摘要
目的探讨前列腺癌来源的外泌体(exosomes)是否通过上调骨髓源性免疫抑制细胞(MDSCs)的金属基质蛋白酶(matrix metalloproteinase)MMP9、MMP2的分泌,进而增强MDSCs向外周迁移的能力。方法通过超速离心法提取鼠源性前列腺癌RM-1细胞上清中的外泌体,电镜观察外泌体的典型形态,Western blot鉴定外泌体的表面标记蛋白。流式细胞术检测荷瘤小鼠不同时间段肿瘤组织中MDSCs的比例,以及外泌体注射至正常小鼠体内后骨髓、脾脏中MDSCs的比例变化。免疫磁珠提取正常小鼠骨髓中的MDSCs,将MDSCs与RM-1-exosomes(50μg)共培养24 h后,免疫荧光观察MDSCs对外泌体的摄取情况。Western blot检测MDSCs表达MMP9、MMP2蛋白的情况,并用侵袭迁移实验检测MDSC与外泌体共培养前后的穿透能力。结果电镜下观察RM-1来源的外泌体呈典型椭圆碟形结构,直径约为30~100 nm。其表面标志蛋白CD63、HSP70、TSG101高表达验证了提取物质为外泌体。MDSCs与外泌体共培养后,免疫荧光可观察到MDSCs能大量摄取RM-1-exosome,侵袭迁移实验显示共培养后的MDSCs穿透基底膜的能力显著增强(P<0.05)。Western blot验证了共培养后的MDSCs表达MMP9、MMP2蛋白较空白组明显升高(P<0.05)。结论前列腺癌细胞来源的外泌体可通过增加MDSCs分泌MMP9、MMP2蛋白从而增强MDSCs向外周迁移的能力。
Tumor-derived exosomes, as a signal carrier of bioactive substances, could regulate the immune response of tumors. This study was aimed to investigate the mechanism of prostate cancer derived exosomes in mediating the migration of bone marrow-derived immunosuppressive cells(MDSCs) to peripheral immune organs by increasing the secretion of matrix metalloproteinase MMP9 and MMP2. Exosomes in the supernatant of RM-1 cells were extracted by ultracentrifugation. The typical morphology of exosomes was observed by electron microscopy, and the surface marker proteins of the exosomes were identified by Western blotting. Flow cytometry was used to detect the proportion of MDSCs in tumor tissues of tumor bearing mice at different times. In addition, after exosomes injection into normal mice, the proportion of MDSCs in bone marrow and spleen were measured by flow cytometry.MDSCs were extracted from bone marrow of normal mice by immunomagnetic beads, then co-cultivation with Rm-1-exosomes(50 μg) for 24 h. The uptake of exosomes by MDSCs was observed by immunofluorescence. The expression of MMP9 and MMP2 in MDSCs were detected by Western blotting, and the penetration ability of MDSCs before and after co-cultivation with exosomes was detected by invasion test. Data showed that exosomes from RM-1 demonstrated typical saucer like structure with a diameter of 30-100 nm. The high expression of CD63, HSP70 and TSG101 confirmed that the extract was exosomes. After co-cultivation with exosomes for 24 h,immunofluorescence showed that exosomes could be uptaken. Invasion and migration experiments showed that the ability of MDSCs to penetrate the basement membrane was significantly enhanced after co-cultivation(P<0.05). Western blot showed that the expression of MMP9 and MMP2 from MDSCs in exosome group was significantly higher than those in the blank group(P<0.05). In conclusion,exosomes derived from RM-1 cell line could promote migration of MDSCs to peripheral immune organs by increasing the secretion of MMP9 and MMP2 proteins.
作者
黎楠
徐浩宇
王和西
张尧
LI Nan;XU Haoyu;WANG Hexi;ZHANG Yao(Department of Urology,First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2020年第6期461-467,共7页
Immunological Journal
基金
国家自然科学基金面上项目(81272572)。
