摘要
Background： Tumor-derived exosomes were considered to be potential candidates for tumor vaccines because they are abundant in immune-regulating proteins, whereas tumor exosomal miRNAs may induce immune tolerance, thereby having an opposite immune function. Objective： This study was designed to separate exosomal protein and depleted exosomal microRNAs （miRNAs）, increasing the immune activity of exosomes for activating dendritic cell/cytokine-induced killer cells （DC/CIKs） against pancreatic cancer （PC）. Methods： PC-derived exosomes （PEs） were extracted from cultured PANC-1 cell supernatants and then ruptured; this was followed by ultrafiltered exosome lysates （UELs）. DCs were stimulated with lipopolysaccharide （LPS）, PE, and UEL, followed by co-culture with CIKs. The anti-tumor effects of DC/CIKs against PC were evaluated by proliferation and killing rates, tumor ne- crosis factor-a （TNF-a） and perforin secretion. Exosomal miRNAs were depleted after lysis and ultrafiltration, while 128 proteins were retained, including several immune-activating proteins. Results： UEL-stimulated DC/CIKs showed a higher killing rate than LPS- and PE-stimulated DC/CIKs. Conclusions： miRNA-depleted exosome proteins may be promising agonists for specifically activating DC/CIKs against PC.
目的:本文通过分离提取无小RNA(miRNA)的外来体(exosome)刺激树突细胞/细胞因子活化杀伤细胞(DC/CIKs),激活其对于胰腺癌细胞的免疫杀伤作用。创新点:无miRNA的exosome超速离心裂解产物可以通过激活DC/CIKs细胞增强其对肿瘤细胞的杀伤作用。方法:通过收集PANC-1细胞的上清并超速离心提取其中的exosome。提取的DC细胞分别通过脂多糖、肿瘤来源exosome及无miRNA的exosome刺激后,与CIK细胞共培养。通过计算增值与杀伤效率,肿瘤坏死因子-α(TNF-α)及穿孔素的分泌,比较各组间CIK细胞对胰腺癌细胞的杀伤作用。结论:经无miRNA的exosome刺激后的CIK细胞比其他两组表现出更高的杀伤效应。实验结果表明无mi RNA的exosome蛋白在DC/CIKs细胞的胰腺癌治疗中是有相当前景的激动剂。
基金
Project supported by the National Natural Science Foundation of China(No.81272671)
the Health Bureau of Zhejiang Province(No.WKJ2013-2-018),China
