| 郑宾宾,刘友东,庄晓鹏,管冰杰,严东旺.结直肠癌细胞外泌体激活肝星状细胞为癌相关成纤维细胞[J].,2020,(16):3001-3005 |
| 结直肠癌细胞外泌体激活肝星状细胞为癌相关成纤维细胞 |
| Hepatic Stellate Cell are Activated into Cancer-associated Fibroblasts by Colorectal Cancer Cells Exosomes |
| 投稿时间：2020-02-23 修订日期：2020-03-18 |
| DOI：10.13241/j.cnki.pmb.2020.16.001 |
| 中文关键词: 结直肠癌细胞 外泌体 肝星状细胞 癌相关成纤维细胞 肿瘤微环境 |
| 英文关键词: Colorectal cancer cell Exosomes Hepatic stellate cell Cancer-associated fibroblasts Tumor microenvironment |
| 基金项目:国家自然科学基金项目（81871931）；上海市科委医学引导项目（17411968200） |
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| 中文摘要: |
| 摘要 目的：探讨结直肠癌(CRC)细胞上清外泌体对肝星状细胞（HSC）细胞的影响。方法：通过超高速离心结合过滤法提取和纯化CRC细胞上清外泌体，然后以透射电电子显微镜(TEM)、纳米颗粒跟踪分析仪(NTA)和蛋白免疫印迹(WB)实验鉴定所提取的外泌体的形态、大小、粒径分布，以及外泌体表面标志蛋白HSP90和TSG101。再通过激光共聚焦显微镜(LSCM）观察荧光标记的外泌体被HSC细胞摄取的情况。以WB实验验证CRC细胞上清外泌体处理后的HSC中成纤维细胞活化蛋白（FAP）和α-平滑肌肌动蛋白(α-SMA)的表达水平。结果：TEM显示结直肠癌细胞外泌体呈"茶托样"杯型或类圆形囊泡样结构；NTA分析发现结直肠癌细胞外泌体直径峰值和大小分布范围分别为57 nm和30-150 nm；WB显示外泌体表面标志蛋白HSP90和TSG101均为阳性。LSCM观察发现DiO标记的外泌体（绿色），能够被Dil标记的HSC（红色）摄取。CRC细胞上清外泌体处理后的HSC中FAP和α-SMA表达水平较对照组显著升高。结论：HSC与CRC细胞上清外泌体共孵育后能够被激活成癌相关成纤维细胞。 |
| 英文摘要: |
| ABSTRACT Objective: To explore the impact of exosomes isolated from colorectal cancer cells supernatants on hepatic stellate cell. Methods: The exosomes from supernatants of CRC cells were extraction and purification via Ultracentrifugation and filtration. Then, the morphological appearance, size and particle distribution of exosomes extracted from CRC cells supernatants, as well as the exosome maker proteins HSP90 and TSG101, were identified by transmission electron microscope (TEM), nanoparticle tracking system (NTA) and western blot (WB). The endocytosis of fluorescently-labeled exosomes from CRC cells supernatants was observed by laser scanning confocal microscope (LSCM). The Protein expression levels of fibroblast activated protein (FAP) and alpha smooth muscle actin(α-SMA) in hepatic stellate cell (HSC) co-cultured with exosomes of CRC cells supernatants were verified by western blot. Results: TEM revealed that the exosomes derived from CRC cells presented "saucer like" cup-shaped morphology or rather round membrane vesicles; NTA analysis showed that the peak diameter of exosomes isolated from CRC cells supernatants were 57 nm and particle size distribution ranged from 30 to 150 nm; WB analysis indicated that exosome protein makers HSP90 and TSG101 were expressed in exosomes extracted from CRC cells supernatants; LSCM observed that DiO-labeled exosomes (green) secreted from supernatants of CRC cells were ingested by Dil-labeled HSC(red); Expression levels of FAP and α-SMA in HSC after exosomes released from CRC cells treatment were significantly higher than those in control group. Conclusion: HSC can be activated into cancer-associated fibroblasts after co-incubation with exosomes derived from supernatants of CRC cells. |
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