去甲斑蝥素联合IL-12、IL-15处理增强PBMC对Raja细胞的杀伤效应

Combining norcantharidin with IL-2 and IL-15 enhances the cytotoxicity of PBMC on Raji cells

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.