Objective: To investigate the release of exosome (Exo) from leukocyte-depleted red cell suspension (LDRCS) at different storage time and its regulation on proliferation of hematological tumor cells and possible mechanism.
Methods: The Exo (RBC-Exo) in LDRCS at different storage time was obtained by ultracentrifugation, and the morphology and immunological marker of RBC-Exo were detected by transmission electron microscopy and Western blot, respectively. The particle size distribution of RBC-Exo in LDRCS at different storage time was detected by Dynamic Light Scattering. CCK-8 assay was used to explore the effect of RBC-Exo on hematological tumor cell proliferation. Western blot was used to detect the expression of proliferation-related proteins in hematological tumor cells after co-culture with RBC-Exo.
Results: RBC-Exo was isolated, which was characterized by cup-like shape, particle size distribution ranged from 20 to 200 nm, CD63/TSG101 enriched, Calnexin negative, CD235a positive and CD41 negative. The particle size distribution of RBC-Exo from LDRCS between middle was not significantly different and late stored stage. But the particle size distribution of RBC-Exo at middle-late stored stage(>14 d) was larger than that at early stored stage (≤14 days). Compared with the control group, RBC-Exo could significantly promote the proliferation of HBL1, U2932 and Jurkat cells. Compared with the control group, the cycle-related protein P21 was significantly down-regulated in HBL1, U2932 and Jurkat cells after co-culture with RBC-Exo for 3 days, while the anti-apoptotic protein BCL-2 was not changed significantly.
Conclusion: The morphology of RBC-Exo from LDRCS at middle-late stored stage was different from that at early stored stage. RBC-Exo could promote the proliferation of hematological tumor cells, possibly by regulating the expression of cycle-associated protein P21.
题目: 去白细胞悬浮红细胞来源外泌体的释放及其对血液肿瘤细胞的调控.
目的: 探讨不同储存时间去白细胞悬浮红细胞（LDRCS）中外泌体（exosome，Exo）的释放及其对血液肿瘤细胞增殖的调控与可能机制.
方法: 应用超高速离心方法获得不同储存时间的LDRCS中Exo（RBC-Exo），采用透射电镜术、蛋白免疫印迹法分别对RBC-Exo的形态、免疫标记进行检测。采用动态光散射检测不同储存时间LDRCS中RBC-Exo的粒径分布变化。采用CCK-8法检测RBC-Exo对血液肿瘤细胞增殖的影响。采用蛋白免疫印迹法检测与RBC-Exo共培养后血液肿瘤细胞内增殖相关蛋白的表达变化.
结果: 分离出呈类杯状、粒径分布于20-200 nm、CD63/TSG101富集、Calnexin阴性、CD235a阳性、CD41阴性的RBC-Exo。储存中晚期（>14 d）LDRCS中RBC-Exo粒径分布无显著差异，但显著大于储存早期（储存≤14 d）的RBC-Exo。与对照组相比，RBC-Exo可以明显促进血液肿瘤细胞HBL1、U2932和Jurkat的增殖。与RBC-Exo共培养3 d的HBL1、U2932和Jurkat细胞中周期相关蛋白P21的表达较对照组明显下调，而抗凋亡蛋白BCL-2并无明显变化.
结论: 储存中晚期的LDRCS中RBC-Exo形态与储存早期存在差异。RBC-Exo可以促进血液肿瘤细胞的增殖，其机制可能是通过调节周期相关蛋白P21的表达.
Keywords: exosome; leukocyte-depleted red cell suspension; proliferation; tumor cell.