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作 者:董超[1] 刘迪[1] 冯业童[1] 刘朋飞[1] 吴璇[1] 吴昊昱[1] 周余来[1]
机构地区:[1]吉林大学药学院生物工程实验中心,吉林长春130021
出 处:《吉林大学学报(医学版)》2013年第3期472-476,共5页Journal of Jilin University:Medicine Edition
基 金:吉林省卫生厅科研基金资助课题(2009Z043);吉林大学博士研究生交叉学科科研基金资助课题(2011J023)
摘 要:目的:从结直肠癌细胞系中分离鉴定外泌体,初步研究结直肠癌来源的外泌体潜在的免疫学特性,为外泌体在结直肠癌免疫学治疗中的应用提供依据。方法:超速离心法从HCT116和SW480 2种结直肠癌细胞系培养上清中分离提取正常细胞外泌体和热休克细胞外泌体,进一步用220nm的滤膜进行纯化获得纯度较高的外泌体。采用透射电镜观察所提取外泌体表征形态,聚丙烯酰胺凝胶电泳法分析细胞和外泌体的蛋白组成,检测外泌体促进外周血单个核细胞(PBMCs)的增殖功能。结果:透射电镜下正常的外泌体和细胞热休克获得的外泌体形态相近,均呈现典型的圆形或椭圆形外泌体结构,直径为50~100nm。聚丙烯酰胺凝胶电泳,结直肠癌细胞裂解物与结直肠癌细胞系来源外泌体的蛋白组成不同,与未处理和经热休克处理的结直肠癌细胞比较,外泌体对PBMCs的增殖有更强的促进作用(P<0.05),热休克作用可使外泌体诱导的PBMCs的增殖程度显著升高(P<0.05)。结论 :超速离心法和滤膜过滤方法可有效地从结直肠癌细胞系中提取外泌体,细胞裂解物和外泌体的蛋白组成差异可能是造成免疫学特性差异的主要原因,将细胞经热休克处理可增强外泌体的免疫原性。Objective To separate and identify the exosomes from colorectal carcinoma cells and to preliminarily study their immunological characteristics,and to provide basis for the application of exosomes in colorectal carcinoma immunotherapy.Methods The normal exosomes and heat shock exosomes of HCT116 and SW480 cells were separated by ultracentrifugation and purified by 220 nm microspore film.The morphology of exosomes were observed under transmission electron microscope.The protein compositions of cells and exosomes were analyzed by SDS-PAGE method.The promoting function of exosomes in proliferation of peripheral blood monouclear cells(PBMCs) was detected.Results Under transmission electron microscope,the normal exosomes and heat shock exosomes had similar appearance.They were either spherical or oval membrane vesicles,whose diameters were about 50-100 nm.The results of SDS-PAGE showed the differences of protein compositions between colorectal cell lysates and exosomes derived from colorectal carcinoma cells.Compared with untreated and heat shock treated colorectal carcinoma cells,the exosomes had stronger promotion effect on PBMCs proliferation(P0.05).The proliferaton of PBMCs could be reinforced with heat shock(P0.05).Conclusion For separation and identification of the exosomes derived from colorectal carcinoma cells,the method of ultracentrifugation and filtration is simple and feasible.The differences of protein composition between colorectal cell lysates and exosomes may be the major reason of their differences in immunological characteristics.The immuogenicity of exosomes could be enhanced by treating the cells with heat shock.
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