外泌体介导多发性骨髓瘤细胞对硼替佐米的耐药

Peripheral blood exosomes from patients with multiple myeloma mediate bortezomib resistance in cultured multiple myeloma cells

目的探索由外泌体介导的多发性骨髓瘤细胞对硼替佐米(Btz)耐药的作用机制。方法在首次确诊骨髓瘤的患者中,针对Btz耐受的类型采集外周血,行超速离心法分离血清中的外泌体,并用电镜技术、NTA法以及WB技术鉴定。用不同浓度的Btz预处理骨髓瘤细胞,选择合适的药物浓度,再用不同浓度外泌体处理骨髓瘤细胞,通过MTS法检测经外泌体处理的瘤细胞对Btz的敏感程度。搜索外泌体数据库exocarta,运用STRING绘制网络图和蛋白互作表格。结果外周血中分离出的囊泡主要分布在200 nm以下,颗粒均值153 nm,众数140.1 nm。WB检测外泌体标志蛋白CD63、Tsg101和Alix表达,外泌体表达呈阳性。经外泌体预处理的骨髓瘤细胞对Btz的敏感度下降。在相同浓度Btz预处理条件下,随着外泌体处理时间、浓度的增加,骨髓瘤细胞耐药能力愈强。数据库搜索到人血清外泌体蛋白ABCB1、ABCB4、PDCD6IP、EGFR等,其中EGFR为网络节点。结论一定浓度的外泌体可作为信号载体介导多发性骨髓瘤细胞对Btz的耐药。EGFR可能是引起外泌体促进骨髓瘤细胞Btz耐药的关键蛋白。

Objective To investigate the role of exosome in mediating bortezomib(Btz) resistance in multiple myeloma cells in vitro and explore the underlying mechanisms. Methods Peripheral blood samples were collected from 15 patients with multiple myeloma with Btz tolerance, and serum exosomes were isolated by ultracentrifugation and identified with electron microscopy, NTA and Western blotting. In vitro cultured multiple myeloma cells were treated with gradient concentrations of Btz to determine the optimal drug concentration for subsequent experiment. The cells were pretreated with different concentrations of exosomes, and their sensitivity to BTZ was assessed using MTS assay. We searched the exosome database Exocarta and used STRING to generate the network map and the protein interaction graph. Results The diameters of the vesicles isolated from the peripheral blood of the patients were mostly below 200 nm with a mean particle size of 153 nm and a mode of 140.1 nm. The results of Western blotting showed that the isolated exosomes expressed the marker proteins CD63,Tsg101 and Alix. In cultured multiple myeloma cells, pretreatment with exosomes resulted in a decreased sensitivity of the cells to bortezomib, and longer treatment durations and higher exosome concentrations consistently enhanced the resistance of the cells to the same Btz concentration. Analysis of the Exocarta database identified human serum exosomal proteins ABCB1,ABCB4, PDCD6 IP, and EGFR, among which EGFR served as a network node. Conclusion Exosome within a specific concentration range may serve as a signal carrier to mediate the resistance of multiple myeloma cells to Btz. EGFR likely plays a key role to promote exosome-mediated Btz resistance in myeloma cells.