[摘要] 目的：探讨前列腺癌外泌体对基质细胞WPMY-1 迁移和侵袭能力的影响及其作用机制。方法：超速离心法提取前列腺癌LNCaP-AI+F细胞上清中的外泌体，电镜观察外泌体的典型形态结构，Zetaview 检测外泌体的粒径分布，Wb鉴定外泌体标志蛋白及其他相关蛋白。将WPMY-1 细胞与前列腺癌外泌体（40 μg/ml）共孵育后，激光共聚焦显微镜观察WPMY-1 细胞对PKH67 标记的外泌体的摄取情况，Transwell 实验检测WPMY-1 细胞迁移和侵袭能力，qPCR检测IL-8、PDGFB和MMP9 等三种肿瘤相关成纤维细胞(cancer-associated fibroblast，CAF)分子表达水平，Wb检测EGFR和ERK1/2 蛋白磷酸化水平。结果：电镜下可观察到典型茶托状外泌体结构，外泌体粒径分布集中在100 nm左右，其标志蛋白CD63 和ALIX的表达证实了所提取颗粒为外泌体。此外，外泌体还表达EGFR、HER2 和SRC 等三种与前列腺癌进展相关的蛋白。WPMY-1 细胞与外泌体共孵育后，共聚焦显微镜下可看到该细胞摄取大量外泌体，明显促进WPMY-1 细胞的迁移和侵袭能力（均P<0.01）；与对照组比较，外泌体（40 μg/ml）处理后WPMY-1 IL-8、PDGFB及MMP9 表达水平增高（P<0.05 或P<0.01）；且外泌体促进了WPMY-1 细胞EGFR和ERK1/2 的磷酸化（P<0.01）。结论：前列腺癌细胞可通过外泌体作用于基质细胞WPMY-1，使其高表达多种CAF 相关分子，促进EGFR 和ERK1/2 的磷酸化，增强其迁移和侵袭能力。

[Abstract] Objective: To investigate the effects of prostate cancer exosomes on the migration and invasion ability of stromal cells (WPMY-1), and to explore its mechanism. Methods: Exosomes in LNCaP-AI+F prostate cancer cell supernatant were isolated by ultracentrifugation and the typical structure of exosome was captured by electron microscope. The particle size distribution was analyzed by Zetaview, and Wb was used to identify the marker proteins and other proteins. After co-incubation of WPMY-1 cells and prostate cancer exosomes (40 μg/ml), laser confocal microscope was used to observe the uptake of PKH67-labeled exosomes by WPMY-1 cells; Transwell assay was used to detect the migration and invasion ability of WPMY-1 cells; qPCR was performed to detect the expression of three cancer-associated fibroblast (CAF)-related molecules (IL-8, PDGFB and MMP9) at mRNA level; and the phosphorylation of EGFR and ERK1/2 was analyzed by Wb. Results: Typical cup-shaped structure of exosomes was observed under electron microscope. The Zetaview results showed that the particle size distribution was concentrated at about 100 nm. The expression of exosome marker proteins CD63 and ALIX further verified that the isolated particles were exosomes. Besides, EGFR, HER2 and SRC, which were related to the progression of prostate cancer, were also enriched in exosomes. After co-incubation, confocal microscope imaging showed a number of PKH67 labeled exosomes in recipient WPMY-1 cells. Transwell experiments showed that exosomes could significantly enhance the migration and invasion ability of WPMY-1 cells (all P<0.01). Compared with the control group, increased secretion of IL-8,PDGFB and MMP9 was observed after exosome treatment (40 μg/ml) (P<0.05 or P<0.01). Wb indicated that exosomes could promote the phosphorylation of EGFR and ERK1/2 of WPMY-1 cells (P<0.01). Conclusion: Prostate cancer cell exosomes could act on the stromal cell WPMY-1 to highly express multiple CAF-related molecules, promote the phosphorylation of EGFR and ERK1/2 and enhance the migration and invasion ability of WPMY-1 cells.

国家自然科学基金资助项目（No.81872347)