepared by spraydrying and its stability to detergents[J].Journal of Agricultural and Food Chemistry,2013,61(13):3311-3319.
[18]SUN Y,GU L,GAO Y.Preparation and characterization of 5-Fluorouracil loaded chitosan microspheres by a twostep solidification method[J].Chemical&Harmaceutical Bulletin,2010,58(7):891-895.
[19]PAMUJULA S,GRAVES R A,MOISEYEV R,et al.Preparation of polylactide-co-glycolide and chitosan hybrid microcapsules of amifostine using coaxial ultrasonic atomizer with solvent evaporation[J].The Journal of Pharmacy and Pharmacology,2008,60(3):283-289.
[20]屈娜,郭立玮,朱华旭,等.离子交联-喷雾干燥联用技术制备鼻用黄芩苷微球及其体外性能评价[J].中国实验方剂学杂志,2016,22(7):1-5.
[21]HUANG Y C,CHIANG C H.Optimizing formulation factors in preparing chitosan microparticles by spray-drying method[J].Journal of Microencapsulation,2003,20(2):247-260.
[22]徐菀佚,乔建斌,马波,等.冻融-冻干法制备的流感疫苗脂质体的细胞免疫研究[J].中国药科大学学报,2015,46(6):730-733.
[23]刘洁,马波,鲁卫东,等.单价流感疫苗脂质体干粉细胞免疫研究[J].南京工业大学学报(自然科学版),2011,33(6):102-106.
[24]叶红艳,徐菀佚,张益丽,等.狂犬疫苗脂质体冻干粉制备及免疫原性研究[J].扬州大学学报(农业与生命科学版),2016,37(3):11-14.