膀胱移行细胞癌来源的exosome诱导体外细胞毒性T细胞杀伤效应

Exosomes derived form bladder transitional cell carcinoma cells induce CTL cytotoxicity in vitro

目的观察膀胱移行细胞癌T24细胞来源的exosome体外诱导细胞毒性特异性T淋巴细胞（CTL）对肿瘤细胞的杀伤效应。方法采用超滤和蔗糖密度梯度离心法分离T24细胞释放的exosome，电镜、Western blot观察exosome的特征。将exosome和肿瘤细胞负载到人外周血分离培养的树突状细胞（DC）上，并与T细胞体外共同培养，分为exosome致敏DC组、未致敏DC组和对照组，Alamar blue检测CTL对T24细胞的细胞毒活性。结果T24细胞分泌的exosome为直径约30～90nm的类圆碟形小囊泡。Western blot证实，exosome表达热休克蛋白70（HSP70）、细胞间黏附分子1（ICAM-1）和人细胞角蛋白20（CK20）分子。与未致敏DC组和对照组比较，exosome致敏DC组活化的T细胞对T24细胞有更强的细胞毒活性（P〈0．01）。结论T24细胞来源的exosome负载了HSP70、ICAM-1等免疫相关蛋白；exosome经DC负载后活化CTL产生抗肿瘤活性。

Objective To isolate and purify exosomes derived from human bladder transitional cell carcinoma T24 cells, analyze the morphology and protein composition, and investigate the antitumor effect of specific cytotoxic T lymphocytes induced by exosomes. Methods Exosomes were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and characterized by electron microscopy and Western blot. Dendritic ceils were amplified and purified from peripheral blood and pulsed with exosomes. Then they were co-cultured with T ceils, and divided into 3 groups： exosome-pulsed DC group, unplused DC group and control group. Alamar-Blue assay was used to evaluate the specific cytolytic activity. Results The exosomes were in size about 30 -90 nm saucer-shaped membranous vesicles. HSP70, ICAM-1 and CK20 were detected by Western blot. The CTL induced by DC pulsed with exosomes had significant cytolytic activity （ P 〈 0.01 ）. Conclusion The exosomes derived from T24 cells are loaded with immunoprotein HSP70 and ICAM-1, and DC pulsed with exosomes can promote the anti-tumor effect of CTLs in vitro.