目的：探讨细胞因子IL-2、IL-15激活的人外周血单个核细胞（peripheral blood mononuclear cell，PBMC）对去甲斑蝥素（norcantharidin，NCTD）处理后的人Burkitt淋巴瘤Raji细胞的杀伤效应及其可能机制。方法：锥虫蓝拒染法检测NCTD对Raji细胞和PBMC细胞增殖的影响；LDH释放法检测IL-2、IL-15激活后的PBMC对K562细胞和Raji细胞的杀伤；流式细胞术（flow cytometry，FCM）检测IL-2、IL-15诱导后PBMC表面NKG2D的表达，以及NCTD作用前后Raji细胞表面NKG2D配体（MICA、MICB、ULBP1、ULBP2、ULBP3）的表达。结果：NCTD抑制Raji细胞的增殖，具有剂量、时间依赖性（P<0.05），但对PBMC增殖无影响（P>0.05）。在效靶比为10∶1、20∶1时，IL-2、IL-15激活的PBMC对K562细胞的杀伤率较未激活的PBMC明显增高\[（52.42±3.89）% vs （15.82±512）%，（79.55±9.22）% vs（27.67±3.66）%，P<0.05\]；PBMC对NCTD处理后Raji细胞杀伤率较未经NCTD处理的Raji细胞明显增高\[（23.63±6.20）% vs （5.04±1.25）%，（41.80±4.09）% vs （8.59±2.19）% ；P<005\]，且IL-2、IL-15激活的PBMC对NCTD处理后Raji细胞的杀伤率较NCTD处理前明显提高\[（38.97±276）% vs（13.19±367）% ，（63.09±7.30）% vs（19.89±4.15）%；P<0.05\]。激活后PBMC表面NKG2D表达率升高\[（44.91±585）% vs （25.28±7.69）%，P<0.05\]。NCTD作用后Raji细胞表面NKG2D的配体ULBP2表达显著升高\[（12.69±3.99）% vs（1.03±0.42）%，P<0.05\]，而其他配体MICA、MICB、ULBP1、ULBP3的表达无明显变化（P>0.05）。结论：NCTD联合细胞因子IL-2、IL-15处理能增强PBMC对Raji细胞的杀伤效应，其机制与NCTD上调Raji细胞表面ULBP2表达和细胞因子上调PBMC表面NKG2D表达有关。

Objective:To explore the cytotoxicity and the underlying mechanisms of peripheral blood mononuclear cells (PBMCs) activated by IL-2 and IL-15 on human Burkitt lymphoma Raji cells after being treated by norcantharidin (NCTD).Methods: Trypan blue assay was used to detect the inhibitory effect of NCTD on Raji cells and PBMC. The cytotoxic effects of PBMC activated by IL-2 and IL-15 against K562 cells and Raji cells were analyzed by LDH releasing assay. The expression of NKG2D on the surface of PBMC activated by IL-2 and IL-15 was assayed by flow cytometry (FCM). The expressions of NKG2D ligands (MICA, MICB, ULBP1, ULBP2, ULBP3) on Raji cells were assayed by FCM before and after NCTD treatment. Results: NCTD inhibited the proliferation of Raji cells significantly in a dose- and time-dependent manner (P<0.05), on the contrary, it showed no significant effect on PBMC proliferation (P>0.05). The cytotoxic rates of IL-2 and IL-15 activated PBMC on K562 cells were more than that of inactivated PBMC when the ratio of effect/target (E∶T) were 10∶1 and 20∶1 (\[52.42±3.89\]% vs \[15.82±5.12\]%, \[79.55±922\]% vs \[2767±366\]%, P<0.05), respectively. The cytotoxic rates of inactivated PBMC against Raji cells after being treated by NCTD were increased compared with untreated Raji cells (\[23.63±6.20\]% vs \[5.04±1.25\]%, \[41.80±409\]% vs \[8.59±2.19\]%, P<0.05) with E∶T of 10∶1 and 20∶1 respectively. Meanwhile, the cytotoxic rates of PBMC after being activated by IL-2 and IL-15 on NCTD-treated Raji cells were also increased compared with that of untreated PBMC (\[38.97±2.76\]% vs \[13.19±3.67\]%, \[63.09±7.30\]% vs \[19.89±4.15\]%, P<0.05). The expression of NKG2D on PBMC activated by IL-2 and IL-15 was increased compared with untreated PBMC (\[4491±585\]% vs \[25.28±769\]%, P<0.05). The expressions of NKG2D ligands on Raji cells, especially ULBP2 were up-regulated (\[12.69±3.99\]% vs \[1.03±0.42\]%, P<0.05) after being treated by NCTD, while no significant changes were found in other ligands (P>0.05). Conclusion: Combining NCTD with IL-2 and IL-15 can increase the cytotoxicity of PBMC on Raji cells, which may be related with NCTD-increased expression of ULBP2 on Raji cell surface and cytokine-increased expression of NKG2D on PBMC surface.

国家自然科学基金资助项目（No.30973454）