淋巴瘤细胞来源性的外泌体对人淋巴瘤细胞恶性生物学行为的影响

Effects of lymphoma cell-derived exosomes on malignant biological behaviors of human lymphoma cells

目的:观察淋巴瘤细胞来源的外泌体对淋巴瘤细胞株增殖、耐药、迁移及侵袭能力的影响。方法:诱导建立对多柔比星(doxorubicin,DOX)耐药的弥漫大B细胞淋巴瘤Su-DHL-4/DOX细胞及Burkitt淋巴瘤Raji/DOX细胞。应用商品化试剂盒Total Exosome Isolation(from cell culture media)从亲本Su-DHL-4和Raji细胞以及耐药Su-DHL-4/DOX和Raji/DOX细胞的培养上清液中分离提取外泌体(分别命名为Su-EXO、Raji-EXO、Su/DOX-EXO及Raji/DOX-EXO)。用透射电子显微镜观察提取获得的外泌体的形态及大小,采用蛋白质印迹法检测外泌体表面标志蛋白凋亡相关基因-2(apoptosis-linked gene-2,ALG-2)相互作用蛋白X(ALG-2 interacting protein-X,ALIX)和肿瘤易感基因101(tumor susceptibility gene 101,TSG101)的表达,以及纳米颗粒跟踪分析仪Zetaview检测外泌体粒径。将不同浓度的外泌体(Su-EXO和Raji-EXO)与其亲本Su-DHL-4和Raji细胞共培养后,应用锥虫蓝拒染法绘制细胞增殖曲线;不同浓度的Su-EXO、Raji-EXO以及Su/DOX-EXO(40μg/mL)和Raji/DOX-EXO(40μg/mL)与亲本Su-DHL-4和Raji细胞共培养后,CCK-8法检测DOX对淋巴瘤细胞的半数抑制浓度(half maximal inhibitory concentration,IC50),Transwell小室法检测淋巴瘤细胞的迁移和侵袭能力。结果:成功建立对DOX耐药的Su-DHL-4/DOX和Raji/DOX细胞株。透射电子显微镜下观察结果显示,分离获得的外泌体呈典型的茶托样、双层膜囊泡结构,且大小均一。蛋白质印迹法结果显示,分离获得的外泌体上有外泌体标志蛋白ALIX及TSG101的表达;Zetaview粒径分析结果显示,所提取的外泌体粒径均在30~150 nm之内。Su-DHL-4和Raji细胞与其各自来源的外泌体Su-EXO和Raji-EXO共培养后,细胞增殖能力均显著增强(P值均<0.01)。Su-DHL-4细胞与其DOX耐药Su-DHL-4/DOX细胞株来源的外泌体Su/DOX-EXO共培养后,Su-DHL-4细胞对的DOX敏感性显著降低(P<0.01)。Su-EXO和Raji-EXO能显著增强淋巴瘤细胞株Su-DHL-4与Raji细胞的迁移(P<0.05和P<0.001)和侵袭(P<0.05和P<0.01)能力。结论:淋巴瘤细胞来源性外泌体能促进淋巴瘤细胞增殖,降低其对化疗药物DOX的敏感性,并增强其迁移及侵袭能力。

Objective: To investigate the effects of lymphoma-derived exosomes on the proliferation, drug resistance, migration and invasion of lymphoma cells. Methods: The doxorubicin(DOX)-resistant diffuse large B cell lymphoma Su-DHL-4/DOX cells and Burkitt lymphoma Raji/DOX cells were induced and established. Total Exosome Isolation(from cell culture media) was used to isolate exosomes from the culture supernatants of parent Su-DHL-4 and Raji cells as well as DOX-resistant Su-DHL-4/DOX and Raji/DOX cells. The morphology and size of exosomes were observed by transmission electron microscopy, the expression levels of exosome surface markers apoptosis-linked gene-2(ALG-2) interacting protein-X(ALIX) and tumor susceptibility gene 101(TSG101) were detected by Western blotting, and the size distributions of exosomes were measured by Zetaview nanoparticle tracing analyzer. The proliferation curve was drawn by trypan blue exclusion method to detect the proliferations of Su-DHL-4 and Raji cells co-cultured with different concentrations of lymphoma-derived exosomes, and the half maximal inhibitory concentration(IC50) in DOX on lymphoma cells was detected by CCK-8 method. The migration and invasion capabilities of lymphoma cells were detected by Transwell chamber assay.Results: DOX-resistant Su-DHL-4/DOX and Raji/DOX cell lines were successfully established. The exosomes isolated from Su-DHL-4, Raji, Su-DHL-4/DOX and Raji/DOX cells were named as Su-EXO, Raji-EXO, Su/DOX-EXO and Raji/DOX-EXO, respectively. Transmission electron microscopy showed that the isolated exosomes were typical saucer-like bilayer membrane vesicles with uniform size. The isolated exosomes expressed exosome markers ALIX and TSG101 proteins. Zetaview particle size analysis showed that the size of the extracted exosomes ranged from 30 to 150 nm. After cocultured with different concentrations of exosomes Su-EXO and Raji-EXO, the proliferations of lymphoma Su-DHL-4 and Raji cells were significantly enhanced(all P < 0.01). The susceptibility of Su-DHL-4 cells to DOX was significantly reduced after co-cultured with Su/DOX-EXO derived from the DOX-resistant Su-DHL-4/DOX cells(P < 0.01). Lymphoma cell-derived exosomes Su-EXO and Raji-EXO could significantly enhance the migration(P < 0.05, P < 0.001) and invasion(P < 0.05, P < 0.01) of lymphoma Su-DHL-4 and Raji cells, respectively.Conclusion: Lymphoma-derived exosomes can promote the proliferation of lymphoma cells, reduce the sensitivity of lymphoma cells to chemotherapeutic drug DOX, and enhance the migration and invasion capabilities of lymphoma calls.